技术领域Technical field
本发明涉及遗传工程领域中提高苜蓿产量和/或分枝数的方法及其所用蛋白质与相关生物材料。The present invention relates to methods for improving alfalfa yield and/or branch number in the field of genetic engineering, as well as proteins and related biological materials used therein.
背景技术Background technique
在自然环境下，植物由于不可移动性，其生长经常遭受不良环境的影响。随着全球变暖的加剧，干旱胁迫成为了影响植物生长最严重的非生物胁迫。紫花苜蓿在我国主要种植在一些干旱和半干旱地区。植物在长期驯化中，形成了许多应对干旱胁迫的分子、生理以及生化机制，其中分子机制是控制生理和生化机制的关键决定因子，从植物中鉴定和克隆关键的干旱胁迫响应基因，通过生物技术手段操纵关键基因表达，尤其是转录因子基因的表达，可以综合调控多个生理响应过程，对增强植物抗旱性具有重要意义。研究表明，干旱胁迫下，植物常常通过主动限制生长，减少水分散失响应干旱胁迫。因此，从植物中挖掘可以促进植物生长并显著增强抗旱性的关键基因，可以为利用生物育种技术改良作物抗旱性提供基因资源，是解决干旱地区作物产量减产严重等问题的重要基础。In natural environments, plants often suffer from adverse environmental impacts due to their immobility. With the intensification of global warming, drought stress has become the most serious abiotic stress affecting plant growth. Alfalfa is mainly grown in some arid and semi-arid areas in my country. During the long-term domestication of plants, many molecular, physiological and biochemical mechanisms have been formed to respond to drought stress. Molecular mechanisms are key determinants in controlling physiological and biochemical mechanisms. Key drought stress response genes have been identified and cloned from plants, and through biotechnology Methods to manipulate the expression of key genes, especially the expression of transcription factor genes, can comprehensively regulate multiple physiological response processes, which is of great significance for enhancing plant drought resistance. Research shows that under drought stress, plants often respond to drought stress by actively limiting growth and reducing water loss. Therefore, mining key genes from plants that can promote plant growth and significantly enhance drought resistance can provide genetic resources for using biological breeding technology to improve crop drought resistance, and is an important basis for solving problems such as severe crop yield reduction in arid areas.
发明内容Contents of the invention
本发明解决的技术问题是如何提高苜蓿产量。The technical problem solved by the invention is how to increase alfalfa yield.
为了解决上述问题，本发明提供了提高苜蓿产量和/或分枝数的方法。In order to solve the above problems, the present invention provides a method for increasing alfalfa yield and/or branch number.
所述方法包括上调或增强或提高苜蓿中蛋白质的编码基因的表达和/或所述蛋白质的活性和/或含量，以提高苜蓿的产量和/或分枝数，所述蛋白质为如下任一种蛋白质：The method includes up-regulating or enhancing or increasing the expression of a gene encoding a protein in alfalfa and/or the activity and/or content of the protein to increase the yield and/or branch number of alfalfa, and the protein is any one of the following protein:
B1)氨基酸序列是序列2所示的蛋白质；B1) The amino acid sequence is the protein shown in sequence 2;
B2）将B1)所述蛋白质的经过氨基酸残基的取代和/或缺失和/或添加得到的与B1)所示的蛋白质具有80％以上的同一性且具有相同功能的蛋白质；B2) A protein with more than 80% identity and the same function as the protein shown in B1) obtained by substituting and/or deleting and/or adding amino acid residues of the protein described in B1);
B3）将B1)或B2）的N末端或/和C末端连接蛋白质标签得到的融合蛋白质。B3) A fusion protein obtained by connecting a protein tag to the N-terminus or/and C-terminus of B1) or B2).
上述的方法中，所述上调或增强或提高苜蓿中蛋白质的编码基因的表达包括向苜蓿中导入所述蛋白质的编码基因。In the above method, the up-regulating or enhancing or increasing the expression of the protein-coding gene in alfalfa includes introducing the protein-coding gene into alfalfa.
为了解决上述问题，本发明还提供了培育产量高和/或分枝数多的苜蓿方法。In order to solve the above problems, the present invention also provides a method for cultivating alfalfa with high yield and/or large number of branches.
所述方法包括上调或增强或提高目的苜蓿中蛋白质的编码基因的表达和/或所述蛋白质的活性和/或含量，得到产量高和/或分枝数多的苜蓿，所述产量高和/或分枝数多的苜蓿的产量和/或分枝数高于所述目的苜蓿；The method includes up-regulating or enhancing or increasing the expression of the gene encoding the protein in the target alfalfa and/or the activity and/or content of the protein, to obtain alfalfa with high yield and/or large number of branches, and the high yield and/or high number of branches. Or the yield and/or number of branches of alfalfa with a large number of branches is higher than that of alfalfa of the stated purpose;
所述蛋白质为如下任一种蛋白质：The protein is any of the following proteins:
B1)氨基酸序列是序列2所示的蛋白质；B1) The amino acid sequence is the protein shown in sequence 2;
B2）将B1)所述蛋白质的经过氨基酸残基的取代和/或缺失和/或添加得到的与B1)所示的蛋白质具有80％以上的同一性且具有相同功能的蛋白质；B2) A protein with more than 80% identity and the same function as the protein shown in B1) obtained by substituting and/or deleting and/or adding amino acid residues of the protein described in B1);
B3）将B1)或B2）的N末端或/和C末端连接蛋白质标签得到的融合蛋白质。B3) A fusion protein obtained by connecting the N-terminus or/and C-terminus of B1) or B2) to a protein tag.
为了解决上述问题，本发明还提供了提高苜蓿抗旱性的方法In order to solve the above problems, the present invention also provides a method for improving the drought resistance of alfalfa
所述方法包括上调或增强或提高苜蓿中蛋白质的编码基因的表达和/或所述蛋白质的活性和/或含量，以提高苜蓿的抗旱性；The method includes up-regulating or enhancing or increasing the expression of a gene encoding a protein in alfalfa and/or the activity and/or content of the protein to improve the drought resistance of alfalfa;
所述蛋白质为如下任一种蛋白质：The protein is any of the following proteins:
B1)氨基酸序列是序列2所示的蛋白质；B1) The amino acid sequence is the protein shown in sequence 2;
B2）将B1)所述蛋白质的经过氨基酸残基的取代和/或缺失和/或添加得到的与B1)所示的蛋白质具有80％以上的同一性且具有相同功能的蛋白质；B2) A protein with more than 80% identity and the same function as the protein shown in B1) obtained by substituting and/or deleting and/or adding amino acid residues of the protein described in B1);
B3）将B1)或B2）的N末端或/和C末端连接蛋白质标签得到的融合蛋白质。B3) A fusion protein obtained by connecting a protein tag to the N-terminus or/and C-terminus of B1) or B2).
上述的方法中，所述上调或增强或提高苜蓿中蛋白质的编码基因的表达包括向苜蓿中导入所述蛋白质的编码基因。In the above method, the up-regulating or enhancing or increasing the expression of the protein-coding gene in alfalfa includes introducing the protein-coding gene into alfalfa.
为了解决上述问题，本发明还提供了培育抗旱性强的苜蓿方法。In order to solve the above problems, the present invention also provides a method for cultivating alfalfa with strong drought resistance.
所述方法包括上调或增强或提高目的苜蓿中蛋白质的编码基因的表达和/或所述蛋白质的活性和/或含量，得到抗旱性强的苜蓿，所述抗旱性强的苜蓿的抗旱性高于所述目的苜蓿；The method includes up-regulating or enhancing or increasing the expression of a gene encoding a protein in a target alfalfa and/or the activity and/or content of the protein to obtain alfalfa with strong drought resistance. The drought resistance of the alfalfa with strong drought resistance is higher than The purpose of alfalfa;
所述蛋白质为如下任一种蛋白质：The protein is any of the following proteins:
B1)氨基酸序列是序列2所示的蛋白质；B1) The amino acid sequence is the protein shown in sequence 2;
B2）将B1)所述蛋白质的经过氨基酸残基的取代和/或缺失和/或添加得到的与B1)所示的蛋白质具有80％以上的同一性且具有相同功能的蛋白质；B2) A protein with more than 80% identity and the same function as the protein shown in B1) obtained by substituting and/or deleting and/or adding amino acid residues of the protein described in B1);
B3）将B1)或B2）的N末端或/和C末端连接蛋白质标签得到的融合蛋白质。B3) A fusion protein obtained by connecting a protein tag to the N-terminus or/and C-terminus of B1) or B2).
上述任一所述的方法中，所述上调或增强或提高苜蓿中蛋白质的编码基因的表达包括向苜蓿中导入所述蛋白质的编码基因。In any of the above methods, the up-regulating or enhancing or increasing the expression of the protein-coding gene in alfalfa includes introducing the protein-coding gene into alfalfa.
上述方法中，所述蛋白质的编码基因可通过下述任一种物质导入苜蓿：In the above method, the gene encoding the protein can be introduced into alfalfa through any of the following substances:
C1)、含有所述编码基因的表达盒；C1), an expression cassette containing the encoding gene;
C3)、含有所述编码基因的重组载体；C3), a recombinant vector containing the encoding gene;
B4)、含有所述编码基因的重组微生物。B4), recombinant microorganism containing the encoding gene.
所述编码基因可为编码序列（CDS）是SEQ ID No.1的DNA分子。The coding gene may be a DNA molecule whose coding sequence (CDS) is SEQ ID No. 1.
所述苜蓿可为紫花苜蓿。所述紫花苜蓿可为中苜1号。The alfalfa may be alfalfa. The alfalfa may be Zhonglu No. 1.
上述任一所述的方法中，所述上调或增强或提高苜蓿中蛋白质的编码基因的表达和/或所述蛋白质的活性和/或含量的物质为下述任一种：In any of the above methods, the substance that upregulates or enhances or increases the expression of the gene encoding the protein in alfalfa and/or the activity and/or content of the protein is any of the following:
B1)、编码上述蛋白质的核酸分子；B1), nucleic acid molecules encoding the above proteins;
B2)、含有B1)所述核酸分子的表达盒；B2), an expression cassette containing the nucleic acid molecule described in B1);
B3)、含有B1)所述核酸分子的重组载体、或含有B2)所述表达盒的重组载体；B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);
B4)、含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物；B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);
B5)、含有B1)所述核酸分子的转基因植物细胞系、或含有B2)所述表达盒的转基因植物细胞系、或含有B3)所述重组载体的转基因植物细胞系；B5), a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3);
B6)、含有B1)所述核酸分子的转基因植物组织、或含有B2)所述表达盒的转基因植物组织、或含有B3)所述重组载体的转基因植物组织；B6), transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3);
B7)、含有B1)所述核酸分子的转基因植物器官、或含有B2)所述表达盒的转基因植物器官、或含有B3)所述重组载体的转基因植物器官。B7), a transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3).
B1）所述核酸分子中，本领域普通技术人员可以很容易地采用已知的方法，例如定向进化或点突变的方法，对本发明的编码蛋白质MsMYBH的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明分离得到的蛋白质MsMYBH的核苷酸序列80％或80％以上同一性的核苷酸，只要编码蛋白质MsMYBH且具有蛋白质MsMYBH功能，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。Among the nucleic acid molecules described in B1), those of ordinary skill in the art can easily use known methods, such as directed evolution or point mutation methods, to mutate the nucleotide sequence encoding the protein MsMYBH of the present invention. Those artificially modified nucleotides that have 80% or more identity with the nucleotide sequence of the protein MsMYBH isolated in the present invention, as long as they encode the protein MsMYBH and have the function of the protein MsMYBH, are derived from the present invention. Nucleotide sequences and are equivalent to the sequences of the present invention.
上述80％或80％以上同一性，可为81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%的同一性。The above 80% or above identity can be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98% or 99% identity.
本文中，同一性是指氨基酸序列或核苷酸序列的同一性。可使用国际互联网上的同源性检索站点测定氨基酸序列的同一性，如NCBI主页网站的BLAST网页。例如，可在高级BLAST2.1中，通过使用blastp作为程序，将Expect值设置为10，将所有Filter设置为OFF，使用BLOSUM62作为Matrix，将Gap existence cost，Per residue gap cost和Lambda ratio分别设置为11，1和0.85（缺省值）并进行检索以对氨基酸序列的同一性进行计算，然后即可获得同一性的值（%）。As used herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI homepage. For example, in advanced BLAST2.1, you can use blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as the Matrix, and set the Gap existence cost, Per residue gap cost, and Lambda ratio to respectively 11, 1 and 0.85 (default value) and perform a search to calculate the identity of the amino acid sequence, and then obtain the identity value (%).
本文中，所述载体是本领域技术人员公知的，包括但不限于：质粒、噬菌体（如λ噬菌体或M13丝状噬菌体等）、黏粒（即柯斯质粒）、Ti质粒或病毒载体。具体可为pROKII载体；Here, the vector is well known to those skilled in the art, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e., cosmid), Ti plasmid or viral vector. Specifically, it can be pROKII vector;
上述生物材料中，B2）或B10）所述的表达盒是指能够在宿主细胞中表达所述基因的DNA，该DNA不但可包括启动基因转录的启动子，还可包括终止基因转录的终止子。进一步，所述表达盒还可包括增强子序列。可用于本发明的启动子包括但不限于：组成型启动子，组织、器官和发育特异的启动子，和诱导型启动子。启动子的例子包括但不限于：花椰菜花叶病毒的组成型启动子 35S; 来自西红柿的创伤诱导型启动子，亮氨酸氨基肽酶 ("LAP", Chao 等人（1999) Plant Physiol 120:979-992)；来自烟草的化学诱导型启动子，发病机理相关 (PR1) (由水杨酸和 BTH (苯并噻二唑 -7-硫代羟酸 S-甲酯)诱导)；西红柿蛋白酶抑制剂 II启动子 (PIN2)或 LAP启动子 (均可用茉莉酮酸曱酯诱导)； 热休克启动子 (美国专利 5，187，267)；四环素诱导型启动子 (美国专利 5，057,422)；种子特异性启动子，如谷子种子特异性启动子pF128（CN101063139B(中国专利2007 1 0099169.7)），种子贮存蛋白质特异的启动子 (例如，菜豆球蛋白、napin, oleosin和 大豆 betaconglycin 的启动子（Beachy 等人 (1985) EMBO J. 4:3047-3053))。它们可单独使用或与其它的植物启动子结合使用。此处引用的所有参考文献均全文引用。合适的转录终止子包括但不限于：农杆菌胭脂碱合成酶终止子（NOS终止子）、花椰菜花叶病毒CaMV 35S终止子、tml终止子、 豌豆rbcS E9终止子和胭脂氨酸和章鱼氨酸合酶终止子（参见， 例如：Odell 等人 （I985) Nature 313:810； Rosenberg等人（1987) Gene, 56:125； Guerineau等人 (1991) Mol. Gen. Genet , 262:141 ; Proudfoot (1991) Cell, 64:671;Sanfacon等人 Genes Dev. , 5:141; Mogen等人 (1990) Plant Cell, 2:1261； Munroe等人 （1990) Gene, 91:151； Ballad等人 (1989) Nucleic Acids Res. 17:7891； Joshi等人 (1987) Nucleic Acid Res., 15:9627)。Among the above biological materials, the expression cassette described in B2) or B10) refers to DNA capable of expressing the gene in the host cell. The DNA may not only include a promoter that initiates gene transcription, but also may include a terminator that terminates gene transcription. . Furthermore, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: the constitutive promoter 35S from cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); tomato protease Inhibitor II promoter (PIN2) or LAP promoter (both can be induced by methyl jasmonate); heat shock promoter (U.S. Patent 5,187,267); tetracycline inducible promoter (U.S. Patent 5,057,422); Seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 2007 1 0099169.7)), seed storage protein-specific promoters (for example, promoters of phaseolin, napin, oleosin and soybean betaconglycin (Beachy et al. (1985) EMBO J. 4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are cited in their entirety. Suitable transcription terminators include, but are not limited to: Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and nopaline and octopine Synthase terminator (see, e.g., Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot ( 1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).
上述B3)中，所述重组载体可为用植物表达载体构建含有所述基因表达盒的重组表达载体。所述植物表达载体可为Gateway 系统载体或双元农杆菌载体等，如pGWB411、pGWB412、pGWB405、pBin438、pCAMBIA1302、pCAMBIA2301、pCAMBIA1301、pCAMBIA1300、pBI121、pCAMBIA1391-Xa、pMDC85或pCAMBIA1391-Xb。使用MsMYBH构建重组表达载体时，在其转录起始核苷酸前可加上任何一种增强型、组成型、组织特异型或诱导型启动子，如花椰菜花叶病毒（CAMV）35S启动子、泛生素基因Ubiqutin启动子（pUbi）等，它们可单独使用或与其它的植物启动子结合使用；此外，使用本发明的基因构建植物表达载体时，还可使用增强子，包括翻译增强子或转录增强子，这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等，但必需与编码序列的阅读框相同，以保证整个序列的正确翻译。所述翻译控制信号和起始密码子的来源是广泛的，可以是天然的，也可以是合成的。翻译起始区域可以来自转录起始区域或结构基因。作为一个具体实施例，本申请使用pBI121载体作为表达载体。作为一个具体实施例，本申请使用pK7WIWG2I载体作为RNAi载体。作为一个具体实施例，所述重组微生物中的微生物菌株可为农杆菌EHA105。In the above B3), the recombinant vector may be a recombinant expression vector containing the gene expression cassette constructed using a plant expression vector. The plant expression vector can be a Gateway system vector or a binary Agrobacterium vector, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, pMDC85 or pCAMBIA1391-Xb. When using MsMYBH to construct a recombinant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before the transcription start nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter, Ubiquitin gene Ubiqutin promoter (pUbi), etc., which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or Transcription enhancers, these enhancer regions can be ATG start codons or adjacent region start codons, etc., but they must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence. The translation control signals and initiation codons come from a wide range of sources, and may be natural or synthetic. The translation initiation region can be derived from the transcription initiation region or from a structural gene. As a specific example, this application uses pBI121 vector as an expression vector. As a specific example, this application uses the pK7WIWG2I vector as the RNAi vector. As a specific example, the microorganism strain in the recombinant microorganism can be Agrobacterium EHA105.
上述的方法中，B1)所述的核酸分子为核苷酸序列为序列1所示的DNA分子。In the above method, the nucleic acid molecule described in B1) is a DNA molecule whose nucleotide sequence is shown in Sequence 1.
如上任一所述的方法中，所述上调或增强或提高苜蓿中蛋白质的编码基因的表达和/或所述蛋白质的活性和/或含量包括向所述目的苜蓿中导入权利要求6或7中B1）所述的核酸分子、B2）所述的表达盒或B3）所述的重组载体。In any of the above methods, the up-regulating or enhancing or increasing the expression of the gene encoding the protein in alfalfa and/or the activity and/or content of the protein includes introducing the method of claim 6 or 7 into the target alfalfa. The nucleic acid molecule described in B1), the expression cassette described in B2) or the recombinant vector described in B3).
上文中，所述核酸分子可为序列1所述的核酸分子。In the above, the nucleic acid molecule may be the nucleic acid molecule described in Sequence 1.
为了解决上述问题，本发明还提供了下述应用。In order to solve the above problems, the present invention also provides the following applications.
蛋白质、调控所述蛋白质的编码基因表达的物质或调控所述蛋白质的活性或含量的物质在下任一种中的应用；The use of proteins, substances that regulate the expression of genes encoding said proteins, or substances that regulate the activity or content of said proteins in any of the following;
A1）调控植物抗旱性中的应用和/或制备调控植物抗旱性产品中的应用；A1) Application in regulating plant drought resistance and/or application in preparing products for regulating plant drought resistance;
A2）调控植物株高中的应用和/或制备调控植物株高产品中的应用；A2) The application of regulating plant height and/or the preparation of products for regulating plant height;
A3）调控植物分枝数中的应用和/或制备调控植物分枝数产品中的应用；A3) Application in regulating the number of plant branches and/or application in preparing products for regulating the number of plant branches;
A4）调控植物丙二醛含量中的应用和/或制备调控植物丙二醛含量产品中的应用；A4) Application in regulating malondialdehyde content in plants and/or application in preparing products for regulating malondialdehyde content in plants;
A5）调控植物脯氨酸含量中的应用和/或制备调控植物脯氨酸含量产品中的应用；A5) Application in regulating the proline content of plants and/or application in preparing products for regulating the proline content of plants;
A6）调控植物叶片相对含水量中的应用和/或制备调控植物叶片相对含水量产品中的应用；A6) Application in regulating the relative water content of plant leaves and/or application in preparing products for regulating the relative water content of plant leaves;
A7）调控植物抗氧化能力中的应用和/或制备调控抗氧化能力产品中的应用；A7) Application in regulating the antioxidant capacity of plants and/or application in preparing products for regulating antioxidant capacity;
A8）调控植物干重中的应用和/或制备调控植物干重产品中的应用；A8) Application in regulating plant dry weight and/or application in preparing products for regulating plant dry weight;
A9）调控植物鲜重中的应用和/或制备调控植物鲜重产品中的应用；A9) Application in regulating the fresh weight of plants and/or application in preparing products for regulating the fresh weight of plants;
A10）调控植物叶片相对含水量中的应用和/或制备调控植物叶片相对含水量产品中的应用；A10) Application in regulating the relative water content of plant leaves and/or application in preparing products for regulating the relative water content of plant leaves;
所述蛋白质为如下任一种蛋白质：The protein is any of the following proteins:
B1)氨基酸序列是序列2所示的蛋白质；B1) The amino acid sequence is the protein shown in sequence 2;
B2）将B1)所述蛋白质的经过氨基酸残基的取代和/或缺失和/或添加得到的与B1)所示的蛋白质具有80％以上的同一性且具有相同功能的蛋白质；B2) A protein with more than 80% identity and the same function as the protein shown in B1) obtained by substituting and/or deleting and/or adding amino acid residues of the protein described in B1);
B3）将B1)或B2）的N末端或/和C末端连接蛋白质标签得到的融合蛋白质；B3) A fusion protein obtained by connecting the N-terminus or/and C-terminus of B1) or B2) to a protein tag;
所述物质为下述任一种：The substance is any of the following:
D1)、编码所述蛋白质的核酸分子；D1), a nucleic acid molecule encoding the protein;
D2)、含有D1)所述核酸分子的表达盒；D2), an expression cassette containing the nucleic acid molecule described in D1);
D3)、含有D1)所述核酸分子的重组载体、或含有D2)所述表达盒的重组载体；D3), a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);
D4)、含有D1)所述核酸分子的重组微生物、或含有D2)所述表达盒的重组微生物、或含有D3)所述重组载体的重组微生物；D4), a recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3);
D5)、含有D1)所述核酸分子的转基因植物细胞系、或含有D2)所述表达盒的转基因植物细胞系、或含有D3)所述重组载体的转基因植物细胞系；D5), a transgenic plant cell line containing the nucleic acid molecule described in D1), or a transgenic plant cell line containing the expression cassette described in D2), or a transgenic plant cell line containing the recombinant vector described in D3);
D6)、含有D1)所述核酸分子的转基因植物组织、或含有D2)所述表达盒的转基因植物组织、或含有D3)所述重组载体的转基因植物组织；D6), transgenic plant tissue containing the nucleic acid molecule described in D1), or transgenic plant tissue containing the expression cassette described in D2), or transgenic plant tissue containing the recombinant vector described in D3);
D7)、含有D1)所述核酸分子的转基因植物器官、或含有D2)所述表达盒的转基因植物器官、或含有D3)所述重组载体的转基因植物器官。D7), a transgenic plant organ containing the nucleic acid molecule described in D1), or a transgenic plant organ containing the expression cassette described in D2), or a transgenic plant organ containing the recombinant vector described in D3).
为了解决上述问题，本发明还提供了下述生物材料。In order to solve the above problems, the present invention also provides the following biological materials.
所述生物材料为如上所述蛋白质或物质。The biological material is a protein or substance as described above.
上述蛋白质中，所述蛋白标签（protein-tag）是指利用DNA体外重组技术，与目的蛋白一起融合表达的一种多肽或者蛋白，以便于目的蛋白的表达、检测、示踪和/或纯化。所述蛋白标签可为Flag标签、His标签、MBP标签、HA标签、myc标签、GST标签和/或SUMO标签等。Among the above-mentioned proteins, the protein-tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and/or SUMO tag, etc.
上述蛋白质中，同一性是指氨基酸序列的同一性。可使用国际互联网上的同源性检索站点测定氨基酸序列的同一性，如NCBI主页网站的BLAST网页。例如，可在高级BLAST2.1中，通过使用blastp作为程序，将Expect值设置为10，将所有Filter设置为OFF，使用BLOSUM62作为Matrix，将Gap existence cost，Per residue gap cost和Lambda ratio分别设置为11，1和0.85（缺省值）并进行检索一对氨基酸序列的同一性进行计算，然后即可获得同一性的值（%）。In the above-mentioned proteins, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI homepage. For example, in advanced BLAST2.1, you can use blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as the Matrix, and set the Gap existence cost, Per residue gap cost, and Lambda ratio to respectively 11, 1 and 0.85 (default value) and search the identity of a pair of amino acid sequences for calculation, and then obtain the identity value (%).
上述蛋白质中，所述80％以上的同一性可为至少81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、95%、96%、98%、99%或100%的同一性。Among the above proteins, the identity of more than 80% can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 95%, 96%, 98%, 99% or 100% identity.
上述蛋白质中，序列2（SEQ ID No.2）由个274个氨基酸残基组成。将其命名为MsMYBH蛋白。其编码基因为MsMYBH基因。Among the above proteins, sequence 2 (SEQ ID No. 2) consists of 274 amino acid residues. Name it MsMYBH protein. The encoding gene is MsMYBH gene.
本申请中，所述调控可为过上调或增强或提高，和/或，敲除或降低或减少。In this application, the regulation may be over-up-regulation or enhancement or improvement, and/or knock-out or reduction or reduction.
本申请中，所述过上调或增强或提高所述蛋白质的编码基因表达的物质或所述蛋白质的活性或含量可提高植物抗旱性。In this application, the substance that over-regulates or enhances or increases the expression of the gene encoding the protein or the activity or content of the protein can improve plant drought resistance.
敲除或降低或减少所述蛋白质的编码基因表达的物质或所述蛋白质的活性或含量可降低植物抗旱性。Knocking out or reducing or reducing the expression of the gene encoding the protein or the activity or content of the protein can reduce plant drought resistance.
本申请中，所述过上调或增强或提高所述蛋白质的编码基因表达的物质或所述蛋白质的活性或含量可提高植物分枝数。In this application, the substance that over-regulates or enhances or increases the expression of the gene encoding the protein or the activity or content of the protein can increase the number of plant branches.
敲除或降低或减少所述蛋白质的编码基因表达的物质或所述蛋白质的活性或含量可降低植物分枝数。Knocking out or reducing or reducing the expression of the gene encoding the protein or the activity or content of the protein can reduce the number of plant branches.
本申请中，所述过上调或增强或提高所述蛋白质的编码基因表达的物质或所述蛋白质的活性或含量可降低植物丙二醛含量。In this application, the substance that over-regulates or enhances or increases the expression of the gene encoding the protein or the activity or content of the protein can reduce the malondialdehyde content of plants.
敲除或降低或减少所述蛋白质的编码基因表达的物质或所述蛋白质的活性或含量可提高植物丙二醛含量。Knocking out or reducing or reducing the expression of the gene encoding the protein or the activity or content of the protein can increase plant malondialdehyde content.
本申请中，所述过上调或增强或提高所述蛋白质的编码基因表达的物质或所述蛋白质的活性或含量可降增加植物脯氨酸含量。In this application, the substance that over-regulates or enhances or increases the expression of the gene encoding the protein or the activity or content of the protein can reduce and increase the proline content of the plant.
敲除或降低或减少所述蛋白质的编码基因表达的物质或所述蛋白质的活性或含量可降低植物脯氨酸含量。Knocking out or reducing or reducing the expression of the gene encoding the protein or the activity or content of the protein can reduce plant proline content.
本申请中，所述过上调或增强或提高所述蛋白质的编码基因表达的物质或所述蛋白质的活性或含量可降增加植物鲜重。In this application, the substance that upregulates or enhances or increases the expression of the gene encoding the protein or the activity or content of the protein can reduce and increase the fresh weight of the plant.
敲除或降低或减少所述蛋白质的编码基因表达的物质或所述蛋白质的活性或含量可降低植物鲜重。Knocking out or reducing or reducing the expression of the gene encoding the protein or the activity or content of the protein can reduce plant fresh weight.
本申请中，所述过上调或增强或提高所述蛋白质的编码基因表达的物质或所述蛋白质的活性或含量可降增加植物干重。In this application, the substance that upregulates or enhances or increases the expression of the gene encoding the protein or the activity or content of the protein can reduce and increase plant dry weight.
敲除或降低或减少所述蛋白质的编码基因表达的物质或所述蛋白质的活性或含量可降低植物干重。Knocking out or reducing or reducing the expression of the gene encoding the protein or the activity or content of the protein can reduce plant dry weight.
本申请中，在干旱胁迫条件下，所述过上调或增强或提高所述蛋白质的编码基因表达的物质或所述蛋白质的活性或含量可降增加植物叶片相对含水量。In this application, under drought stress conditions, the substance that over-regulates or enhances or increases the expression of the gene encoding the protein or the activity or content of the protein can reduce and increase the relative water content of plant leaves.
在干旱胁迫条件下，敲除或降低或减少所述蛋白质的编码基因表达的物质或所述蛋白质的活性或含量可降低植物叶片相对含水量。Under drought stress conditions, knocking out or reducing or reducing the expression of the gene encoding the protein or the activity or content of the protein can reduce the relative water content of plant leaves.
本申请中，在干旱胁迫条件下，所述过上调或增强或提高所述蛋白质的编码基因表达的物质或所述蛋白质的活性或含量可降增加植物抗氧化酶活性。In this application, under drought stress conditions, the substance that over-regulates or enhances or increases the expression of the gene encoding the protein or the activity or content of the protein can decrease and increase plant antioxidant enzyme activity.
在干旱胁迫条件下，敲除或降低或减少所述蛋白质的编码基因表达的物质或所述蛋白质的活性或含量可降低植物抗氧化酶活性。所述抗氧化为可为POD活性（过氧化物酶活性）、CAT活性（过氧化氢酶活性）或SOD活性（超氧化物歧化酶活性）。Under drought stress conditions, knocking out or reducing or reducing the expression of the gene encoding the protein or the activity or content of the protein can reduce plant antioxidant enzyme activity. The antioxidant may be POD activity (peroxidase activity), CAT activity (catalase activity) or SOD activity (superoxide dismutase activity).
本申请中，在干旱胁迫条件下，所述过上调或增强或提高所述蛋白质的编码基因表达的物质或所述蛋白质的活性或含量可降增加植物Pn。In this application, under drought stress conditions, the substance that over-regulates or enhances or increases the expression of the gene encoding the protein or the activity or content of the protein can decrease and increase plant Pn.
在干旱胁迫条件下，敲除或降低或减少所述蛋白质的编码基因表达的物质或所述蛋白质的活性或含量可降低植物Pn。Under drought stress conditions, knocking out or reducing or reducing the expression of the gene encoding the protein or the activity or content of the protein can reduce plant Pn.
本申请中，在干旱胁迫条件下，所述过上调或增强或提高所述蛋白质的编码基因表达的物质或所述蛋白质的活性或含量可降增加植物Fm。In this application, under drought stress conditions, the substance that over-regulates or enhances or increases the expression of the gene encoding the protein or the activity or content of the protein can decrease and increase plant Fm.
在干旱胁迫条件下，敲除或降低或减少所述蛋白质的编码基因表达的物质或所述蛋白质的活性或含量可降低植物Fm。Under drought stress conditions, knocking out or reducing or reducing the expression of the gene encoding the protein or the activity or content of the protein can reduce plant Fm.
上述的应用中，所述植物为如下任一种：In the above application, the plant is any of the following:
J1）双子叶植物；J1) Dicotyledonous plants;
J2）豆科植物；J2) Legumes;
J3）苜蓿属植物；J3) Alfalfa;
J4）苜蓿。J4) Alfalfa.
本申请中，所述苜蓿可为苜蓿品种中苜1号。In this application, the alfalfa may be alfalfa variety No. 1.
上文中，所述调控基因表达的物质可为进行如下6种调控中至少一种调控的物质：1）在所述基因转录水平上进行的调控；2）在所述基因转录后进行的调控（也就是对所述基因的初级转录物的剪接或加工进行的调控）；3）对所述基因的RNA转运进行的调控（也就是对所述基因的mRNA由细胞核向细胞质转运进行的调控）；4）对所述基因的翻译进行的调控；5）对所述基因的mRNA降解进行的调控；6）对所述基因的翻译后的调控（也就是对所述基因翻译的蛋白质的活性进行调控）。As mentioned above, the substance that regulates gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcription level of the gene; 2) regulation after the gene is transcribed ( That is, the regulation of the splicing or processing of the primary transcript of the gene); 3) the regulation of the RNA transport of the gene (that is, the regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) Regulation of translation of the gene; 5) Regulation of mRNA degradation of the gene; 6) Post-translational regulation of the gene (that is, regulation of the activity of the protein translated by the gene) ).
上述应用中，所述调控所述蛋白质的编码基因表达的物质为下述任一种：In the above application, the substance that regulates the expression of the gene encoding the protein is any of the following:
B1)、编码上述蛋白质的核酸分子；B1), nucleic acid molecules encoding the above-mentioned proteins;
B2)、含有B1)所述核酸分子的表达盒；B2), an expression cassette containing the nucleic acid molecule described in B1);
B3)、含有B1)所述核酸分子的重组载体、或含有B2)所述表达盒的重组载体；B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);
B4)、含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物；B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);
B5)、含有B1)所述核酸分子的转基因植物细胞系、或含有B2)所述表达盒的转基因植物细胞系、或含有B3)所述重组载体的转基因植物细胞系；B5), a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3);
B6)、含有B1)所述核酸分子的转基因植物组织、或含有B2)所述表达盒的转基因植物组织、或含有B3)所述重组载体的转基因植物组织；B6), transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3);
B7)、含有B1)所述核酸分子的转基因植物器官、或含有B2)所述表达盒的转基因植物器官、或含有B3)所述重组载体的转基因植物器官；B7), a transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3);
B8)、抑制或降低或下调权利要求1或2所述蛋白质的编码基因的表达或抑制或降低或下调所述蛋白质的活性或含量的核酸分子；B8), a nucleic acid molecule that inhibits, reduces or down-regulates the expression of the gene encoding the protein of claim 1 or 2 or inhibits, reduces or down-regulates the activity or content of the protein;
B9)、表达B8)所述核酸分子的编码基因；B9), expressing the coding gene of the nucleic acid molecule described in B8);
B10)、含有B9)所述基因的表达盒；B10), an expression cassette containing the gene described in B9);
B11)、含有B9)所述基因的重组载体、或含有B10)所述表达盒的重组载体；B11), a recombinant vector containing the gene described in B9), or a recombinant vector containing the expression cassette described in B10);
B12)、含有B9)所述基因的重组微生物、或含有B10)所述表达盒的重组微生物、或含有B11)所述重组载体的重组微生物；B12), a recombinant microorganism containing the gene described in B9), or a recombinant microorganism containing the expression cassette described in B10), or a recombinant microorganism containing the recombinant vector described in B11);
B13)、含有B9)所述基因的转基因植物细胞系、或含有B10)所述表达盒的转基因植物细胞系、或含有B11)所述重组载体的转基因植物细胞系；B13), a transgenic plant cell line containing the gene described in B9), or a transgenic plant cell line containing the expression cassette described in B10), or a transgenic plant cell line containing the recombinant vector described in B11);
B14)、含有B9)所述基因的转基因植物组织、或含有B10)所述表达盒的转基因植物组织、或含有B11)所述重组载体的转基因植物组织；B14), transgenic plant tissue containing the gene described in B9), or transgenic plant tissue containing the expression cassette described in B10), or transgenic plant tissue containing the recombinant vector described in B11);
B15)、含有B9)所述基因的转基因植物器官、或含有B10)所述表达盒的转基因植物器官、或含有B11)所述重组载体的转基因植物器官。B15), a transgenic plant organ containing the gene described in B9), or a transgenic plant organ containing the expression cassette described in B10), or a transgenic plant organ containing the recombinant vector described in B11).
B1）或B9)所述核酸分子中，本领域普通技术人员可以很容易地采用已知的方法，例如定向进化或点突变的方法，对本发明的编码蛋白质MsMYBH的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明分离得到的蛋白质MsMYBH的核苷酸序列80％或80％以上同一性的核苷酸，只要编码蛋白质MsMYBH且具有蛋白质MsMYBH功能，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。Among the nucleic acid molecules described in B1) or B9), those of ordinary skill in the art can easily use known methods, such as directed evolution or point mutation methods, to mutate the nucleotide sequence encoding the protein MsMYBH of the present invention. Those artificially modified nucleotides that have 80% or more identity with the nucleotide sequence of the protein MsMYBH isolated in the present invention, as long as they encode the protein MsMYBH and have the function of the protein MsMYBH, are derived from the present invention. Nucleotide sequences and are equivalent to the sequences of the present invention.
上述80％或80％以上同一性，可为81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%的同一性。The above 80% or above identity can be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98% or 99% identity.
本文中，同一性是指氨基酸序列或核苷酸序列的同一性。可使用国际互联网上的同源性检索站点测定氨基酸序列的同一性，如NCBI主页网站的BLAST网页。例如，可在高级BLAST2.1中，通过使用blastp作为程序，将Expect值设置为10，将所有Filter设置为OFF，使用BLOSUM62作为Matrix，将Gap existence cost，Per residue gap cost和Lambda ratio分别设置为11，1和0.85（缺省值）并进行检索以对氨基酸序列的同一性进行计算，然后即可获得同一性的值（%）。As used herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI homepage. For example, in advanced BLAST2.1, you can use blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as the Matrix, and set the Gap existence cost, Per residue gap cost, and Lambda ratio to respectively 11, 1 and 0.85 (default value) and perform a search to calculate the identity of the amino acid sequence, and then obtain the identity value (%).
本文中，所述载体是本领域技术人员公知的，包括但不限于：质粒、噬菌体（如λ噬菌体或M13丝状噬菌体等）、黏粒（即柯斯质粒）、Ti质粒或病毒载体。具体可为pROKII载体；Here, the vector is well known to those skilled in the art, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e., cosmid), Ti plasmid or viral vector. Specifically, it can be pROKII vector;
上述生物材料中，B2）或B10）所述的表达盒是指能够在宿主细胞中表达所述基因的DNA，该DNA不但可包括启动基因转录的启动子，还可包括终止基因转录的终止子。进一步，所述表达盒还可包括增强子序列。可用于本发明的启动子包括但不限于：组成型启动子，组织、器官和发育特异的启动子，和诱导型启动子。启动子的例子包括但不限于：花椰菜花叶病毒的组成型启动子 35S; 来自西红柿的创伤诱导型启动子，亮氨酸氨基肽酶 ("LAP", Chao 等人（1999) Plant Physiol 120:979-992)；来自烟草的化学诱导型启动子，发病机理相关 (PR1) (由水杨酸和 BTH (苯并噻二唑 -7-硫代羟酸 S-甲酯)诱导)；西红柿蛋白酶抑制剂 II启动子 (PIN2)或 LAP启动子 (均可用茉莉酮酸曱酯诱导)； 热休克启动子 (美国专利 5，187，267)；四环素诱导型启动子 (美国专利 5，057,422)；种子特异性启动子，如谷子种子特异性启动子pF128（CN101063139B(中国专利2007 1 0099169.7)），种子贮存蛋白质特异的启动子 (例如，菜豆球蛋白、napin, oleosin和 大豆 betaconglycin 的启动子（Beachy 等人 (1985) EMBO J. 4:3047-3053))。它们可单独使用或与其它的植物启动子结合使用。此处引用的所有参考文献均全文引用。合适的转录终止子包括但不限于：农杆菌胭脂碱合成酶终止子（NOS终止子）、花椰菜花叶病毒CaMV 35S终止子、tml终止子、 豌豆rbcS E9终止子和胭脂氨酸和章鱼氨酸合酶终止子（参见， 例如：Odell 等人 （I985) Nature 313:810； Rosenberg等人（1987) Gene, 56:125； Guerineau等人 (1991) Mol. Gen. Genet , 262:141 ; Proudfoot (1991) Cell, 64:671;Sanfacon等人 Genes Dev. , 5:141; Mogen等人 (1990) Plant Cell, 2:1261； Munroe等人 （1990) Gene, 91:151； Ballad等人 (1989) Nucleic Acids Res. 17:7891； Joshi等人 (1987) Nucleic Acid Res., 15:9627)。Among the above biological materials, the expression cassette described in B2) or B10) refers to DNA capable of expressing the gene in the host cell. The DNA may not only include a promoter that initiates gene transcription, but also may include a terminator that terminates gene transcription. . Furthermore, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: the constitutive promoter 35S from cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); tomato protease Inhibitor II promoter (PIN2) or LAP promoter (both can be induced by methyl jasmonate); heat shock promoter (U.S. Patent 5,187,267); tetracycline inducible promoter (U.S. Patent 5,057,422); Seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 2007 1 0099169.7)), seed storage protein-specific promoters (for example, promoters of phaseolin, napin, oleosin and soybean betaconglycin (Beachy et al. (1985) EMBO J. 4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are cited in their entirety. Suitable transcription terminators include, but are not limited to: Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and nopaline and octopine Synthase terminator (see, e.g., Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot ( 1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).
上述B3)或B11)中，所述重组载体可为用植物表达载体构建含有所述基因表达盒的重组表达载体。所述植物表达载体可为Gateway 系统载体或双元农杆菌载体等，如pGWB411、pGWB412、pGWB405、pBin438、pCAMBIA1302、pCAMBIA2301、pCAMBIA1301、pCAMBIA1300、pBI121、pCAMBIA1391-Xa、pMDC85或pCAMBIA1391-Xb。使用MsMYBH构建重组表达载体时，在其转录起始核苷酸前可加上任何一种增强型、组成型、组织特异型或诱导型启动子，如花椰菜花叶病毒（CAMV）35S启动子、泛生素基因Ubiqutin启动子（pUbi）等，它们可单独使用或与其它的植物启动子结合使用；此外，使用本发明的基因构建植物表达载体时，还可使用增强子，包括翻译增强子或转录增强子，这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等，但必需与编码序列的阅读框相同，以保证整个序列的正确翻译。所述翻译控制信号和起始密码子的来源是广泛的，可以是天然的，也可以是合成的。翻译起始区域可以来自转录起始区域或结构基因。作为一个具体实施例，本申请使用pBI121载体作为表达载体。作为一个具体实施例，本申请使用pK7WIWG2I载体作为RNAi载体。作为一个具体实施例，所述重组微生物中的微生物菌株可为农杆菌EHA105。In the above B3) or B11), the recombinant vector may be a recombinant expression vector containing the gene expression cassette constructed using a plant expression vector. The plant expression vector can be a Gateway system vector or a binary Agrobacterium vector, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, pMDC85 or pCAMBIA1391-Xb. When using MsMYBH to construct a recombinant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before the transcription start nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter, Ubiquitin gene Ubiqutin promoter (pUbi), etc., which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or Transcription enhancers, these enhancer regions can be ATG start codons or adjacent region start codons, etc., but they must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence. The translation control signals and initiation codons come from a wide range of sources, and may be natural or synthetic. The translation initiation region can be derived from the transcription initiation region or from a structural gene. As a specific example, this application uses pBI121 vector as an expression vector. As a specific example, this application uses the pK7WIWG2I vector as the RNAi vector. As a specific example, the microorganism strain in the recombinant microorganism can be Agrobacterium EHA105.
上述的应用中，B1)所述的核酸分子为核苷酸序列为序列1所示的DNA分子。In the above application, the nucleic acid molecule described in B1) is a DNA molecule whose nucleotide sequence is shown in Sequence 1.
上述的应用中，B9)所述核酸分子为由如式(I)所示的DNA分子转录得到的RNA：In the above application, the nucleic acid molecule described in B9) is RNA transcribed from the DNA molecule represented by formula (I):
SEQ正向-X-SEQ反向 (I)；SEQ forward-X-SEQ reverse (I);
所述SEQ 正向是序列1的部分片段；所述SEQ反向的序列与所述SEQ正向的序列反向互补；所述X是所述SEQ正向与所述SEQ反向之间的间隔序列，使如式(I)所示的DNA分子转录得到的RNA分子形成茎环结构。The forward direction of the SEQ is a partial fragment of sequence 1; the reverse sequence of the SEQ is reverse complementary to the sequence of the forward direction of the SEQ; the X is the interval between the forward direction of the SEQ and the reverse direction of the SEQ The sequence causes the RNA molecule obtained by transcribing the DNA molecule represented by formula (I) to form a stem-loop structure.
一种培育高抗旱性植物的方法，包括上调或增强或提高目的植物中上述蛋白质的编码基因的表达量，和/或，所述蛋白质的活性和/或含量得到高抗旱性植物，所述高抗旱性植物的抗旱性高于所述目的植物。A method for cultivating highly drought-resistant plants, including up-regulating or enhancing or increasing the expression of the gene encoding the above-mentioned protein in the target plant, and/or the activity and/or content of the protein to obtain highly drought-resistant plants, the high Drought-resistant plants have higher drought resistance than the target plants.
一种提高植物抗旱性的方法，包括将通过上调或增强或提高植物中上述蛋白质的编码基因的表达，和/或，所述上述蛋白质的活性和/或含量来提高植物抗旱性。A method for improving plant drought resistance includes improving plant drought resistance by up-regulating or enhancing or increasing the expression of genes encoding the above-mentioned proteins in plants, and/or the activity and/or content of the above-mentioned proteins.
本申请中，所述植物可为苜蓿。所述苜蓿可为苜蓿品种中苜1号。In this application, the plant may be alfalfa. The alfalfa may be alfalfa variety No. 1.
上述的方法中，所述上调或增强或提高植物中上述蛋白质的编码基因的表达包括向所述目的植物中导入权利要求3中B1）所述的核酸分子、B2）所述的表达盒或B3）所述的重组载体。In the above method, the up-regulating or enhancing or improving the expression of the gene encoding the above-mentioned protein in the plant includes introducing into the target plant the nucleic acid molecule described in B1), the expression cassette described in B2) or B3 in claim 3. ) the recombinant vector described.
上文中，所述核酸分子可为序列1所述的核酸分子。In the above, the nucleic acid molecule may be the nucleic acid molecule described in Sequence 1.
一种培育低抗旱性植物的方法，包括敲除或降低或减少目的植物中上述蛋白质的编码基因的表达量，和/或，所述蛋白质的活性和/或含量得到低抗旱性植物，所述低抗旱性植物的抗旱性低于所述目的植物。A method of cultivating plants with low drought resistance, including knocking out or reducing or reducing the expression of the gene encoding the above-mentioned protein in the target plant, and/or the activity and/or content of the protein to obtain plants with low drought resistance, said Low drought resistance plants have lower drought resistance than the target plants.
上述的应用或上述的方法中，所述植物为如下任一种：In the above-mentioned application or the above-mentioned method, the plant is any one of the following:
J1）双子叶植物；J1) Dicotyledonous plants;
J2）豆科植物；J2) Legumes;
J3）苜蓿属植物；J3) Alfalfa;
J4）苜蓿。J4) Alfalfa.
本申请中，所述苜蓿可为苜蓿品种中苜1号。In this application, the alfalfa may be alfalfa variety No. 1.
蛋白质，所述蛋白质为下述任一种：Protein, the protein is any of the following:
A1）氨基酸序列是序列2所示的蛋白质；A1) The amino acid sequence is the protein shown in sequence 2;
A2）将A1）的氨基酸序列经过氨基酸残基的取代和/或缺失和/或添加得到的与A1）所示的蛋白质具有80％以上的同一性且具有相同功能的蛋白质；A2) A protein with more than 80% identity and the same function as the protein shown in A1) obtained by substituting and/or deleting and/or adding amino acid residues to the amino acid sequence of A1);
A3）在A1）或A2）的N端和/或C端连接标签得到的具有相同功能的融合蛋白质。A3) A fusion protein with the same function obtained by connecting a tag to the N-terminus and/or C-terminus of A1) or A2).
与权利要求上述蛋白质相关的生物材料，所述生物材料为下述任一种：Biological material related to the above-mentioned protein of the claim, said biological material is any of the following:
B1)、编码如上任一所述蛋白质的核酸分子；B1), nucleic acid molecules encoding proteins as described in any one of the above;
B2)、含有B1)所述核酸分子的表达盒；B2), an expression cassette containing the nucleic acid molecule described in B1);
B3)、含有B1)所述核酸分子的重组载体、或含有B2)所述表达盒的重组载体；B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);
B4)、含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物；B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);
B5)、含有B1)所述核酸分子的转基因植物细胞系、或含有B2)所述表达盒的转基因植物细胞系、或含有B3)所述重组载体的转基因植物细胞系；B5), a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3);
B6)、含有B1)所述核酸分子的转基因植物组织、或含有B2)所述表达盒的转基因植物组织、或含有B3)所述重组载体的转基因植物组织；B6), transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3);
B7)、含有B1)所述核酸分子的转基因植物器官、或含有B2)所述表达盒的转基因植物器官、或含有B3)所述重组载体的转基因植物器官。B7), a transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3).
有益效果beneficial effects
本发明公开了提高苜蓿产量和/或分枝数的方法及其所用蛋白质与相关生物材料。本发明解决的技术问题是调控植物抗旱性。具体公开了蛋白质、调控所述蛋白质的编码基因表达的物质或调控所述蛋白质的活性或含量的物质在调控植物抗旱性中的应用和/或制备调控植物抗旱性产品中的应用，所述蛋白为氨基酸序列是序列2所示，在植物中过表达MsMYBH蛋白编码基因能够增加植物的抗旱性且植物中对MsMYBH蛋白编码基因表达进行沉默能够降低植物的抗旱性缺，可用于植物育种及抗旱性研究。The invention discloses a method for increasing alfalfa yield and/or branch number, as well as proteins and related biological materials used therein. The technical problem solved by the invention is to regulate plant drought resistance. Specifically disclosed are the applications of proteins, substances that regulate the expression of genes encoding said proteins, or substances that regulate the activity or content of said proteins in regulating plant drought resistance and/or in preparing products that regulate plant drought resistance. The protein The amino acid sequence is shown in Sequence 2. Overexpression of the MsMYBH protein-coding gene in plants can increase the drought resistance of plants, and silencing the expression of the MsMYBH protein-coding gene in plants can reduce the drought resistance of plants. It can be used for plant breeding and drought resistance. Research.
本发明构建了MsMYBH基因的过表达载体：重组载体MsMYBH-pBI121和MsMYBH基因的RNAi载体重组载体MsMYBH-pK7WIWG2I，通过转基因方法获得超表达MsMYBH阳性转基因植株（OE-1、OE-2、OE-3、OE-4、OE-5、OE-6和OE-7）和RNAi MsMYBH阳性阳性转基因植株（RNAi-1、RNAi-2、RNAi-3、RNAi-4、RNAi-5、RNAi-6和RNAi-7），选择效果明显的OE-1、OE-2、OE-3、RNAi-1、RNAi-2和RNAi-3用于后续实验。以WT（中苜1号）为对照，选择超表达MsMYBH阳性转基因株系（OE-1、OE-2和OE-3）和RNi MsMYBH阳性转基因株系（RNAi-1、RNAi-2和RNAi-3）进行抗旱试验。The present invention constructs an overexpression vector of the MsMYBH gene: the recombinant vector MsMYBH-pBI121 and the RNAi vector recombinant vector MsMYBH-pK7WIWG2I of the MsMYBH gene, and obtains overexpressed MsMYBH positive transgenic plants (OE-1, OE-2, OE-3) through transgenic methods. , OE-4, OE-5, OE-6 and OE-7) and RNAi MsMYBH-positive transgenic plants (RNAi-1, RNAi-2, RNAi-3, RNAi-4, RNAi-5, RNAi-6 and RNAi -7), select OE-1, OE-2, OE-3, RNAi-1, RNAi-2 and RNAi-3 with obvious effects for subsequent experiments. Using WT (Zhonglu No. 1) as a control, we selected overexpressing MsMYBH positive transgenic lines (OE-1, OE-2 and OE-3) and RNi MsMYBH positive transgenic lines (RNAi-1, RNAi-2 and RNAi- 3) Conduct drought resistance tests.
结果表明：在植物中过表达MsMYBH蛋白编码基因能够增加植物的抗旱性且在植物中对MsMYBH蛋白编码基因表达进行沉默能够降低植物的抗旱性。The results show that overexpression of the MsMYBH protein-encoding gene in plants can increase the plant's drought resistance, and silencing the MsMYBH protein-encoding gene expression in plants can reduce the plant's drought resistance.
尤其是体现在，正常培养条件下：1.过表达MsMYBH阳性转基因植株的分枝数高于WT，WT的分枝数高于阳性转基因株系；2.超表达MsMYBH阳性转基因株系的鲜重和干重都显著高于WT，而WT显著高于RNAi MsMYBH阳性转基因株系。In particular, under normal culture conditions: 1. The number of branches of overexpressing MsMYBH positive transgenic plants is higher than that of WT, and the number of branches of WT is higher than that of positive transgenic lines; 2. The fresh weight of overexpressing MsMYBH positive transgenic lines and dry weight were significantly higher than WT, while WT was significantly higher than RNAi MsMYBH-positive transgenic lines.
干旱处条件下：1.过表达MsMYBH阳性转基因植株的抗旱性高于WT，WT的抗旱性高于RNAi MsMYBH阳性转基因株系；2.过表达MsMYBH阳性转基因植株的分枝数高于WT，WT的分枝数高于RNAi MsMYBH阳性转基因株系；3.过表达MsMYBH阳性转基因植株的叶片萎蔫程度低于WT，WT的叶片萎蔫程度低于RNAi MsMYBH阳性转基因株系；4.过表达MsMYBH阳性转基因植株的叶片光合作用和水分利用效率高于WT，WT的叶片光合作用和水分利用效率高于RNAiMsMYBH阳性转基因株系；5.过表达MsMYBH阳性转基因植株的抗氧化酶活性高于WT，WT的抗氧化酶活性高于RNAi MsMYBH阳性转基因株系。Under drought conditions: 1. The drought resistance of overexpressing MsMYBH positive transgenic plants is higher than that of WT, and the drought resistance of WT is higher than that of RNAi MsMYBH positive transgenic lines; 2. The number of branches of overexpressing MsMYBH positive transgenic plants is higher than that of WT, WT The number of branches is higher than that of the RNAi MsMYBH-positive transgenic line; 3. The degree of leaf wilting of over-expressing MsMYBH-positive transgenic plants is lower than that of WT, and the degree of leaf wilting of WT is lower than that of RNAi MsMYBH-positive transgenic lines; 4. Over-expressing MsMYBH-positive transgenic plants The leaf photosynthesis and water use efficiency of the plant are higher than those of WT, and the leaf photosynthesis and water use efficiency of WT are higher than those of RNAiMsMYBH positive transgenic lines; 5. The antioxidant enzyme activity of overexpressing MsMYBH positive transgenic plants is higher than that of WT, and the resistance of WT The oxidase activity was higher than that of RNAi MsMYBH positive transgenic lines.
附图说明Description of drawings
图1为MsMYBH的克隆图。Figure 1 shows the cloning diagram of MsMYBH.
图2为超表达MsMYBH（A）和RNAi MsMYBH（B）的载体构建图。Figure 2 shows the vector construction diagram for overexpression MsMYBH (A) and RNAi MsMYBH (B).
图3为超表达（A、B）和RNAi （C、D）MsMYBH转基因阳性紫花苜蓿植株在基因水平和转录水平鉴定图。Figure 3 shows the identification of overexpressed (A, B) and RNAi (C, D) MsMYBH transgene-positive alfalfa plants at the gene level and transcription level.
图4为MsMYBH组织特异性表达（A）和亚细胞定位（B）图。Figure 4 shows the tissue-specific expression (A) and subcellular localization (B) of MsMYBH.
图5为干旱胁迫下，MsMYBH的表达模式图。Figure 5 shows the expression pattern of MsMYBH under drought stress.
图6为对照及干旱处理下，野生型及转基因植株的表型图。Figure 6 shows the phenotypes of wild-type and transgenic plants under control and drought treatments.
图7为干旱胁迫下野生型和转基因株系的光合生理变化（A、B）以及对照条件下的生物量(C、D)和品质指标(E-H)图。Figure 7 shows photosynthetic physiological changes (A, B) of wild-type and transgenic lines under drought stress, as well as biomass (C, D) and quality indicators (E-H) under control conditions.
图8为干旱胁迫及对照条件下野生型和转基因株系的H2O2积累（a）以及抗氧化物酶活性（b-d）图。Figure 8 shows the H2 O2 accumulation (a) and antioxidant enzyme activity (bd) of wild-type and transgenic lines under drought stress and control conditions.
具体实施方式Detailed ways
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。The present invention will be described in further detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in literature in the field or in accordance with product instructions. Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.
下述实施例采用SPSS11.5统计软件对数据进行处理，实验结果以平均值±标准偏差表示，采用One-way ANOVA检验，P＜0.05（*）表示具有显著性差异，P＜0.01（**）表示具有极显著性差异，另外多重比较中不同小写字母代表了具有显著差异（P<0.05）。The following examples use SPSS11.5 statistical software to process data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA test is used. P<0.05 (*) indicates a significant difference, and P<0.01 (** ) indicates a highly significant difference, and different lowercase letters in multiple comparisons indicate significant differences (P<0.05).
实施例1 基因的克隆及转基因载体构建Example 1 Cloning of genes and construction of transgenic vectors
提取“中苜1号”紫花苜蓿的RNA，以“中苜1号”紫花苜蓿的RNA为模板反转录获得“中苜1号”紫花苜蓿的cDNA，以“中苜1号”紫花苜蓿的cDNA为模板使用克隆引物（F:ATGGGGAGAAGAAAGTGTTCGCATTG，序列表中序列4；R: TTAAGTGACACTAATTGGGCCAAGA，序列表中序列5) 进行扩增，反应体系：95℃预变性5min；95℃变性30s，58℃退火30s，72℃延伸1min，34个循环；72℃延伸7min。克隆到MsMYBH基因的cDNA（图1，图1中，第一泳道为marker，第二泳道为MsMYBH基因的cDNA），通过单克隆测序，成功获得了MsMYBH 的CDS序列（如序列1所示）。其编码的氨基酸序列如序列2所示，将其命名为MsMYBH蛋白。其基因组序列如序列3所示。将其命名为MsMYBH基因。Extract the RNA of alfalfa "Zhongli No. 1", and reverse-transcribe the RNA of alfalfa "Zhongli No. 1" as a template to obtain the cDNA of alfalfa "Zhongli No. 1". cDNA was used as a template using cloning primers (F: ATGGGGAGAAGAAAGTGTTCGCATTG, sequence 4 in the sequence listing; R: TTAAGTGACACTAATTGGGCCAAGA, sequence 5 in the sequence listing) for amplification. Reaction system: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, Extension at 72°C for 1 min, 34 cycles; extension at 72°C for 7 min. The cDNA of the MsMYBH gene was cloned (Figure 1. In Figure 1, the first lane is the marker, and the second lane is the cDNA of the MsMYBH gene). Through single cloning sequencing, the CDS sequence of MsMYBH was successfully obtained (as shown in Sequence 1). The encoded amino acid sequence is shown in Sequence 2, and it is named MsMYBH protein. Its genome sequence is shown in Sequence 3. Name it MsMYBH gene.
序列1具体如下：Sequence 1 is detailed as follows:
ATGGGGAGAAGAAAGTGTTCGCATTGTGGTAAGATAGGACATAATTGTAGGACATGCACATCCTTCACTACCCTTGGAGGACTTCGTCTCTTTGGGGTCCAACTATCATCATCCTCCTCGTCATCATCTAGTACCATGATCAAGAAAAGCTTTAGCATGGACACCTTTCCCTCACCATCCTCTCCATCTTCCTCATTCTCTTCATCAACATCATTAACCAATATTGATGAAAATTATTATCACAAACCCACTTCTAATATTTCATATCTATCAGATTGTTTTATAGGTCCACCCCAAGAGAGAAAGAAAGGAGTTCCATGGACAGAAGAAGAGCACAGAACATTCCTTGTTGGATTAGAGAAGCTAGGAAAAGGAGACTGGAGAGGCATCTCTAAAAACTTTGTCACTTCAAGAACCCCTACACAAGTCGCTAGCCATGCTCAAAAATACTTTCTTAGATTGGCTACTATCAACAAGAAGCGACGTTCAAGTCTCTTCGACTTGGTTGGGAGCAAGAACACCAACACAAAAGAACAAGGGTATGCTAATTCAGTAGTAAATTTGGGTCATAAATTTGAGGATAAGTGTAAATGTGAAGTTGAGATGAATGATGGCACCACTTTGTCCTACTTTAAACAAGAAGAAGCAGCCAAATCAGAAAAGCAAGAAAATAATTACTCTACAGATAATTGGCTACATGACTCTTCAAATTGTGCAGCAGTGCCTAATTTGGACCTCACACTTTCAGTTGCATCCCCTAAGCTTGAACAAAATCAACCCTCCTCTGCTGGCTCATTCCTTCTTGGCCCAATTAGTGTCACTTAA。ATGGGGAGAAGAAAGTGTTCGCATTGTGGTAAGATAGGACATAATTGTAGGACATGCACATCCTTCACTACCCTTGGAGGACTTCGTCTCTTTGGGGTCCAACTATCATCATCCTCCTCGTCATCATCTAGTACCATGATCAAGAAAAGCTTTAGCATGGACACCTTTCCCTCACCATCCTCTCCATCTTCCTCATTCTCTTCATCAACATCATTAACCAATATTGATGAAAATTATTATCACAAACCCACTTCTAATATTTCATATCTATCAGATTG TTTTATAGGTCCACCCCAAGAGAGAAAGAAAGGAGTTCCATGGACAGAAGAAGAGCACAGAACATTCCTTGTTGGATTAGAGAAGCTAGGAAAAGGAGACTGGAGAGGCATCTCTAAAAACTTTGTCACTTCAAGAACCCCTACACAAGTCGCTAGCCATGCTCAAAAATACTTTCTTAGATTGGCTACTATCAACAAGAAGCGACGTTCAAGTCTCTTCGACTTGGTTGGGAGCAAGAACACCAACACAAAAGAACAAGGGTATGC TAATTCAGTAGTAAATTTGGGTCATAAATTTGAGGATAAGTGTAAATGTGAAGTTGAGATGAATGATGGCACCACTTTGTCCTACTTTAAACAAGAAGAAGCAGCCAAATCAGAAAAGCAAGAAAATAATTACTCTACAGATAATTGGCTACATGACTCTTCAAATTGTGCAGCAGTGCCTAATTTGGACCTCACACTTTCAGTTGCATCCCCTAAGCTTGAACAAAATCAACCCTCCTCTGCTGGCTCATTCCTTCTTGGCCCAATTAGTGTC ACTTAA.
序列2具体如下：Sequence 2 is as follows:
MGRRKCSHCGKIGHNCRTCTSFTTLGGLRLFGVQLSSSSSSSSSTMIKKSFSMDTFPSPSSPSSSFSSSTSLTNIDENYYHKPTSNISYLSDCFIGPPQERKKGVPWTEEEHRTFLVGLEKLGKGDWRGISKNFVTSRTPTQVASHAQKYFLRLATINKKRRSSLFDLVGSKNTNTKEQGYANSVVNLGHKFEDKCKCEVEMNDGTTLSYFKQEEAAKSEKQENNYSTDNWLHDSSNCAAVPNLDLTLSVASPKLEQNQPSSAGSFLLGPISVT*。MGRRKCSHCGKIGHNCRTCTSFTTLGGLRLFGVQLSSSSSSSTMIKKSFSMDTFPSPSSPSSSFSSSTSLTNIDENYYHKPTSNISYLSDCFIGPPQERKKGVPWTEEEHRTFLVGLEKLGKGDWRGISKNFVTSRTPTQVASHAQKYFLRLATINKKRRSSLFDLVGSKNTNTKEQGYANSVVNLGHKFEDKCKCEVEMNDGTTLSYFK QEEAAKSEKQENNYSTDNWLHDSSNCAAVPNLDLTLSVASPKLEQNQPSSAGSFLLGPISVT*.
序列3具体如下：Sequence 3 is detailed as follows:
ATGGGGAGAAGAAAGTGTTCGCATTGTGGTAAGATAGGACATAATTGTAGGACATGCACATCCTTCACTACCCTTGGAGGACTTCGTCTCTTCGGGGTCCAACTATCATCTTCCTCCTCATCATCATCTAGTACCATGATCAAGAAAAGCTTTAGCATGGACACCTTTCCCTCACCATCCTCTCCATCTTCCTCATTCTCTTCATCAACATCATTAACCAATATTGATGAAAATTATTATCACAAACCCACTTCTAATATTTCATATCTATCAGATTGTTTTATAGGTCCACCCCAAGAGAGAAAGAAAGGTATATTTATACATATGTTAGCGGAAAGATGGTCACTAATTAATGTTGTTTACGTTTTCTATTAATTTAATGTGTATTTTTTTATAGTATTTGTTGTATAGTTCATCAATTCAAAATTAATGAAAAATAAAATATATTTTTTTTTACAAATTTTATTGTTTGTAGTTGAAATGAAACTTCTTTCAAAACTAATTTATCATGTTCAAATTTCCAACAGCTTCCTTCATTTGTTTTTTTATTTATAATTTGTAATATACTAATATGTAACTACCAATAAGAATAGAATTGAAAGAAAAATGCTACACCAAAATCTTATATTCCTTTATATATTGGTATGGATGTATAGTCATGATCAATGTTAAGTAGTTATTCATACATGTATACTTCTAATTTGAGCTAAGGGAGTTAATGGATAAGTGTTTGAATCATGATGAATGAGAAAATACTTGGGATCTCTAGTCTAACATATATTATATACTGATTGATTGATCAGGAGTTCCATGGACAGAAGAAGAGCACAGAACATTCCTTGTTGGATTAGAGAAGCTAGGAAAAGGAGACTGGAGAGGCATCTCTAAAAACTTTGTCACTTCAAGAACCCTACACAAGTCGCTAGCCATGCTCAAAAATACTTTCTTAGATTGGCTACTATCAACAAGAAGCGACGTTCAAGTCTCTTTGACTTGGTATATACACTATACTATGTTAGACAAATTCAATTCATTCATGATCATCAATTGCTTAATTATTTAGCTATCTCTTGTCGTTCACCACTGAATATAAAGTAAGTCTTACATGGCCACAAGCCCATAACCACAAATAAAAATTATTTAATTAGTTACATTTCTTAATCTTTTCCTCTTTTAACTTTTTTTATTTGTGTGTTCAAGTAAGTATTTTTCCTAAATAAATATTTTATTATTTTTATTTTTATGGATCCTTGTCATTGAATTAATAATATGATATATTTGGCTGCAGGTTGGGAGCAAGAACACCAACACAAAAGAACAAGGGTATGCTAATTCAGTAGTAAATTTGGGTCATAAATTTGAGGATAAGTGTAAATGTGAAGTTGAGATGAATGATGGCACCACTTTGTCCTACTTTAAACAAGAAGAAGCAGCCAAATCAGAAAAGCAAGAAAATAATTACTCTACAGATAGTTGGCTACATGACTCTTCAAATTGTGCAGCAGTGCCTAATTTGGACCTCACACTTTCAGTTGCATCCCCTAAGCTTGAACAAAATCAACCCTCCCCTGCTGGCTCATTCCTTCTTGGCCCAATTAGCGTCACTTAA。ATGGGGAGAAGAAAGTGTTCGCATTGTGGTAAGATAGGACATAATTGTAGGACATGCACATCCTTCACTACCCTTGGAGGACTTCGTCTCTTCGGGGTCCAACTATCATCTCCTCCTCATCATCTAGTACCATGATCAAGAAAAGCTTTAGCATGGACACCTTTCCCTCACCATCCTCTCCATCTTCCTCATTCTCTTCATCAACATCATTAACCAATATTGATGAAAATTATTATCACAAACCCACTTCTAATATTTCATATCTATCAGATTG TTTTATAGGTCCACCCCAAGAGAGAAAGAAAGGTATATTTATACATATGTTAGCGGAAAGATGGTCACTAATTAATGTTGTTTACGTTTTCTATTAATTTAATGTGTATTTTTTTATAGTATTTGTTGTATAGTTCATCAATTCAAAATTAATGAAAAATAAAATATATTTTTTTTTACAAATTTTATTGTTTGTAGTTGAAATGAAACTTCTTTCAAAACTAATTTATCATGTTCAAATTTCCAACAGCTTCCTTCATTTGTTTTTTTATT TATAATTTGTAATATACTAATATGTAACTACCAATAAGAATAGAATTGAAAGAAAAATGCTACACCAAAATCTTATATTCCTTATATATTGGTATGGATGTATAGTCATGATCAATGTTAAGTAGTTATTCATACATGTATACTTCTAATTTGAGCTAAGGGAGTTAATGGATAAGTGTTTGAATCATGATGAATGAGAAAATACTTGGGATCTCTAGTCTAACATATATTATATACTGATTGATTGATCAGGAGTTCCATGGACAGA AGAAGAGCACAGAACATTCCTTGTTGGATTAGAGAAGCTAGGAAAAGGAGACTGGAGAGGCATCTCTAAAAACTTTGTCACTTCAAGAACCCTACACAAGTCGCTAGCCATGCTCAAAAATACTTTCTTAGATTGGCTACTATCAACAAGAAGCGACGTTCAAGTCTCTTTGACTTGGTATATACACTATACTATGTTAGACAAATTCAATTCATTCATGATCATCAATTGCTTAATTATTTAGCTATCTCTTGTCGTTCACCACTGAATA TAAAGTAAGTCTTACATGGCCACAAGCCCATAACCACAAATAAAAATTATTTAATTAGTTACATTTCTTAATCTTTTCCTCTTTTAACTTTTTTTATTTGTGTGTTCAAGTAAGTATTTTTCCTAAATAAATATTTTATTTTTTATTTTTATGGATCCTTGTCATTGAATTAATAATATGATATATTTGGCTGCAGGTTGGGAGCAAGAACACCAACACAAAAGAACAAGGGTATGCTAATTCAGTAGTAAAATTTGGGTCATAAATTTGAGG ATAAGTGTAAATGTGAAGTTGAGATGAATGATGGCACCACTTTGTCCTACTTTAAACAAGAAGAAGCAGCCAAATCAGAAAAGCAAGAAAATAATTACTCTACAGATAGTTGGCTACATGACTCTTCAAATTGTGCAGCAGTGCCTAATTTGGACCTCACACTTTCAGTTGCATCCCCTAAGCTTGAACAAAATCAACCCTCCCCTGCTGGCTCATTCCTTCTTGGCCCAATTAGCGTCACTTAA.
进一步通过克隆到的MsMYBH的CDS序列分别设计了超表达MsMYBH引物（OEMsMYBH-F和OEMsMYBH-R，具体如表1所示）和RNAiMsMYBH引物(RNAi MsMYBH-F和RNAi MsMYBH-R，具体如表1所示) 。利用同源重组的方法成功构建到了重组质粒MsMYBH-pBI121（图2中A，第一泳道为重组质粒MsMYBH-pBI121，第二泳道为重组质粒MsMYBH-pBI121经限制性内切酶Xbal和Xmal双酶切后的结果，第三泳道为Marker）。Further, overexpressionMsMYBH primers (OEMsMYBH-F and OEMsMYBH-R, as shown in Table 1) and RNAiMsMYBH primers (RNAi MsMYBH-F and RNAi MsMYBH-R, as shown in Table1 ) were further designed based on the cloned CDS sequence of MsMYBH. 1). The recombinant plasmid MsMYBH-pBI121 was successfully constructed using the method of homologous recombination (A in Figure 2, the first lane is the recombinant plasmid MsMYBH-pBI121, the second lane is the recombinant plasmid MsMYBH-pBI121 after restriction endonucleases Xbal and Xmal double enzymes The result after cutting, the third lane is Marker).
重组质粒MsMYBH-pBI121是将pBI121质粒（GenBank号为AF485783.1）的限制性内切酶Xbal和Xmal之间的小片段替换为序列1的DNA片段，保持pBI121质粒其他序列不变得到的重组载体，将重组载体命名为重组质粒MsMYBH-pBI121。The recombinant plasmid MsMYBH-pBI121 is a recombinant vector obtained by replacing the small fragment between the restriction endonucleases Xbal and , the recombinant vector was named recombinant plasmid MsMYBH-pBI121.
重组载体MsMYBH-pK7WIWG2I是通过gateway技术将pK7WIWG2I质粒（购自索莱宝公司，货号VT001143）中核苷酸序列为5’-GCGGCCGCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTAGGATCCGGCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAACGCGTGGATCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATACCCGAAGTATGTCAAAAAGAGGTGTGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGACTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCCTTATACACAGCCAGTCTGCAGGTCGAC-3’（序列17）的DNA片段替换为核苷酸序列为5’-ATGGGGAGAAGAAAGTGTTCGCATTGTGGTAAGATAGGACATAATTGTAGGACATGCACATCCTTCACTACCCTTGGAGGACTTCGTCTCTTTGGGGTCCAACTATCATCATCCTCCTCGTCATCATCTAGTACCATGATCAAGAAAAGCTTTAGCATGGACACCTTTCCCTCACCATCCTCTCCATCTTCCTCATTCTCTTCATCAACATCATTAACCAATATTGATGAAAATTATTATCACAAACCCACTTCTAATATTTCA-3’（序列18）的DNA片段且将核苷酸序列为5’-GTCGACCTGCAGACTGGCTGTGTATAAGGGAGCCTGACATTTATATTCCCCAGAACATCAGGTTAATGGCGTTTTTGATGTCATTTTCGCGGTGGCTGAGATCAGCCACTTCTTCCCCGATAACGGAGACCGGCACACTGGCCATATCGGTGGTCATCATGCGCCAGCTTTCATCCCCGATATGCACCACCGGGTAAAGTTCACGGGAGACTTTATCTGACAGCAGACGTGCACTGGCCAGGGGGATCACCATCCGTCGCCCGGGCGTGTCAATAATATCACTCTGTACATCCACAAACAGACGATAACGGCTCTCTCTTTTATAGGTGTAAACCTTAAACTGCATTTCACCAGTCCCTGTTCTCGTCAGCAAAAGAGCCGTTCATTTCAATAAACCGGGCGACCTCAGCCATCCCTTCCTGATTTTCCGCTTTCCAGCGTTCGGCACGCAGACGACGGGCTTCATTCTGCATGGTTGTGCTTACCAGACCGGAGATATTGACATCATATATGCCTTGAGCAACTGATAGCTGTCGCTGTCAACTGTCACTGTAATACGCTGCTTCATAGCACACCTCTTTTTGACATACTTCGGGTATACATATCAGTATATATTCTTATACCGCAAAAATCAGCGCGCAAATACGCATACTGTTATCTGGCTTTTAGTAAGCCGGATCCACGCGTTTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAGACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGCCGGATCCTAACTCAAAATCCACACATTATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGCGGCCGC-3’（序列19）的DNA片段替换为核苷酸序列为5’-TGAAATATTAGAAGTGGGTTTGTGATAATAATTTTCATCAATATTGGTTAATGATGTTGATGAAGAGAATGAGGAAGATGGAGAGGATGGTGAGGGAAAGGTGTCCATGCTAAAGCTTTTCTTGATCATGGTACTAGATGATGACGAGGAGGATGATGATAGTTGGACCCCAAAGAGACGAAGTCCTCCAAGGGTAGTGAAGGATGTGCATGTCCTACAATTATGTCCTATCTTACCACAATGCGAACACTTTCTTCTCCCCAT-3’（序列20）的DNA片段，保持pK7WIWG2I质粒其他序列不变得到的重组载体，将重组载体命名为重组载体MsMYBH-pK7WIWG2I。重组载体MsMYBH-pK7WIWG2I的电泳结果如图2中B所示（图2中B，第一泳道为重组载体MsMYBH-pK7WIWG2I，第二泳道为MsMYBH-pK7WIWG2I鉴定前段（使用序列表中引物9和引物11的扩增结果），第三用泳道为MsMYBH-pK7WIWG2I鉴定后段（使用序列表中引物10和引物11的扩增结果），第四泳道为Marker）。The recombinant vector MsMYBH-pK7WIWG2I uses gateway technology to replace the DNA fragment with the nucleotide sequence 5'--3' (sequence 17) in the pK7WIWG2I plasmid (purchased from Soleba Corporation, Cat. No. VT001143) with the nucleotide sequence 5' -ATGGGGAGAAGAAAGTGTTCGCATTGTGGTAAGATAGGACATAATTGTAGGACATGCACATCCTTCACTACCCTTGGAGGACTTCGTCTCTTTGGGGTCCAACTATCATCATCCTCCTCGTCATCATCTAGTACCATGATCAAGAAAAGCTTTAGCATGGACACCTTTCCCTCACCATCCTCTCCATCTTCCTCATTCTCTTCATCAACATCATTAACCAATATTGATGAAAATTATTATCACAAACCCACTTCTAATATTTCA-3' (sequence 18) and replace the DNA fragment with the nucleotide sequence 5'--3' (Sequence 19) with the nucleotide sequence 5'-TGAAATATTAGAAGTGGGTTTGTGATAATAATTTTCATCAATATTGGTTAATGATGTTGATGAAGAGAATGAGGAAGATGGAGAGGATGGTGAGGGAAAGGTGTCCATGCTAAAGCTTTTCTTGATCATGGTACTAGATGATGACGAGGAGGATGATGATAGTTGGACCCCAAAGAGACGAAGTCCTCCAAG GGTAGTGAAGGATGTGCATGTCCTACAATTATGTCCTATCTTACCACAATGCGAACACTTTCTTCTCCCCAT-3' (sequence 20) The recombinant vector obtained by keeping the other sequences of the pK7WIWG2I plasmid unchanged was named the recombinant vector MsMYBH-pK7WIWG2I. The electrophoresis results of the recombinant vector MsMYBH-pK7WIWG2I are shown in B in Figure 2 (B in Figure 2, the first lane is the recombinant vector MsMYBH-pK7WIWG2I, and the second lane is the identification front section of MsMYBH-pK7WIWG2I (use primer 9 and primer 11 in the sequence list) The amplification results), the third lane is the MsMYBH-pK7WIWG2I identification back segment (the amplification results using primer 10 and primer 11 in the sequence list), the fourth lane is Marker).
实施例2：培育超表达和RNAi MsMYBH的转基因紫花苜蓿植株Example 2: Cultivation of transgenic alfalfa plants overexpressing and RNAi MsMYBH
分别将实施例1中重组载体MsMYBH-pBI121和重组载体MsMYBH-pK7WIWG2I转化到农杆菌EHA105菌株，然后利用农杆菌介导的遗传转化方法转化“中苜1号”紫花苜蓿叶片，经过脱分化、再分化、生根等过程，获得了超表达MsMYBH和RNi MsMYBH阳性转基因株系各7个株系，分别命名为将OE-1、OE-2、OE-3、OE-4、OE-5、OE-6、OE-7、RNAi-1、RNAi-2、RNAi-3、RNAi-4、RNAi-5、RNAi-6和RNAi-7。The recombinant vector MsMYBH-pBI121 and the recombinant vector MsMYBH-pK7WIWG2I in Example 1 were transformed into Agrobacterium tumefaciens EHA105 strain respectively, and then the "Zhongalu No. 1" alfalfa leaves were transformed using the Agrobacterium-mediated genetic transformation method. After dedifferentiation and re-transformation, Differentiation, rooting and other processes, 7 overexpressing MsMYBH and RNi MsMYBH positive transgenic lines were obtained, which were named OE-1, OE-2, OE-3, OE-4, OE-5, OE- 6. OE-7, RNAi-1, RNAi-2, RNAi-3, RNAi-4, RNAi-5, RNAi-6 and RNAi-7.
超表达MsMYBH阳性转基因株系（OE-1、OE-2、OE-3、OE-4、OE-5、OE-6和OE-7）分别用艾德莱DNA提取试剂盒、RNA提取试剂盒和反转录试剂盒，获得超表达MsMYBH阳性转基因株系的DNA模板和超表达MsMYBH阳性转基因株系的cDNA模板。使用基因水平鉴定引物（OEMsMYBH-F和OEMsMYBH-R）对超表达MsMYBH阳性转基因株系的DNA模板采用艾德莱DNA提取试剂盒（DN14）、RNA提取试剂盒(RN09)在基因水平和转录水平鉴定，结果如图3中A（第一泳道为Maker，第二泳道为ddH2O对照，第三泳道为重组载体MsMYBH-pBI121，第四和五泳道为WT（中苜1号），第六至十二泳道为超表达MsMYBH阳性转基因株系（OE-1、OE-2、OE-3、OE-4、OE-5、OE-6和OE-7）的DNA模板）所示，扩增出825bp的条带，为阳性转基因植株。Overexpressing MsMYBH positive transgenic lines (OE-1, OE-2, OE-3, OE-4, OE-5, OE-6 and OE-7) were used with Adelaide DNA extraction kit and RNA extraction kit respectively. and a reverse transcription kit to obtain the DNA template of the over-expressing MsMYBH positive transgenic strain and the cDNA template of the over-expressing MsMYBH positive transgenic strain. Use gene-level identification primers (OEMsMYBH-F and OEMsMYBH-R) to identify the DNA template of the overexpressing MsMYBH-positive transgenic line and use Adelaide DNA extraction kit (DN14) and RNA extraction kit (RN09) at the gene level and transcription level. Identification, the results are shown in Figure 3 A (the first lane is Maker, the second lane is ddH2 O control, the third lane is recombinant vector MsMYBH-pBI121, the fourth and fifth lanes are WT (Zhonglu No. 1), the sixth lane Lanes to lane 12 are shown as DNA templates of overexpressing MsMYBH-positive transgenic lines (OE-1, OE-2, OE-3, OE-4, OE-5, OE-6 and OE-7), amplified A band of 825bp is found, indicating a positive transgenic plant.
使用转录水平鉴定引物（MsMYBH-F和MsMYBH-R）、以actin基因为内参基因（检测引物：MsActin-qPCR-F：5｀-CAAAAGATGGCAGATGCTGAGGAT-3｀；序列表中的序列15；MsActin-qPCR-R：5｀-CATGACACCAGTATGACGAGGTCG-3｀；序列表中的序列16），和Taq酶以及诺唯赞qPCRmix转录水平鉴定，结果如图3中B（纵坐标为MsMYBH基因的相对表达量，WT为中苜1号，OE-1、OE-2、OE-3、OE-4、OE-5、OE-6和OE-7分别为超表达MsMYBH阳性转基因株系（OE-1、OE-2、OE-3、OE-4、OE-5、OE-6和OE-7））所示。Use transcription level identification primers (MsMYBH-F and MsMYBH-R), and actin gene as the internal reference gene (detection primer: MsActin-qPCR-F: 5｀-CAAAAGATGGCAGATGCTGAGGAT-3｀; sequence 15 in the sequence list; MsActin-qPCR- R: 5｀-CATGACACCAGTATGACGAGGTCG-3｀; sequence 16 in the sequence list), and Taq enzyme and Novozin qPCRmix transcription level identification, the results are shown in Figure 3 B (the ordinate is the relative expression of the MsMYBH gene, WT is the middle Mulberry No. 1, OE-1, OE-2, OE-3, OE-4, OE-5, OE-6 and OE-7 are respectively over-expressing MsMYBH positive transgenic lines (OE-1, OE-2, OE -3, OE-4, OE-5, OE-6 and OE-7)).
RNAi MsMYBH阳性转基因株系（RNAi-1、RNAi-2、RNAi-3、RNAi-4、RNAi-5、RNAi-6和RNAi-7）分别用艾德莱DNA提取试剂盒（DN14）、RNA提取试剂盒(RN09)和反转录试剂盒（PC5801），获得RNAi MsMYBH阳性转基因株系的DNA模板和RNAi MsMYBH阳性转基因株系的cDNA模板。使用基因水平鉴定引物（pK7WIWG2I-F、pK7WIWG2I-R和RNAi MsMYBH-R2）对RNAiMsMYBH阳性转基因株系的DNA模板在基因水平鉴定，结果如图3中C所示（第一泳道为Maker，第二泳道和第三泳道为重组载体MsMYBH-pK7WIWG2I，第四和五泳道为WT（中苜1号），第六至十九泳道依次为RNAi MsMYBH阳性转基因株系的DNA模板（RNAi-1、RNAi-2、RNAi-3、RNAi-4、RNAi-5、RNAi-6和RNAi-7），其中2-19中的偶数泳道使用序列表中引物9和11扩增出后段条带（444bp），2-19中的奇数泳道使用序列表中引物10和11扩增出前段条带（564bp）），结果如图3C所示，WT无条带，转基因植株扩增出与重组质粒相同的条带，所以均为阳性转基因植株。RNAi MsMYBH positive transgenic lines (RNAi-1, RNAi-2, RNAi-3, RNAi-4, RNAi-5, RNAi-6 and RNAi-7) were extracted using Adelaide DNA extraction kit (DN14) and RNA extraction respectively. Kit (RN09) and reverse transcription kit (PC5801) to obtain the DNA template of the RNAi MsMYBH positive transgenic line and the cDNA template of the RNAi MsMYBH positive transgenic line. Gene-level identification primers (pK7WIWG2I-F, pK7WIWG2I-R and RNAi MsMYBH-R2) were used to identify the DNA template of the RNAiMsMYBH-positive transgenic line at the gene level. The results are shown in C in Figure 3 (the first lane is Maker, the second lane The swimming lanes and the third lane are the recombinant vector MsMYBH-pK7WIWG2I, the fourth and fifth lanes are WT (Zhonglu No. 1), and the sixth to nineteenth lanes are the DNA templates of the RNAi MsMYBH positive transgenic lines (RNAi-1, RNAi- 2. RNAi-3, RNAi-4, RNAi-5, RNAi-6 and RNAi-7), among which the even-numbered lanes in 2-19 use primers 9 and 11 in the sequence list to amplify the back band (444bp), The odd-numbered lanes in 2-19 use primers 10 and 11 in the sequence list to amplify the front band (564 bp). The results are shown in Figure 3C. WT has no band, and the transgenic plant amplifies the same band as the recombinant plasmid. , so they are all positive transgenic plants.
使用转录水平鉴定引物（MsMYBH-F和 MsMYBH-R）、以actin基因为内参基因（检测引物：MsActin-qPCR-F和MsActin-qPCR-R），和Taq酶（PC0902）以及诺唯赞qPCRmix（R323-01）转录水平鉴定，结果如图3中D（纵坐标为MsMYBH基因的相对表达量，WT为中苜1号，RNAi-1、RNAi-2、RNAi-3、RNAi-4、RNAi-5、RNAi-6和RNAi-7分别为RNAi MsMYBH阳性阳性转基因株系（RNAi-1、RNAi-2、RNAi-3、RNAi-4、RNAi-5、RNAi-6和RNAi-7））所述。Use transcription level identification primers (MsMYBH-F and MsMYBH-R), actin gene as the internal reference gene (detection primers: MsActin-qPCR-F and MsActin-qPCR-R), Taq enzyme (PC0902) and Novozyme qPCRmix ( R323-01) transcription level identification, the results are as shown in D in Figure 3 (the ordinate is the relative expression of the MsMYBH gene, WT is Zhongli No. 1, RNAi-1, RNAi-2, RNAi-3, RNAi-4, RNAi- 5. RNAi-6 and RNAi-7 are described in the RNAi MsMYBH positive transgenic lines (RNAi-1, RNAi-2, RNAi-3, RNAi-4, RNAi-5, RNAi-6 and RNAi-7) respectively. .
进一步利用扦插繁殖的方式，将WT（中苜1号）、OE-1、OE-2、OE-3、RNAi-1、RNAi-2和RNAi-3等转基因株系进行了扩繁，为MsMYBH表型鉴定提供足够材料。Further using cutting propagation, transgenic lines such as WT (Zhonglu No. 1), OE-1, OE-2, OE-3, RNAi-1, RNAi-2 and RNAi-3 were propagated to form MsMYBH Sufficient material is provided for phenotypic identification.
实施例3 MsMYBH的组织特异性表达和亚细胞定位Example 3 Tissue-specific expression and subcellular localization of MsMYBH
采集“中苜1号”紫花苜蓿的根、茎、叶、花，提取RNA，反转录为CDNA。利用MsMYBH的qPCR引物（MsMYBH-F和MsMYBH-R），以actin基因为内参基因（检测引物：MsActin-qPCR-F和MsActin-qPCR-R），检测了不同组织中MsMYBH的表达量，结果显示在叶中表达量最高，其次为根、茎，花中表达量最低（图4中A）。通过洋葱表皮的亚细胞定位显示，MsMYBH定位在细胞核中（图4中B）。The roots, stems, leaves, and flowers of alfalfa "Zhonglu No. 1" were collected, RNA was extracted, and reverse transcribed into CDNA. Using the qPCR primers of MsMYBH (MsMYBH-F and MsMYBH-R) and the actin gene as the internal reference gene (detection primers: MsActin-qPCR-F and MsActin-qPCR-R), the expression levels of MsMYBH in different tissues were detected. The results show The expression level is highest in leaves, followed by roots and stems, and the lowest expression level is in flowers (A in Figure 4). Subcellular localization through onion epidermis showed that MsMYBH was localized in the nucleus (Fig. 4, B).
实施例4: 干旱胁迫下，MsMYBH表达模式Example 4: MsMYBH expression pattern under drought stress
通过对“中苜1号”紫花苜蓿幼苗进行300mM甘露醇处理，在处理不同时间下取样，提取RNA，反转录为CDNA，然后检测MsMYBH在干旱胁迫下的表达模式。结果显示：干旱胁迫下，MsMYBH在叶和根中的表达模式类似，但叶中的表达量显著高于根中，在胁迫处理的4h前MsMYBH的表达量受到干旱胁迫的显著诱导（图5，横坐标为干旱胁迫处理时间，纵坐标为相对表达量；●为根，◆为叶），表明了MsMYBH在干旱胁迫的早期响应中具有重要作用。The alfalfa seedlings of "Zhonglu No. 1" were treated with 300mM mannitol, samples were taken at different treatment times, RNA was extracted, reverse transcribed into CDNA, and then the expression pattern of MsMYBH under drought stress was detected. The results showed that under drought stress, the expression pattern of MsMYBH in leaves and roots was similar, but the expression level in leaves was significantly higher than that in roots. The expression level of MsMYBH was significantly induced by drought stress 4 hours before stress treatment (Figure 5, The abscissa is the drought stress treatment time, and the ordinate is the relative expression amount; ● is the root, ◆ is the leaf), indicating that MsMYBH plays an important role in the early response to drought stress.
实施例5:MsMYBH促进紫花苜蓿的生长并增强抗旱性Example 5: MsMYBH promotes alfalfa growth and enhances drought resistance
以WT（中苜1号）为对照，选择超表达MsMYBH阳性转基因株系（OE-1、OE-2和OE-3）和RNAi MsMYBH阳性转基因株系（RNAi-1、RNAi-2和RNAi-3）进行抗旱试验，具体如下：Using WT (Zhonglu No. 1) as a control, we selected overexpressing MsMYBH positive transgenic lines (OE-1, OE-2 and OE-3) and RNAi MsMYBH positive transgenic lines (RNAi-1, RNAi-2 and RNAi- 3) Conduct a drought resistance test, as follows:
将WT、OE-1、OE-2、OE-3、RNAi-1、RNAi-2和RNAi-3通过扦插繁殖的方式进行扩繁（栽培基质为营养土：蛭石体=1:1），播种前栽培基质（栽培基质为营养土：蛭石体=1:1）浇透（种植盆底部有孔洞多余水分会随孔洞排出），之后每隔4天浇水100ml/100g栽培基质，获得WT、OE-1、OE-2、OE-3、RNAi-1、RNAi-2和RNAi-3的扦插子代，扦插后一个月对上述生根的扦插子代进行一次刈割（统一进行刈割，留茬高度在15cm左），刈割后一个月，获得WT、OE-1、OE-2、OE-3、RNAi-1、RNAi-2和RNAi-3的扦插子代幼苗，将上述扦插子代幼苗（WT、OE-1、OE-2、OE-3、RNAi-1、RNAi-2和RNAi-3的扦插子代幼苗）随机分成两组，即对照组和干旱处理组，每组WT扦插子代幼苗、OE-1扦插子代幼苗、OE-2扦插子代幼苗、OE-3扦插子代幼苗、RNAi-1扦插子代幼苗、RNAi-2扦插子代幼苗和RNAi-3扦插子代幼苗各3株为1个重复，共3次重复，进行拍照记录生长表型，记作处理前（具体如图6中A，OE MYBH中的1、OE MYBH中的2和OEMYBH中的3分别代表OE-1扦插子代、OE-2扦插子代和OE-3扦插子代，Ri MYBH中的1、Ri MYBH中的2和Ri MYBH中的3分别为RNAi-1扦插子代、RNAi-2扦插子代和RNAi-3扦插子代；WT为中苜1号扦插子代；对照为对照组，干旱为干旱处理组，处理前为干旱处理前表型，处理后为干旱处理后表型）继续下述操作：Propagate WT, OE-1, OE-2, OE-3, RNAi-1, RNAi-2 and RNAi-3 through cutting propagation (the cultivation medium is nutrient soil: vermiculite = 1:1), Before sowing, water the cultivation medium (the cultivation medium is nutrient soil: vermiculite = 1:1) thoroughly (there are holes at the bottom of the planting pot, excess water will be discharged with the holes), and then water 100ml/100g of the cultivation medium every 4 days to obtain WT , OE-1, OE-2, OE-3, RNAi-1, RNAi-2 and RNAi-3 cutting progeny, the above-mentioned rooted cutting progeny were cut once one month after cutting (cutting was carried out uniformly, The height of the stubble is about 15cm). One month after cutting, the cutting progeny seedlings of WT, OE-1, OE-2, OE-3, RNAi-1, RNAi-2 and RNAi-3 were obtained. The above cuttings were The seedlings (the cutting progeny seedlings of WT, OE-1, OE-2, OE-3, RNAi-1, RNAi-2 and RNAi-3) were randomly divided into two groups, namely the control group and the drought treatment group. Each group was WT. Cutting progeny seedlings, OE-1 cutting progeny seedlings, OE-2 cutting progeny seedlings, OE-3 cutting progeny seedlings, RNAi-1 cutting progeny seedlings, RNAi-2 cutting progeny seedlings and RNAi-3 cuttings Each generation of seedlings has 3 plants as 1 repetition, with a total of 3 repetitions. Photographs are taken to record the growth phenotype, which is recorded as before treatment (specifically A in Figure 6, 1 in OE MYBH, 2 in OE MYBH and 3 in OEMYBH). represent the progeny of OE-1 cutting, OE-2 cutting and OE-3 respectively. The 1 in Ri MYBH, the 2 in Ri MYBH and the 3 in Ri MYBH represent the progeny of RNAi-1 cutting and RNAi respectively. -2 cutting progeny and RNAi-3 cutting progeny; WT is the cutting progeny of Zhonglu No. 1; control is the control group, drought is the drought treatment group, before treatment is the phenotype before drought treatment, and after treatment is the phenotype after drought treatment. type) continue as follows:
干旱处理组：将上述扦插子代幼苗（WT、OE-1、OE-2、OE-3、RNAi-1、RNAi-2和RNAi-3的扦插子代幼苗）在日光温室（温度25±2℃，光16小时/暗8小时条件下进行自然干旱处理（停止浇水）20天，干旱处理后进行拍照记录生长表型，记作处理后（具体如图6中A，OE MYBH中的1、OE MYBH中的2和OE MYBH中的3分别代表OE-1扦插子代、OE-2扦插子代和OE-3扦插子代，Ri MYBH中的1、Ri MYBH中的2和Ri MYBH中的3分别为RNAi-1扦插子代、RNAi-2扦插子代和RNAi-3扦插子代；WT为中苜1号扦插子代；对照为对照组，干旱为干旱处理组，处理前为干旱处理前表型，处理后为干旱处理后表型），干旱处理后检测和记录检测苜蓿株高、分枝数、叶片相对含水量、丙二醛含量、脯氨酸含量（结果如图6中B-F所示，OE MYBH中的1、OE MYBH中的2和OE MYBH中的3分别代表OE-1扦插子代、OE-2扦插子代和OE-3扦插子代，Ri MYBH中的1、Ri MYBH中的2和Ri MYBH中的3分别为RNAi-1扦插子代、RNAi-2扦插子代和RNAi-3扦插子代；WT为中苜1号扦插子代；对照为对照组，干旱为干旱处理组）以及POD活性、CAT活性、SOD活性（结果如图8中B-D所示，OE MYBH中的1、OE MYBH中的2和OE MYBH中的3分别代表OE-1扦插子代、OE-2扦插子代和OE-3扦插子代，Ri MYBH中的1、Ri MYBH中的2和Ri MYBH中的3分别为RNAi-1扦插子代、RNAi-2扦插子代和RNAi-3扦插子代；WT为中苜1号扦插子代；对照为对照组，干旱为干旱处理组），以及净光合速率Pn和最大荧光产量Fm（结果如图7中A和B所示，OE MYBH1、OE MYBH2和OE MYBH3分别代表OE-1扦插子代、OE-2扦插子代和OE-3扦插子代，Ri MYBH1、Ri MYBH2和Ri MYBH3分别为RNAi-1扦插子代、RNAi-2扦插子代和RNAi-3扦插子代；WT为中苜1号扦插子代），干旱处理后取苜蓿叶片进行H2O2含量检测（结果如图8中A所示，OE MYBH从左至右分别为OE-1扦插子代、OE-2扦插子代和OE-3扦插子代，Ri MYBH从左至右分别为RNAi-1扦插子代、RNAi-2扦插子代和RNAi-3扦插子代；WT为中苜1号扦插子代；对照为对照组，干旱为干旱处理组），全程进行表型观察，采用SPSS11.5统计软件对数据进行处理，实验结果以平均值±标准偏差表示，采用One-way ANOVA检验，不同字母表示具有显著性差异(P<0.05)。Drought treatment group: The above-mentioned cutting progeny seedlings (the cutting progeny seedlings of WT, OE-1, OE-2, OE-3, RNAi-1, RNAi-2 and RNAi-3) were placed in a solar greenhouse (temperature 25±2 ℃, 16 hours of light/8 hours of darkness for natural drought treatment (stop watering) for 20 days. After the drought treatment, take photos to record the growth phenotype, recorded as after treatment (specifically A in Figure 6, 1 in OE MYBH , 2 in OE MYBH and 3 in OE MYBH represent the OE-1 cutting progeny, OE-2 cutting progeny and OE-3 cutting progeny respectively, 1 in Ri MYBH, 2 in Ri MYBH and Ri MYBH 3 are the progeny of RNAi-1 cuttings, RNAi-2 cuttings and RNAi-3 cuttings respectively; WT is the cutting progeny of Zhongli No. 1; the control is the control group, the drought is the drought treatment group, and the drought before treatment is Phenotype before treatment, phenotype after drought treatment after treatment), detection and recording after drought treatment to detect alfalfa plant height, branch number, leaf relative water content, malondialdehyde content, and proline content (the results are shown in Figure 6 As shown in BF, the 1 in OE MYBH, the 2 in OE MYBH and the 3 in OE MYBH represent the OE-1 cutting progeny, OE-2 cutting progeny and OE-3 cutting progeny respectively. The 1 and 3 in Ri MYBH 2 in Ri MYBH and 3 in Ri MYBH are the progeny of RNAi-1 cuttings, RNAi-2 cuttings and RNAi-3 cuttings respectively; WT is the progeny of Zhongmulo No. 1 cuttings; the control is the control group, drought is the drought treatment group) as well as POD activity, CAT activity, and SOD activity (the results are shown in BD in Figure 8. 1 in OE MYBH, 2 in OE MYBH, and 3 in OE MYBH represent the OE-1 cutting progeny, respectively. OE-2 cutting progeny and OE-3 cutting progeny, 1 in Ri MYBH, 2 in Ri MYBH and 3 in Ri MYBH are RNAi-1 cutting progeny, RNAi-2 cutting progeny and RNAi-3 respectively. Cutting progeny; WT is the cutting progeny of Zhongmulo No. 1; control is the control group, and drought is the drought treatment group), as well as the net photosynthetic rate Pn and the maximum fluorescence yield Fm (the results are shown in A and B in Figure 7, OE MYBH1 , OE MYBH2 and OE MYBH3 represent the progeny of OE-1 cutting, OE-2 cutting and OE-3 cutting respectively, Ri MYBH1, Ri MYBH2 and Ri MYBH3 represent the progeny of RNAi-1 cutting and RNAi-2 cutting respectively. progeny and progeny of RNAi-3 cuttings; WT is the progeny of cuttings from Zhonglu No. 1). After drought treatment, alfalfa leaves were taken for H2 O2 content detection (the results are shown in A in Figure 8, OE MYBH from left to right They are the progeny of OE-1 cutting, OE-2 cutting and OE-3 cutting respectively. Ri MYBH from left to right are the progeny of RNAi-1 cutting, RNAi-2 cutting and RNAi-3 cutting respectively. generation; WT is the progeny of cuttings from Zhonglu No. 1; control is the control group, and drought is the drought treatment group). Phenotypic observations were conducted throughout the process. SPSS11.5 statistical software was used to process the data. The experimental results were expressed as mean ± standard deviation. , using One-way ANOVA test, different letters indicate significant differences (P<0.05).
对照组：将幼苗在日光温室（温度25±2℃，光16小时/暗8小时，4200LUX)条件下进行正常培养（每隔4天浇水100ml/100g，营养土与蛭石1:1）20天，正常培养后进行拍照记录生长表型，记作处理后（具体如图6中A，OE MYBH中的1、OE MYBH中的2和OE MYBH中的3分别代表OE-1扦插子代、OE-2扦插子代和OE-3扦插子代，Ri MYBH中的1、Ri MYBH中的2和RiMYBH中的3分别为RNAi-1扦插子代、RNAi-2扦插子代和RNAi-3扦插子代；WT为中苜1号扦插子代；对照为对照组，干旱为干旱处理组，处理前为干旱处理前表型，处理后为干旱处理后表型），正常培养后记录检测苜蓿株高、分枝数、叶片相对含水量、丙二醛含量、脯氨酸含量（结果如图6中B-F所示，OE MYBH中的1、OE MYBH中的2和OE MYBH中的3分别代表OE-1扦插子代、OE-2扦插子代和OE-3扦插子代，Ri MYBH中的1、Ri MYBH中的2和Ri MYBH中的3分别为RNAi-1扦插子代、RNAi-2扦插子代和RNAi-3扦插子代；WT为中苜1号扦插子代；对照为对照组，干旱为干旱处理组）以及鲜重、干重、茎叶比、粗蛋白、NDF和ADF（结果如图7中C-G所示，OE MYBH中的1、OE MYBH中的2和OE MYBH中的3分别代表OE-1扦插子代、OE-2扦插子代和OE-3扦插子代，Ri MYBH中的1、Ri MYBH中的2和Ri MYBH中的3分别为RNAi-1扦插子代、RNAi-2扦插子代和RNAi-3扦插子代；WT为中苜1号扦插子代）以及POD活性、CAT活性、SOD活性（结果如图8中B-D所示，OE MYBH中的1、OE MYBH中的2和OE MYBH中的3分别代表OE-1扦插子代、OE-2扦插子代和OE-3扦插子代，Ri MYBH中的1、Ri MYBH中的2和Ri MYBH中的3分别为RNAi-1扦插子代、RNAi-2扦插子代和RNAi-3扦插子代；WT为中苜1号扦插子代；对照为对照组，干旱为干旱处理组），以及净光合速率Pn和最大荧光产量Fm（结果如图7中A和B所示，OEMYBH1、OE MYBH2和OE MYBH3分别代表OE-1扦插子代、OE-2扦插子代和OE-3扦插子代，RiMYBH1、Ri MYBH2和Ri MYBH3分别为RNAi-1扦插子代、RNAi-2扦插子代和RNAi-3扦插子代；WT为中苜1号扦插子代），正常培养后，取苜蓿叶片进行H2O2含量检测（结果如图8中A所示，OEMYBH从左至右分别为OE-1扦插子代、OE-2扦插子代和OE-3扦插子代，Ri MYBH从左至右分别为RNAi-1扦插子代、RNAi-2扦插子代和RNAi-3扦插子代；WT为中苜1号扦插子代；对照为对照组，干旱为干旱处理组），全程进行表型观察，采用One-way ANOVA检验，不同字母.表示具有显著性差异）。Control group: The seedlings were cultured normally in a solar greenhouse (temperature 25±2°C, 16 hours light/8 hours dark, 4200LUX) (watering 100ml/100g every 4 days, nutrient soil and vermiculite 1:1) After 20 days of normal culture, take photos to record the growth phenotype, which is recorded as after treatment (specifically, A in Figure 6, 1 in OE MYBH, 2 in OE MYBH and 3 in OE MYBH respectively represent the progeny of OE-1 cuttings. , OE-2 cutting progeny and OE-3 cutting progeny, 1 in Ri MYBH, 2 in Ri MYBH and 3 in RiMYBH are RNAi-1 cutting progeny, RNAi-2 cutting progeny and RNAi-3 respectively. Progeny of cuttings; WT is the progeny of cuttings from Zhonglu No. 1; control is the control group, drought is the drought treatment group, before treatment is the phenotype before drought treatment, after treatment is the phenotype after drought treatment), alfalfa is recorded and detected after normal culture Plant height, number of branches, relative water content of leaves, malondialdehyde content, and proline content (the results are shown as BF in Figure 6, 1 in OE MYBH, 2 in OE MYBH, and 3 in OE MYBH represent respectively OE-1 cutting progeny, OE-2 cutting progeny and OE-3 cutting progeny, 1 in Ri MYBH, 2 in Ri MYBH and 3 in Ri MYBH are RNAi-1 cutting progeny and RNAi-2 respectively. Cutting progeny and RNAi-3 cutting progeny; WT is the cutting progeny of Zhongli No. 1; control is the control group, and drought is the drought treatment group) as well as fresh weight, dry weight, stem-to-leaf ratio, crude protein, NDF and ADF ( The results are shown in CG in Figure 7. The 1 in OE MYBH, the 2 in OE MYBH and the 3 in OE MYBH represent the OE-1 cutting progeny, OE-2 cutting progeny and OE-3 cutting progeny respectively, Ri 1 in MYBH, Ri 2 in MYBH and Ri 3 in MYBH are the progeny of RNAi-1 cutting, RNAi-2 cutting and RNAi-3 cutting respectively; WT is the progeny of Zhongli No. 1 cutting) and POD activity, CAT activity, SOD activity (the results are shown in BD in Figure 8. 1 in OE MYBH, 2 in OE MYBH and 3 in OE MYBH represent the progeny of OE-1 cuttings and OE-2 cuttings respectively. and OE-3 cutting progeny, 1 in Ri MYBH, 2 in Ri MYBH and 3 in Ri MYBH are the progeny of RNAi-1 cutting, RNAi-2 cutting and RNAi-3 cutting respectively; WT is The cutting progeny of Zhonglu No. 1; the control is the control group, and the drought is the drought treatment group), as well as the net photosynthetic rate Pn and the maximum fluorescence yield Fm (the results are shown in A and B in Figure 7, OEMYBH1, OE MYBH2 and OE MYBH3 respectively. Represents OE-1 cutting progeny, OE-2 cutting progeny and OE-3 cutting progeny, RiMYBH1, Ri MYBH2 and Ri MYBH3 are RNAi-1 cutting progeny, RNAi-2 cutting progeny and RNAi-3 cutting progeny respectively. generation; WT is the progeny of the cuttings of Zhonglu No. 1). After normal culture, the alfalfa leaves were taken for H2 O2 content detection (the results are shown in A in Figure 8, OEMYBH from left to right are the progeny of OE-1 cuttings). , OE-2 cutting progeny and OE-3 cutting progeny, Ri MYBH from left to right are RNAi-1 cutting progeny, RNAi-2 cutting progeny and RNAi-3 cutting progeny; WT is Zhongli No. 1 Progeny of cuttings; control is the control group, drought is the drought treatment group), phenotypic observations were conducted throughout the process, and One-way ANOVA test was used, different letters (.) indicate significant differences).
株高：对干旱处理组和对照的苜蓿进行根颈部到顶部之间的高度测量，其中顶部是指主茎顶部。Plant height: The height from the root neck to the top of the alfalfa in the drought treatment group and the control group was measured, where the top refers to the top of the main stem.
分枝数：对干旱处理组和对照的苜蓿进行地表根茎部形成分枝数的测定。Number of branches: The number of branches formed on the surface rhizomes of alfalfa in the drought treatment group and the control group was measured.
叶片相对含水量：对干旱处理组和对照组的苜蓿分别称取叶片0.2g，记为Wf，之后将叶片浸入水中 8h，称其饱和鲜重 Wt，再于 105 ℃烘箱中杀青15 min，75 ℃条件下烘干至恒重，称干重，记为 Wd，按照公式计算相对含水量：相对含水量=（Wf-Wd）/（Wt-Wd）×100%。Relative water content of leaves: Weigh 0.2g of alfalfa leaves in the drought treatment group and the control group respectively, recorded as Wf. Then the leaves were immersed in water for 8 hours and their saturated fresh weight Wt was then cured in an oven at 105°C for 15 min, 75 Dry to constant weight under ℃ conditions, weigh the dry weight, record it as Wd, and calculate the relative moisture content according to the formula: relative moisture content = (Wf-Wd)/(Wt-Wd) × 100%.
丙二醛含量：采用索莱宝公司丙二醛(MDA)含量检测试剂盒（BC0020）对干旱处理组和对照组的苜蓿叶片进行检测，每次检测重复3次。Malondialdehyde content: The alfalfa leaves of the drought treatment group and the control group were tested using Solebao's malondialdehyde (MDA) content detection kit (BC0020). Each test was repeated three times.
脯氨酸含量：采用索莱宝公司脯氨酸(Pro)含量检测试剂盒（BC0290）对干旱处理组和对照组的苜蓿叶片进行检测，每次检测重复3次。Proline content: The alfalfa leaves of the drought treatment group and the control group were tested using the Proline (Pro) content detection kit (BC0290) of Solebao Company. Each test was repeated three times.
鲜重：对干旱处理组和对照组的苜蓿进行称重，即为鲜重。Fresh weight: Weigh the alfalfa in the drought treatment group and the control group, which is the fresh weight.
干重：对干旱处理组和对照组的苜蓿进行取样，对样品进行105 ℃烘箱中杀青 15min，75 ℃条件下烘干至恒重，称干重。Dry weight: Sampling alfalfa from the drought treatment group and the control group, incubating the samples in an oven at 105°C for 15 minutes, drying them to constant weight at 75°C, and weighing the dry weight.
茎叶比：对干旱处理组和对照组的整株苜蓿进行105 ℃杀青30 min,然后在60℃-65℃烘干至恒重后，进行茎叶分离并称重，茎与叶片的重量之比即为茎叶比.。Stem-leaf ratio: The whole alfalfa plants in the drought treatment group and the control group were cured at 105°C for 30 minutes, and then dried at 60°C-65°C to constant weight. The stems and leaves were separated and weighed. The weight of the stems and leaves was The ratio is the stem-to-leaf ratio.
粗蛋白：对干旱处理组和对照组的苜蓿按照GB/T 6432—2018《饲料中粗蛋白的测定》测定粗蛋白含量。Crude protein: The crude protein content of alfalfa in the drought treatment group and the control group was determined in accordance with GB/T 6432-2018 "Determination of Crude Protein in Feed".
NDF（中性洗涤纤维.）：采用范氏（Van Soest）的洗涤纤维分析法测定中性洗涤纤维（NDF）。具体测定方法：NDF (Neutral Detergent Fiber.): Neutral detergent fiber (NDF) is measured using Van Soest's detergent fiber analysis method. Specific measurement method:
（1）准确称取40目筛饲料样品0.5-1.0g（m）,置无嘴高型烧杯中，加入中性洗涤剂溶液100ml和2ml十氢化萘和0.5g无水亚硫酸钠。（2）装上冷凝装置，立即置于电炉上煮沸（5-10min）,并微沸1h.（3）煮沸完毕，冷却10min，将已知质量的玻璃坩埚（m1）安装于抽滤瓶上，将残渣全部移入，抽滤，并用沸水冲洗，再抽滤。再用20ml丙酮冲洗，抽滤。（4）取下坩埚，105℃烘干，称重（m2）。NDF：=(m2-m1)/m。(1) Accurately weigh 0.5-1.0g (m) of the 40-mesh sieve feed sample, place it in a tall beaker without a spout, add 100ml of neutral detergent solution, 2ml of decalin and 0.5g of anhydrous sodium sulfite. (2) Install the condensation device, immediately place it on the electric stove and boil (5-10min), and boil slightly for 1h. (3) After boiling, cool for 10min, and install the glass crucible (m1) of known mass on the suction filter bottle , move all the residue in, filter with suction, rinse with boiling water, and then filter with suction. Rinse with 20ml of acetone and filter with suction. (4) Remove the crucible, dry it at 105°C, and weigh it (m2). NDF:=(m2-m1)/m.
ADF（酸性洗涤纤维）：采用范氏（Van Soest）的洗涤纤维分析法测定酸性洗涤纤维（ADF）具体测定方法：ADF (acid detergent fiber): Determination of acid detergent fiber (ADF) using Van Soest's detergent fiber analysis method. The specific determination method:
（1）准确称取样品，0.5-1.0g（m）,置无嘴烧杯中，加入酸性洗涤剂溶液100ml和数滴十氢化萘。（2）同NDF测定。（3）用已知质量的玻璃坩埚（m3）,在抽滤瓶上抽滤残渣，并用沸水洗涤残渣，抽滤反复3次，然后用少量丙酮洗涤残渣，反复冲洗至滤液无色为止，抽净全部丙酮。（4）同NDF测定，坩埚+残渣质量为（m4）。ADF=(m4-m3)/m。(1) Accurately weigh the sample, 0.5-1.0g (m), place it in a spoutless beaker, add 100ml of acidic detergent solution and a few drops of decalin. (2) Same as NDF measurement. (3) Use a glass crucible (m3) of known mass to suction filter the residue on the suction filter bottle, wash the residue with boiling water, repeat the suction filtration three times, then wash the residue with a small amount of acetone, rinse repeatedly until the filtrate is colorless, and pump Net all acetone. (4) Same as NDF measurement, the mass of crucible + residue is (m4). ADF=(m4-m3)/m.
POD活性（过氧化物酶活性）：采用索莱宝公司过氧化物酶（POD）活性检测试剂盒（BC0090）对干旱处理组和对照组的苜蓿叶片进行检测，每次检测重复3次。POD activity (peroxidase activity): The alfalfa leaves of the drought treatment group and the control group were tested using Solebao's peroxidase (POD) activity detection kit (BC0090). Each test was repeated three times.
CAT活性（过氧化氢酶活性）：采用索莱宝公司过氧化氢酶（CAT）活性检测试剂盒（BC0200）对干旱处理组和对照组的苜蓿叶片进行检测，每次检测重复3次。CAT activity (catalase activity): The alfalfa leaves of the drought treatment group and the control group were tested using the Catalase (CAT) activity detection kit (BC0200) of Solebao Company. Each test was repeated three times.
SOD活性（超氧化物歧化酶活性）：采用索莱宝公司超氧化物歧化酶（SOD）活性检测试剂盒（BC0170）对干旱处理组和对照组的苜蓿叶片进行检测，每次检测重复3次。SOD activity (superoxide dismutase activity): The alfalfa leaves in the drought treatment group and the control group were tested using Solebao's superoxide dismutase (SOD) activity detection kit (BC0170). Each test was repeated three times. .
Pn（净光合速率）：使用便携式光合作用测量系统 LI-6400XT 仪器对干旱处理组和对照组的苜蓿叶片进行净光合速率（Photosynthetic rate，Pn）的测定，每次检测3个叶片。Pn (net photosynthetic rate): Use the portable photosynthetic measurement system LI-6400XT instrument to measure the net photosynthetic rate (Photosynthetic rate, Pn) of the alfalfa leaves in the drought treatment group and the control group. Three leaves were tested each time.
Fm（最大荧光产量）：由便携式叶绿素荧光仪MultispeQ（Photosynq，美国）对干旱处理组和对照组和的苜蓿叶片的进行检测，具体方法为：将仪器夹住烟草叶片后，点击测量键，15s左右测量完毕，测量时使植株保持直立，不改变叶片的角度，每次检测3个叶片，每个叶片重复3次。Fm (maximum fluorescence yield): The alfalfa leaves of the drought treatment group and the control group were detected by a portable chlorophyll fluorometer MultispeQ (Photosynq, USA). The specific method is: after clamping the instrument to the tobacco leaves, click the measurement button for 15 seconds After the left and right measurements are completed, keep the plant upright during the measurement without changing the angle of the leaves. Test 3 leaves at a time and repeat each leaf 3 times.
H2O2含量检测：利用DAB法（3,3-二氨基联苯胺四盐酸盐）检测H2O2的含量，H2O2能与DAB迅速反应生成棕红色化合物，从而定位组织中的过氧化氢，颜色越深表明H2O2含量越高。使用塞维尔公司DAB染色试剂盒（servicebio，G1212-200T）检测干旱处理组和对照组苜蓿叶片的H2O2含量，每次检测3个叶片。H2 O2 content detection: Use the DAB method (3,3-diaminobenzidine tetrahydrochloride) to detect the H2 O2 content. H2 O2 can react quickly with DAB to generate a brown-red compound, thereby locating it in the tissue. of hydrogen peroxide, the darker the color, the higher the H2 O2 content. Use Sevier Company DAB staining kit (servicebio, G1212-200T) to detect the H2 O2 content of alfalfa leaves in the drought treatment group and control group, with 3 leaves tested each time.
超表达MsMYBH阳性转基因株系（OE-1、OE-2、OE-3、OE-4、OE-5、OE-6和OE-7）分别采用艾德莱的DNA提取试剂盒、RNA提取试剂盒在基因水平和转录水平鉴定，鉴定引物结果如图3中A和B所示，RNAi MsMYBH阳性阳性转基因株系（RNAi-1、RNAi-2、RNAi-3、RNAi-4、RNAi-5、RNAi-6和RNAi-7）在基因水平和转录水平鉴定结果如图3中C和D所示；WT为中苜1号，相对表达量为MsMYBH基因的相对表达量。The overexpressing MsMYBH positive transgenic lines (OE-1, OE-2, OE-3, OE-4, OE-5, OE-6 and OE-7) used Adelaide's DNA extraction kit and RNA extraction reagent respectively. The cassette was identified at the gene level and transcription level. The identification primer results are shown in Figure 3, A and B. RNAi MsMYBH positive transgenic lines (RNAi-1, RNAi-2, RNAi-3, RNAi-4, RNAi-5, The identification results of RNAi-6 and RNAi-7) at the gene level and transcription level are shown in Figure 3, C and D; WT is Zhongli No. 1, and the relative expression level is the relative expression level of the MsMYBH gene.
结果表明：The results show:
在正常对照条件下（对照组），超表达MsMYBH阳性转基因株系(OE MYBH：OE-1、OE-2和OE-3)、野生性植株（WT）和RNAi MsMYBH阳性转基因植株(Ri MYBH：RNAi-1、RNAi-2和RNAi-3)株高间差异不大，但OE MYBH转基因株系的分枝数显著多于WT，而WT的分枝数又显著多于Ri MYBH转基因植株（图6A-C；图6中，对照为对照组，干旱为干旱处理组，处理后为干旱处理后，处理前为干旱处理前，OE MYBH为超表达MsMYBH阳性转基因株系，Ri MYBH为RNAiMsMYBH阳性转基因植株，OE MYBH中的1、2和3分别为OE-1扦插子代、OE-2扦插子代和OE-3扦插子代，Ri MYBH中的1、2和3分别为RNAi-1扦插子代、RNAi-2扦插子代和RNAi-3扦插子代，WT为中苜1号扦插子代）。同时，在对照条件下，检测了转基因植株和WT的生物量及相关指标数据（鲜重、干重、茎叶比、粗蛋白、DNF和ADF）。Under normal control conditions (control group), overexpressing MsMYBH-positive transgenic lines (OE MYBH: OE-1, OE-2 and OE-3), wild-type plants (WT) and RNAi MsMYBH-positive transgenic plants (Ri MYBH: RNAi-1, RNAi-2 and RNAi-3) There is not much difference in plant height, but the number of branches of OE MYBH transgenic lines is significantly more than that of WT, and the number of branches of WT is significantly more than that of Ri MYBH transgenic plants (Fig. 6A-C; In Figure 6, the control is the control group, drought is the drought treatment group, after treatment is after drought treatment, before treatment is before drought treatment, OE MYBH is the over-expression MsMYBH positive transgenic line, Ri MYBH is the RNAiMsMYBH positive transgene Plants, 1, 2 and 3 in OE MYBH are the progeny of OE-1 cuttings, OE-2 cuttings and OE-3 cuttings respectively, and 1, 2 and 3 in Ri MYBH are RNAi-1 cuttings respectively. generation, RNAi-2 cutting progeny and RNAi-3 cutting progeny, WT is the cutting progeny of Zhongli No. 1). At the same time, under control conditions, the biomass and related index data (fresh weight, dry weight, stem-to-leaf ratio, crude protein, DNF and ADF) of transgenic plants and WT were detected.
而超表达MsMYBH阳性转基因株系的鲜重和干重都显著高于WT，而WT显著高于RNAiMsMYBH阳性转基因株系（图7中C和D；图7C-D中，OE MYBH为超表达MsMYBH阳性转基因株系，Ri MYBH为RNAi MsMYBH阳性转基因植株，OE MYBH中的1、2和3分别为OE-1扦插子代、OE-2扦插子代和OE-3扦插子代，Ri MYBH中的1、2和3分别为RNAi-1扦插子代、RNAi-2扦插子代和RNAi-3扦插子代，WT为中苜1号扦插子代，图7为对照组检测结果图），且其牧草品质并未下降（图7F-H，OE MYBH为超表达MsMYBH阳性转基因株系，Ri MYBH为RNAi MsMYBH阳性转基因植株，OE MYBH中的1、2和3分别为OE-1扦插子代、OE-2扦插子代和OE-3扦插子代，Ri MYBH中的1、2和3分别为RNAi-1扦插子代、RNAi-2扦插子代和RNAi-3扦插子代，WT为中苜1号扦插子代，图7为对照组检测结果图）。以上结果均表明，MsMYBH促进了紫花苜蓿的生长。The fresh weight and dry weight of overexpressing MsMYBH-positive transgenic lines were significantly higher than that of WT, while WT was significantly higher than that of RNAiMsMYBH-positive transgenic lines (Figure 7, C and D; Figure 7C-D, OE MYBH is overexpressing MsMYBH Positive transgenic lines, Ri MYBH are RNAi MsMYBH positive transgenic plants, 1, 2 and 3 in OE MYBH are the progenies of OE-1 cuttings, OE-2 cuttings and OE-3 cuttings respectively, Ri MYBH 1, 2 and 3 are the progeny of RNAi-1 cuttings, RNAi-2 cuttings and RNAi-3 cuttings respectively, WT is the cutting progeny of Zhongli No. 1, Figure 7 is the test result of the control group), and its Forage quality has not declined (Figure 7F-H, OE MYBH is the over-expression MsMYBH positive transgenic line, Ri MYBH is the RNAi MsMYBH positive transgenic plant, 1, 2 and 3 in OE MYBH are the OE-1 cutting progeny, OE respectively. -2 cutting progeny and OE-3 cutting progeny, 1, 2 and 3 in Ri MYBH are RNAi-1 cutting progeny, RNAi-2 cutting progeny and RNAi-3 cutting progeny respectively, WT is Zhonglu1 No. cutting progeny, Figure 7 shows the test results of the control group). The above results all show that MsMYBH promotes the growth of alfalfa.
在干旱胁迫处理后（干旱处理组），与WT相比，OE MYBH转基因株系表现出更强的抗旱性，其分枝数更多，叶片萎蔫程度最小（图6A）；而 Ri MYBH转基因植株抗旱性最弱，其大量叶片萎蔫脱落（图6A）。生理指标的测定也证明了OE MYBH转基因株系具有更强的抗旱性，主要表现为其叶片相对含水量最高，丙二醛含量最低，脯氨酸含量最高（图6D-F，对照为对照组，干旱为干旱处理组，处理后为干旱处理后，处理前为干旱处理前，OE MYBH为超表达MsMYBH阳性转基因株系，Ri MYBH为RNAi MsMYBH阳性转基因植株，OE MYBH中的1、2和3分别为OE-1扦插子代、OE-2扦插子代和OE-3扦插子代，Ri MYBH中的1、2和3分别为RNAi-1扦插子代、RNAi-2扦插子代和RNAi-3扦插子代，WT为中苜1号扦插子代）；干旱处理12d前，叶片光合作用和最大荧光产量也都显著高于WT(图7A和B，OE MYBH1为OE-1，OE MYBH2为OE-2，OEMYBH3为OE-3，Ri MYBH1为RNAi-1，Ri MYBH2为RNAi-2，Ri MYBH3为RNAi-3)；干旱胁迫后，H2O2的积累最少(图8中A)，且抗氧化酶活性（POD活性、CAT活性和SOD活性）最高（图8中B-D，对照为对照组，干旱为干旱处理组，处理后为干旱处理后，处理前为干旱处理前，OE MYBH为超表达MsMYBH阳性转基因株系，Ri MYBH为RNAi MsMYBH阳性转基因植株，OE MYBH中的1、2和3分别为OE-1扦插子代、OE-2扦插子代和OE-3扦插子代，Ri MYBH中的1、2和3分别为RNAi-1扦插子代、RNAi-2扦插子代和RNAi-3扦插子代，WT为中苜1号扦插子代）。Ri MYBH转基因植株的表现相反（图6中D-F、图7中A-B和图8）。以上结果均表明了MsMYBH增强了紫花苜蓿的抗旱性。After drought stress treatment (drought treatment group), compared with WT, OE MYBH transgenic lines showed stronger drought resistance, with more branches and minimal leaf wilting (Figure 6A); while Ri MYBH transgenic plants The drought resistance is the weakest, and a large number of its leaves wilted and fell off (Figure 6A). The measurement of physiological indicators also proved that the OE MYBH transgenic line has stronger drought resistance, which is mainly reflected in the fact that its leaves have the highest relative water content, the lowest malondialdehyde content, and the highest proline content (Figure 6D-F, the control is the control group , drought is the drought treatment group, after treatment is after drought treatment, before treatment is before drought treatment, OE MYBH is the over-expression MsMYBH positive transgenic line, Ri MYBH is the RNAi MsMYBH positive transgenic plant, 1, 2 and 3 in OE MYBH They are the progeny of OE-1 cutting, OE-2 cutting and OE-3 respectively. 1, 2 and 3 in Ri MYBH are the progeny of RNAi-1 cutting, RNAi-2 cutting and RNAi- respectively. 3 cutting progeny, WT is the progeny of Zhongmulo No. 1 cuttings); before 12 days of drought treatment, leaf photosynthesis and maximum fluorescence yield were also significantly higher than WT (Figure 7A and B, OE MYBH1 is OE-1, OE MYBH2 is OE-2, OEMYBH3 is OE-3, Ri MYBH1 is RNAi-1, Ri MYBH2 is RNAi-2, Ri MYBH3 is RNAi-3); after drought stress, the accumulation of H2 O2 is the least (A in Figure 8), And the antioxidant enzyme activity (POD activity, CAT activity and SOD activity) is the highest (BD in Figure 8, the control is the control group, drought is the drought treatment group, after treatment is after drought treatment, before treatment is before drought treatment, OE MYBH is Overexpression of MsMYBH positive transgenic lines, Ri MYBH is RNAi MsMYBH positive transgenic plants, 1, 2 and 3 in OE MYBH are the progeny of OE-1 cuttings, OE-2 cuttings and OE-3 cuttings respectively, Ri 1, 2 and 3 in MYBH are the progeny of RNAi-1 cuttings, RNAi-2 cuttings and RNAi-3 cuttings respectively, and WT is the progeny of Zhongli No. 1 cuttings). The performance of Ri MYBH transgenic plants was opposite (DF in Figure 6, AB in Figure 7, and Figure 8). The above results all show that MsMYBH enhances the drought resistance of alfalfa.
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。The present invention has been described in detail above. For those skilled in the art, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without performing unnecessary experiments. Although specific embodiments of the present invention have been shown, it should be understood that further modifications can be made to the invention. In short, based on the principles of the present invention, this application is intended to include any changes, uses, or improvements to the present invention, including changes that depart from the scope disclosed in this application and are made using conventional techniques known in the art.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104152464A (en)* | 2014-08-01 | 2014-11-19 | 华中师范大学 | Cotton fiber specific MYBL1 gene |
| CN107937413A (en)* | 2017-11-30 | 2018-04-20 | 南京农业大学 | The application of one M. truncatula MYB class transcription factors MtMYB1 |
| CN111087458A (en)* | 2019-12-30 | 2020-05-01 | 上海交通大学 | Alfalfa MYB transcription factor and aluminum toxin resistant application thereof |
| CN113840925A (en)* | 2019-03-14 | 2021-12-24 | 热带生物科学英国有限公司 | Modifying the specificity of non-coding RNA molecules for silencing genes in eukaryotic cells |
| WO2022188290A1 (en)* | 2021-03-10 | 2022-09-15 | 中国农业科学院作物科学研究所 | Osftl1 and application of encoding gene thereof in shortening heading stage of rice |
| CN116515887A (en)* | 2023-04-26 | 2023-08-01 | 中国农业大学 | Application of Medicago MsSPL4 Gene in Improving Stress Resistance of Plants |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104152464A (en)* | 2014-08-01 | 2014-11-19 | 华中师范大学 | Cotton fiber specific MYBL1 gene |
| CN107937413A (en)* | 2017-11-30 | 2018-04-20 | 南京农业大学 | The application of one M. truncatula MYB class transcription factors MtMYB1 |
| CN113840925A (en)* | 2019-03-14 | 2021-12-24 | 热带生物科学英国有限公司 | Modifying the specificity of non-coding RNA molecules for silencing genes in eukaryotic cells |
| CN111087458A (en)* | 2019-12-30 | 2020-05-01 | 上海交通大学 | Alfalfa MYB transcription factor and aluminum toxin resistant application thereof |
| WO2022188290A1 (en)* | 2021-03-10 | 2022-09-15 | 中国农业科学院作物科学研究所 | Osftl1 and application of encoding gene thereof in shortening heading stage of rice |
| CN116515887A (en)* | 2023-04-26 | 2023-08-01 | 中国农业大学 | Application of Medicago MsSPL4 Gene in Improving Stress Resistance of Plants |
| Title |
|---|
| 佚名: "MS.gene06188.t1", pages 1 - 10, Retrieved from the Internet <URL:http://alfalfagedb.liu-lab.com/blastplus/blastp#8926032446675145307>* |
| 李菲等: "蒺藜苜蓿和天蓝苜蓿MYB 转录因子的筛选和特征分析", 《分子植物育种》, vol. 15, no. 5, 31 May 2017 (2017-05-31), pages 1630 - 1638* |
| Publication number | Publication date |
|---|---|
| CN117535342B (en) | 2024-05-07 |
| Publication | Publication Date | Title |
|---|---|---|
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