技术领域Technical Field
本发明属于生物育种技术领域，具体涉及ZmRIN4蛋白在调控玉米株型中的应用。The invention belongs to the technical field of biological breeding, and particularly relates to the application of ZmRIN4 protein in regulating corn plant type.
背景技术Background Art
玉米是世界上种植面积最广的粮、经、饲和工业原料兼用作物，在粮食安全保障体系中占据重要地位。株高和穗位高是玉米重要的农艺性状，与株型、抗倒伏性、群体光合效率、收获指数及产量等密切相关，是玉米理论和应用研究的重点。自1968年Donald提出了作物理想株型的概念之后，株高和穗位高更是玉米理想株型的重要指标，严重影响着玉米产量、抗倒伏性和生态适应性等。理想株型的定向选择有助于提高作物的抗倒伏性和种植密度，是实现玉米产量进一步突破的基础。Corn is the most widely planted crop in the world that can be used as food, cash crop, feed, and industrial raw material, and it occupies an important position in the food security system. Plant height and ear height are important agronomic traits of corn, which are closely related to plant type, lodging resistance, group photosynthetic efficiency, harvest index, and yield, and are the focus of theoretical and applied research on corn. Since Donald proposed the concept of ideal plant type of crops in 1968, plant height and ear height have become important indicators of the ideal plant type of corn, which seriously affect corn yield, lodging resistance, and ecological adaptability. The directional selection of ideal plant type helps to improve the lodging resistance and planting density of crops, and is the basis for achieving further breakthroughs in corn yields.
上世纪六十年代兴起的“绿色革命”其实质就是矮化育种，通过将矮杆基因导入到作物中，增加抗倒伏性、提高收获指数和氮肥耐受力，对全世界粮食产量的提高起了关键作用。在现代玉米育种过程中，中美玉米材料均向着穗位降低、雄穗分枝数减少、叶夹角变小及开花期提前的方向选择。玉米株高和穗位高在过去几十年的育种过程中经历了连续地向矮化方向的高强度选择。玉米株高的升高，尤其是穗位高的升高，会显著升高玉米重心，大大增加其倒伏和茎折的概率。有效降低株高和穗位高，可以有效地降低倒伏率，有利于源库平衡和协调，促进光合产物向穗部转运，是提高玉米耐密性的根本要求。较高的株高不利于群体内的通风透光，影响群体光合效率，直接影响玉米产量。The essence of the "Green Revolution" that emerged in the 1960s is dwarfing breeding. By introducing dwarf genes into crops, lodging resistance, harvest index and nitrogen fertilizer tolerance are increased, which has played a key role in increasing global grain production. In the process of modern corn breeding, both Chinese and American corn materials are selected in the direction of lower ear position, fewer male ear branches, smaller leaf angles and earlier flowering period. In the past few decades of breeding, corn plant height and ear height have undergone continuous high-intensity selection in the direction of dwarfing. The increase in corn plant height, especially the increase in ear height, will significantly increase the center of gravity of corn and greatly increase the probability of lodging and stem breakage. Effectively reducing plant height and ear height can effectively reduce the lodging rate, which is conducive to the balance and coordination of source and sink, and promotes the transport of photosynthetic products to the ear, which is the fundamental requirement for improving corn density tolerance. Higher plant height is not conducive to ventilation and light transmission within the group, affects the photosynthetic efficiency of the group, and directly affects corn yield.
株高作为重要的农艺性状，一直是玉米理论研究和应用研究的重点。大量的研究表明，玉米株高是由一系列微效基因控制的复杂数量性状。早期大量研究利用双亲连锁群体对玉米株高进行QTL定位。随着测序技术的发展，利用大群体的高密度SNPs数据和丰富的表型数据快速和准确地定位了株高和穗位高的主效QTL，在玉米10条染色体上都检测到株高QTL，但大多为微效QTL。MaizeGDB数据库中已收集了二百余个玉米株高QTL，遗传效应均较小，且不同研究者利用不同定位群体亲本所定位的QTL位点不尽一致，表明控制株高与穗位的遗传基础非常复杂。过表达ZmPIN1a，获得了根系发达、植株矮化和抗旱能力增强的突变体。Semi-dwarf3(sdw3)基因编码乙烯合成关键酶ZmACS7，是新的矮杆基因，其功能获得型突变体表现为植株矮化，叶夹角增大。此外，过表达ZmPHYC、改造ZmPHYB1和敲除ZmPIFs也都创制了矮化突变体。As an important agronomic trait, plant height has always been the focus of theoretical and applied research in maize. A large number of studies have shown that maize plant height is a complex quantitative trait controlled by a series of micro-effect genes. In the early stage, a large number of studies used parental linkage populations to locate QTLs for maize plant height. With the development of sequencing technology, the main QTLs of plant height and ear height were quickly and accurately located using high-density SNPs data of large populations and rich phenotypic data. Plant height QTLs were detected on all 10 chromosomes of maize, but most of them were micro-effect QTLs. More than 200 maize plant height QTLs have been collected in the MaizeGDB database. The genetic effects are small, and the QTL loci located by different researchers using different positioning population parents are not consistent, indicating that the genetic basis of controlling plant height and ear position is very complex. Overexpression of ZmPIN1a obtained mutants with developed root system, dwarf plants and enhanced drought resistance. Semi-dwarf3 (sdw3) gene encodes ZmACS7, a key enzyme in ethylene synthesis, and is a new dwarf gene. Its gain-of-function mutants show dwarf plants and increased leaf angles. In addition, overexpression of ZmPHYC, modification of ZmPHYB1 and knockout of ZmPIFs also created dwarf mutants.
玉米是我国的第一大粮食作物，对我国的粮食安全和农业发展有着举足轻重的作用。提高玉米种植密度是玉米产量提高的主要手段，合理的株高可以有效地提高玉米抗倒伏能力。因此挖掘玉米株高调控基因，为培育耐密理想株型玉米提供靶标基因资源。Corn is the largest food crop in my country and plays a vital role in my country's food security and agricultural development. Increasing corn planting density is the main means to increase corn yield, and a reasonable plant height can effectively improve corn's resistance to lodging. Therefore, it is necessary to explore corn plant height regulatory genes to provide target gene resources for cultivating corn with an ideal plant type that is tolerant to density.
发明内容Summary of the invention
本发明要解决的技术问题是如何调控玉米的株型。The technical problem to be solved by the present invention is how to regulate the plant type of corn.
为解决该技术问题，第一个方面，本发明提供蛋白质或调控基因表达的物质或调控所述蛋白质活性或含量的物质在下述任一项中的应用，所述基因编码所述蛋白质，所述蛋白质为ZmRIN4蛋白；To solve the technical problem, in a first aspect, the present invention provides the use of a protein or a substance regulating gene expression or a substance regulating the activity or content of the protein in any of the following items, wherein the gene encodes the protein, and the protein is ZmRIN4 protein;
A1)、在调控植物株高中的应用；A1) Application in regulating plant strains;
A2)、在制备调控植物株高的产品中的应用；A2) Application in the preparation of products for regulating plant height;
A3)、在调控植物穗位高中的应用；A3) Application in regulating plant ear position;
A4)、在制备调控植物穗位高的产品中的应用；A4), application in the preparation of products for regulating plant ear height;
A5)、在调控植物茎节数量中的应用；A5) Application in regulating the number of plant stem nodes;
A6)、在制备调控植物茎节数量的产品中的应用；A6) Application in the preparation of products for regulating the number of plant stem nodes;
A7)、在植物育种或植物辅助育种中应用；A7) Application in plant breeding or plant assisted breeding;
所述ZmRIN4是下述任一种蛋白质：The ZmRIN4 is any of the following proteins:
a1)、氨基酸序列是SEQ ID No.1所示的蛋白质；a1), the amino acid sequence is the protein shown in SEQ ID No.1;
a2)、将a1)所示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)所示的氨基酸序列具有80％以上同一性的蛋白质；a2), a protein having more than 80% identity with the amino acid sequence shown in a1) obtained by substitution and/or deletion and/or addition of amino acid residues in the amino acid sequence shown in a1);
a3)、在a1)或a2)的N端或/和C端连接标签得到的融合蛋白质。a3) A fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of a1) or a2).
SEQ ID No.1由80个氨基酸残基组成。SEQ ID No. 1 consists of 80 amino acid residues.
上述蛋白质可人工合成，也可先合成其编码基因，再进行生物表达得到。The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.
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蛋白标签、His蛋白标签、MBP蛋白标签、HA蛋白标签、myc蛋白标签、GST蛋白标签和/或SUMO蛋白标签等。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 can be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag and/or a SUMO protein tag, etc.
进一步地，所述应用中，所述调控基因表达的物质或调控所述蛋白质活性或含量可为上调或提高或促进基因表达的物质或上调或提高或促进所述蛋白质活性或含量。Furthermore, in the application, the substance that regulates gene expression or regulates the activity or content of the protein may be a substance that upregulates, increases or promotes gene expression or upregulates, increases or promotes the activity or content of the protein.
进一步地，所述应用中，所述调控植物株高或/和穗位高或/和茎节数量可为降低株高或/和穗位高或/和茎节数量。Furthermore, in the application, the regulation of plant height and/or ear height and/or number of stem nodes may be reduction of plant height and/or ear height and/or number of stem nodes.
进一步地，所述应用中，所述植物育种的目的可为培育株型优良的植物。例如培育株高或穗位高降低的玉米。Furthermore, in the application, the purpose of plant breeding may be to cultivate plants with good plant type, such as cultivating corn with reduced plant height or ear height.
进一步地，上述应用中，所述蛋白质来源于玉米。Furthermore, in the above application, the protein is derived from corn.
进一步地，上述的应用中，所述调控基因表达的物质或调控所述蛋白质活性或含量的物质为生物材料，所述生物材料为下述任一种：Furthermore, in the above application, the substance regulating gene expression or the substance regulating the activity or content of the protein is a biological material, and the biological material is any one of the following:
B1)、编码上述蛋白质的核酸分子；B1), a nucleic acid molecule encoding the above protein;
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), a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3);
B8)、抑制或降低上述蛋白质的编码基因的表达或上述蛋白质的活性的核酸分子；B8), a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the above protein or the activity of the above protein;
B9)、含有B8)所述核酸分子的表达盒、重组载体、重组微生物或转基因植物细胞系。B9), an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule described in B8).
上述相关生物材料中，B2)所述的表达盒是指能够在宿主细胞中表达ZmRIN4蛋白的DNA，该DNA不但可包括启动ZmRIN4蛋白编码基因转录的启动子，还可包括终止ZmRIN4蛋白编码基因转录的终止子。In the above-mentioned related biological materials, the expression cassette described in B2) refers to a DNA capable of expressing ZmRIN4 protein in a host cell, and the DNA may include not only a promoter for initiating transcription of a gene encoding ZmRIN4 protein, but also a terminator for terminating transcription of a gene encoding ZmRIN4 protein.
进一步，B2)所述表达盒还可包括增强子序列。Furthermore, the expression cassette B2) may further include an enhancer sequence.
上述相关生物材料中，B2)所述重组载体可含有SEQ ID No.2所示的用于编码ZmRIN4蛋白的DNA分子。上述相关生物材料中，B4)所述重组微生物具体可为酵母、细菌、藻和真菌。In the above-mentioned related biological materials, the recombinant vector in B2) may contain the DNA molecule encoding the ZmRIN4 protein shown in SEQ ID No. 2. In the above-mentioned related biological materials, the recombinant microorganism in B4) may specifically be yeast, bacteria, algae and fungi.
上述相关生物材料中，B6)所述植物组织可来源于根、茎、叶、花、果实、种子、花粉、胚和花药。Among the above-mentioned related biological materials, the plant tissue described in B6) can be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.
上述相关生物材料中，B7)所述转基因植物器官可为转基因植物的根、茎、叶、花、果实和种子。Among the above-mentioned related biological materials, the transgenic plant organs described in B7) can be roots, stems, leaves, flowers, fruits and seeds of transgenic plants.
上述相关生物材料中，所述转基因植物细胞系、转基因植物组织和转基因植物器官可包括繁殖材料，也可不包括繁殖材料。Among the above-mentioned related biological materials, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs may or may not include propagation materials.
上述相关生物材料中，B9)所述的表达盒是指能够在宿主细胞中表达sgRNA的DNA，该DNA不但可包括启动sgRNA基因转录的启动子，还可包括终止sgRNA基因转录的终止子。In the above-mentioned related biological materials, the expression cassette described in B9) refers to a DNA capable of expressing sgRNA in a host cell, and the DNA may include not only a promoter for initiating transcription of the sgRNA gene, but also a terminator for terminating transcription of the sgRNA gene.
进一步地，上述的应用中，B1)所述核酸分子为如下g1)-g3)任一项所述的DNA分子：Furthermore, in the above application, the nucleic acid molecule described in B1) is a DNA molecule described in any one of g1) to g3) below:
g1)、编码链的编码序列是SEQ ID No.2的DNA分子；g1), the coding sequence of the coding strand is a DNA molecule of SEQ ID No. 2;
g2)、编码链的核苷酸序列是SEQ ID No.3的DNA分子；g2), the nucleotide sequence of the coding strand is a DNA molecule of SEQ ID No. 3;
g3)、与g1)或g2)所述DNA分子具有80％以上的同一性，且调控植物抗逆性的DNA分子。g3) A DNA molecule that has more than 80% identity with the DNA molecule described in g1) or g2) and regulates plant stress resistance.
进一步地，上述的应用中，B8)所述核酸分子为表达靶向上述应用中的所述基因的gRNA的DNA分子或为靶向上述应用中的所述基因的gRNA。Furthermore, in the above application, the nucleic acid molecule in B8) is a DNA molecule expressing a gRNA targeting the gene in the above application or is a gRNA targeting the gene in the above application.
进一步地，上述的应用中，上述gRNA的靶标序列的核苷酸序列是SEQ ID No.3第2759-2778位(5’-GGCCGTCCCTTGCCAAAGTT-3’)。Furthermore, in the above application, the nucleotide sequence of the target sequence of the above gRNA is SEQ ID No. 3, positions 2759-2778 (5’-GGCCGTCCCTTGCCAAAGTT-3’).
进一步地，上述的应用中，B1)所述gRNA(gRNA基因)的核苷酸序列是SEQ ID No.4第501-603位。其中SEQ ID No.4第501-520位为sgRNA的spacer序列，第521-603位为sgRNA的骨架序列。Further, in the above application, the nucleotide sequence of the gRNA (gRNA gene) described in B1) is SEQ ID No. 4, positions 501-603. Among them, SEQ ID No. 4, positions 501-520 are the spacer sequence of sgRNA, and positions 521-603 are the backbone sequence of sgRNA.
进一步地，上述的应用中，所述植物选自下述任一种：Furthermore, in the above application, the plant is selected from any one of the following:
C1)、单子叶植物；C1), monocots;
C2)、禾本科植物；C2), Gramineae;
C3)、玉蜀黍属植物；C3), Zea mays;
C4)、玉米(Setaria italica)。C4), corn (Setaria italica).
上述应用中，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Lambdaratio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 application, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST 2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.
上述应用中，所述80％以上的同一性可为至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。In the above applications, the above 80% identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
上述应用中，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)。In the above application, 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 transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).
上述应用中，所述调控基因表达可为抑制或降低所述基因表达，所述抑制或降低所述基因表达可通过基因敲除实现或通过基因沉默实现。In the above application, the regulating gene expression may be to inhibit or reduce the gene expression, and the inhibiting or reducing the gene expression may be achieved by gene knockout or gene silencing.
所述基因敲除(geneknockout)是指通过同源重组使特定靶基因失活的现象。基因敲除是通过DNA序列的改变使特定靶基因失活。The gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout is the inactivation of a specific target gene by changing the DNA sequence.
所述基因沉默是指在不损伤原有DNA的情况下使基因不表达或低表达的现象。基因沉默以不改变DNA序列为前提，使基因不表达或低表达。基因沉默可发生在两种水平上，一种是由于DNA甲基化、异染色质化以及位置效应等引起的转录水平的基因沉默，另一种是转录后基因沉默，即在基因转录后的水平上通过对靶标RNA进行特异性抑制而使基因失活，包括反义RNA、共抑制(co-suppression)、基因压抑(quelling)、RNA干扰(RNAi)和微小RNA(miRNA)介导的翻译抑制等。The gene silencing refers to the phenomenon that the gene is not expressed or is underexpressed without damaging the original DNA. Gene silencing is based on the premise that the DNA sequence is not changed, so that the gene is not expressed or is underexpressed. Gene silencing can occur at two levels, one is gene silencing at the transcriptional level caused by DNA methylation, heterochromatinization and position effect, and the other is post-transcriptional gene silencing, that is, at the level after gene transcription, the gene is inactivated by specifically inhibiting the target RNA, including antisense RNA, co-suppression, gene repression, RNA interference (RNAi) and microRNA (miRNA)-mediated translation inhibition, etc.
上述应用中，所述调控基因表达的物质可为抑制或降低所述基因表达的试剂。所述抑制或降低所述基因表达的试剂可为敲除所述基因的试剂，如通过同源重组敲除所述基因的试剂，或通过CRISPR-Cas9敲除所述基因的试剂。所述抑制或降低所述基因表达的试剂可以包含靶向所述基因的多核苷酸，例如siRNA、shRNA、sgRNA、miRNA或反义RNA。In the above application, the substance regulating gene expression may be an agent that inhibits or reduces the expression of the gene. The agent that inhibits or reduces the expression of the gene may be an agent that knocks out the gene, such as an agent that knocks out the gene by homologous recombination, or an agent that knocks out the gene by CRISPR-Cas9. The agent that inhibits or reduces the expression of the gene may include a polynucleotide that targets the gene, such as siRNA, shRNA, sgRNA, miRNA or antisense RNA.
第二个方面，本发明提供上述应用中的所述蛋白质或/和上述应用中的所述生物材料。In a second aspect, the present invention provides the protein in the above application and/or the biomaterial in the above application.
第三个方面，本发明提供一种调控植物株高或/和穗位高或/和茎节数量的方法，所述方法包括通过调控受体植物中所述ZmRIN4蛋白的表达或调控所述ZmRIN4蛋白的活性或含量，来调控所述受体植物株高或/和穗位高或/和茎节数量。In a third aspect, the present invention provides a method for regulating plant height and/or ear height and/or the number of stem nodes, the method comprising regulating the plant height and/or ear height and/or the number of stem nodes of the recipient plant by regulating the expression of the ZmRIN4 protein in the recipient plant or regulating the activity or content of the ZmRIN4 protein.
进一步地，所述方法包括M1)或M2)，Further, the method comprises M1) or M2),
M1)、通过上调或提高或促进受体植物中所述ZmRIN4蛋白的表达或所述ZmRIN4蛋白的活性或含量，来抑制或降低所述受体植物株高或/和穗位高或/和茎节数量；M1), inhibiting or reducing the plant height and/or ear height and/or stem node number of the recipient plant by up-regulating or increasing or promoting the expression of the ZmRIN4 protein or the activity or content of the ZmRIN4 protein in the recipient plant;
M2)、通过下调或降低或抑制受体植物中所述ZmRIN4蛋白的表达或所述ZmRIN4蛋白的活性或含量，来促进或提高所述受体植物株高或/和穗位高或/和茎节数量。M2), by down-regulating, reducing or inhibiting the expression of the ZmRIN4 protein or the activity or content of the ZmRIN4 protein in the recipient plant, to promote or increase the plant height and/or ear height and/or the number of stem nodes of the recipient plant.
进一步地，所述的方法中，所述植物为玉米，M1)所述方法包括向受体玉米中导入上述ZmRIN4蛋白的编码基因来提高或促进所述受体玉米中所述ZmRIN4蛋白编码基因的表达或提高或促进所述受体玉米中所述ZmRIN4蛋白的活性或含量，得到株高或/和穗位高或/和茎节数量低于(矮于)或少于所述受体玉米的目的玉米。Furthermore, in the method described above, the plant is corn, and M1) the method includes introducing the coding gene of the above-mentioned ZmRIN4 protein into the recipient corn to increase or promote the expression of the coding gene of the ZmRIN4 protein in the recipient corn or to increase or promote the activity or content of the ZmRIN4 protein in the recipient corn, so as to obtain the target corn whose plant height and/or ear height and/or stem node number is lower (shorter) than or less than that of the recipient corn.
进一步地，所述的方法中，所述植物为玉米，M2)所述方法包括向受体玉米中导入上述gRNA的基因和Cas蛋白的编码基因来抑制或降低所述受体玉米中所述ZmRIN4蛋白编码基因的表达或抑制或降低所述受体玉米中所述ZmRIN4蛋白的活性或含量，得到株高或/和穗位高或/和茎节数量高于或多于所述受体玉米的目的玉米。Furthermore, in the method described above, the plant is corn, and M2) the method includes introducing the gene of the above-mentioned gRNA and the gene encoding the Cas protein into the recipient corn to inhibit or reduce the expression of the ZmRIN4 protein encoding gene in the recipient corn or inhibit or reduce the activity or content of the ZmRIN4 protein in the recipient corn, so as to obtain the target corn having a plant height and/or ear height and/or a number of stem nodes higher than or more than that of the recipient corn.
进一步地，M2)所述抑制或降低所述受体玉米中所述ZmRIN4蛋白编码基因的表达或抑制或降低所述受体玉米中所述ZmRIN4蛋白的活性或含量为将受体玉米中的基因组序列进行下述任一种突变：Furthermore, M2) inhibiting or reducing the expression of the gene encoding the ZmRIN4 protein in the recipient corn or inhibiting or reducing the activity or content of the ZmRIN4 protein in the recipient corn is to perform any of the following mutations on the genome sequence in the recipient corn:
M2-1)、将受体玉米中SEQ ID No.3的第2773-2774位的核苷酸AA缺失，导致目的基因发生移码突变，进而导致目的蛋白无法正常表达，从而实现了目基因的敲除；M2-1), deleting the nucleotide AA at position 2773-2774 of SEQ ID No.3 in the recipient corn, resulting in a frameshift mutation in the target gene, which in turn causes the target protein to be unable to be expressed normally, thereby achieving knockout of the target gene;
M2-2)、将受体玉米中SEQ ID No.3的第2776-2796位的核苷酸(5’-AAGTTTGGTGAATGGGATGTC-3’)缺失，导致目的基因发生突变，进而导致目的蛋白无法正常表达，从而实现了目基因的敲除。M2-2), the nucleotides 2776-2796 (5'-AAGTTTGGTGAATGGGATGTC-3') of SEQ ID No. 3 in the recipient corn are deleted, resulting in mutation of the target gene, which in turn causes the target protein to be unable to be expressed normally, thereby achieving knockout of the target gene.
第四个方面，本发明提供一种制备株高降低或/和穗位高降低或/和茎节数量减少的玉米的方法，所述方法包括通过上调或提高或促进受体玉米中所述ZmRIN4蛋白的表达或所述ZmRIN4蛋白的活性或含量，来抑制或降低玉米株高或/和穗位高或/和茎节数量。In a fourth aspect, the present invention provides a method for preparing corn with reduced plant height and/or reduced ear height and/or reduced number of stem nodes, the method comprising inhibiting or reducing the corn plant height and/or ear height and/or the number of stem nodes by upregulating or increasing or promoting the expression of the ZmRIN4 protein or the activity or content of the ZmRIN4 protein in the recipient corn.
进一步地，所述方法包括向受体玉米中导入上述ZmRIN4蛋白的编码基因来提高或促进所述受体玉米中所述ZmRIN4蛋白编码基因的表达或提高或促进所述受体玉米中所述ZmRIN4蛋白的活性或含量，得到株高或/和穗位高或/和茎节数量低于(矮于)或少于所述受体玉米的目的玉米。Furthermore, the method includes introducing the coding gene of the above-mentioned ZmRIN4 protein into the recipient corn to improve or promote the expression of the coding gene of the ZmRIN4 protein in the recipient corn or to improve or promote the activity or content of the ZmRIN4 protein in the recipient corn, thereby obtaining the target corn having plant height and/or ear height and/or stem node number lower (shorter than) or less than that of the recipient corn.
进一步地，所述的方法中，所述ZmRIN4蛋白的编码基因的核苷酸序列是SEQIDNo.2。Furthermore, in the method described above, the nucleotide sequence of the gene encoding the ZmRIN4 protein is SEQ ID No. 2.
进一步地，所述的方法中，所述ZmRIN4蛋白的编码基因通过重组表达载体导入到受体玉米中。Furthermore, in the method described above, the gene encoding the ZmRIN4 protein is introduced into the recipient corn via a recombinant expression vector.
在本发明的一个实施例中，所述重组表达载体为pCAMBIA3301-ZmRIN4。所述ZmRIN4蛋白的编码基因由Ubi启动子启动表达。In one embodiment of the present invention, the recombinant expression vector is pCAMBIA3301-ZmRIN4. The gene encoding the ZmRIN4 protein is expressed by the Ubi promoter.
本发明所取得的有益技术效果如下：The beneficial technical effects achieved by the present invention are as follows:
1、RIN4过表达可降低玉米株高，RIN4基因敲除后株高升高；RIN4过表达可降低玉米穗位高，RIN4基因敲除后穗位升高；RIN4过表达可降低玉米茎节数量，RIN4基因敲除后茎节数量升高；对于玉米理想株型遗传改良中具有重要作用；1. RIN4 overexpression can reduce corn plant height, and RIN4 gene knockout can increase plant height; RIN4 overexpression can reduce corn ear height, and RIN4 gene knockout can increase ear height; RIN4 overexpression can reduce the number of corn nodes, and RIN4 gene knockout can increase the number of nodes; it plays an important role in the genetic improvement of ideal corn plant type;
2、本项目拟首次揭示ZmRIN4在调控玉米株高或/和穗位高或/和茎节数量过程中的作用机制，探索ZmRIN4在玉米株高或/和穗位高或/和茎节数量生物育种的应用前景，为理想株型玉米育种提供新的基因资源；可应用于各大玉米育种单位、玉米种子公司育种部门，实现定向精确改良玉米耐旱性的分子设计育种，有广阔市场前景。2. This project intends to reveal for the first time the mechanism of action of ZmRIN4 in regulating maize plant height and/or ear height and/or number of internodes, explore the application prospects of ZmRIN4 in biological breeding of maize plant height and/or ear height and/or number of internodes, and provide new genetic resources for breeding of ideal plant types of maize; it can be applied to major maize breeding units and breeding departments of corn seed companies to achieve molecular design breeding for targeted and precise improvement of maize drought resistance, and has broad market prospects.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为载体pCAMBIA3301-ZmRIN4的图谱；Fig. 1 is a map of vector pCAMBIA3301-ZmRIN4;
图2转化体T1代目的基因ZmRIN4的PCR检测结果，图2中M为分子量标准，从上到下依次为1.5kb、1kb、900bp、800bp、700bp、600bp、500bp、400bp、300bp、200bp、100bp；CK+为以质粒DNA为模板扩增产物；CK-为以转基因受体自交系KN5585基因组DNA为模板扩增产物；1-20为RIN4转基因玉米基因组DNA。Figure 2 is the PCR detection results of the target gene ZmRIN4 of the T1 generation of transformants. In Figure 2, M is the molecular weight standard, and from top to bottom are 1.5kb, 1kb, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp, 300bp, 200bp, and 100bp; CK+ is the amplification product using plasmid DNA as a template; CK- is the amplification product using the genomic DNA of the transgenic recipient inbred line KN5585 as a template; 1-20 is the genomic DNA of RIN4 transgenic corn.
图3敲除纯合株系测序结果；Figure 3 Knockout homozygous strain sequencing results;
图4RIN4过表达和基因编辑植株株高、穗位高(2021新疆)；Figure 4 Plant height and ear height of RIN4 overexpression and gene-edited plants (2021 Xinjiang);
图5RIN4过表达和基因编辑植株株高、穗位高(2022新疆)；Figure 5 Plant height and ear height of RIN4 overexpression and gene-edited plants (2022 Xinjiang);
图6RIN4过表达和基因编辑植株株高、穗位高(2022海南)。Figure 6 Plant height and ear height of RIN4 overexpression and gene-edited plants (2022 Hainan).
具体实施方式DETAILED DESCRIPTION
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
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。Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
CRISPR/Cas9基因编辑载体CPB由中国农业科学院作物科学研究所谢传晓研究员课题组惠赠，在文献“Qi X,et al.,Genome Editing Enables Next-Generation HybridSeed Production Technology.Molecular Plant,2020,13(9):1262-1269.”中公开，公众可从申请人获得上述生物材料，所得上述生物材料只为重复本发明的实验所用，不可作为其它用途使用；The CRISPR/Cas9 gene editing vector CPB was kindly donated by the research group of Researcher Xie Chuanxiao from the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, and is disclosed in the document "Qi X, et al., Genome Editing Enables Next-Generation HybridSeed Production Technology. Molecular Plant, 2020, 13(9): 1262-1269." The public can obtain the above-mentioned biological material from the applicant. The obtained biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes;
下述实施例中，所述玉米C01来源于国家种质资源库(网址为:http://www.cgris.net/)，公众可从中国农业科学院作物科学研究所获得。In the following examples, the corn C01 is derived from the National Germplasm Resource Bank (website: http://www.cgris.net/), and the public can obtain it from the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences.
下述实施例中，所述玉米KN5585来源于国家种质资源库(网址为:http://www.cgris.net/)，公众可从中国农业科学院作物科学研究所获得。In the following examples, the corn KN5585 is derived from the National Germplasm Resource Bank (website: http://www.cgris.net/), and the public can obtain it from the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences.
下述实施例中，所述P-Easy-blunt载体购自北京全式金生物技术有限公司，货号：CB101-01；In the following examples, the P-Easy-blunt vector was purchased from Beijing Quanshijin Biotechnology Co., Ltd., catalog number: CB101-01;
大肠杆菌Escherichia coli DH5α/EC DH5α购自北京擎科生物科技有限公司，货号：TSC01；Escherichia coli DH5α/EC DH5α was purchased from Beijing Qingke Biotechnology Co., Ltd., catalog number: TSC01;
农杆菌GV3101(pSoup)感受态细胞够自北京博迈德基因技术有限公司，货号：C314-01；Agrobacterium GV3101 (pSoup) competent cells were obtained from Beijing Biomed Gene Technology Co., Ltd., catalog number: C314-01;
FastPfu PCR SuperMix购自北京全式金生物技术有限公司，货号：AS221-11； FastPfu PCR SuperMix was purchased from Beijing Quanshijin Biotechnology Co., Ltd., catalog number: AS221-11;
下述实施例中，所述pCAMBIA3301载体为申请人实验室保存，在文献“Zhang X,etal.,Overexpression of a maize BR transcription factor ZmBZR1 in Arabidopsisenlarges organ and seed size of the transgenic plants.Plant Science,2020,292,110378.”中公开，公众可从申请人获得上述生物材料，所得上述生物材料只为重复本发明的实验所用，不可作为其它用途使用。In the following examples, the pCAMBIA3301 vector is preserved in the applicant's laboratory and is disclosed in the document "Zhang X, et al., Overexpression of a maize BR transcription factor ZmBZR1 in Arabidopsis enlarges organ and seed size of the transgenic plants. Plant Science, 2020, 292, 110378." The public can obtain the above-mentioned biological material from the applicant, and the obtained biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.
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 is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.
实施例1、基因定位Example 1: Gene localization
前期经过多年(2017、2018、2020)田间耐旱性鉴定，鉴定了279份自交系正常灌溉和干旱胁迫下雌穗生物量。一行旱处理(WS)，另一行为正常灌溉(WW)，每行设置3个重复。根据有效积温(GDD)进行干旱处理，旱区至开花前7天时土壤水势达到150cBar，干旱胁迫持续至吐丝后14天，期间土壤水势维持150-180cBar，对照区正常灌溉。通过GWAS关联到与雌穗生物量耐旱系数(WS/WW)相关的SNP(S6_120040853)，位于候选基因Zm00001eb280910，编码RPM1-interacting protein 4(RIN4)。其中RIN4的氨基酸序列是SEQ ID No.1，其编码序列是SEQ ID No.2，基因组序列为SEQ ID No.3。After years of field drought tolerance identification (2017, 2018, and 2020), the female ear biomass of 279 inbred lines under normal irrigation and drought stress were identified. One row was drought treated (WS) and the other row was normally irrigated (WW), with 3 replicates per row. Drought treatment was carried out according to the effective accumulated temperature (GDD). The soil water potential in the drought area reached 150cBar 7 days before flowering, and drought stress lasted until 14 days after silking, during which the soil water potential was maintained at 150-180cBar, and the control area was irrigated normally. Through GWAS, a SNP (S6_120040853) associated with the drought tolerance coefficient of female ear biomass (WS/WW) was found, which was located in the candidate gene Zm00001eb280910, encoding RPM1-interacting protein 4 (RIN4). The amino acid sequence of RIN4 is SEQ ID No.1, its coding sequence is SEQ ID No.2, and the genomic sequence is SEQ ID No.3.
表1：RIN4蛋白及其相关基因的序列Table 1: Sequences of RIN4 protein and its related genes
实施例2、载体构建与转化Example 2: Vector construction and transformation
2.1、过表达载体的构建2.1. Construction of overexpression vector
(1)克隆目的基因(1) Cloning of target gene
以反转录后的cDNA作为克隆Zm00001eb280910基因的模板，以RIN4-FW/RIN4-RV为引物进行PCR扩增。克隆PCR反应使用FastPfu PCR SuperMix进行扩增。The reverse transcribed cDNA was used as a template for cloning the Zm00001eb280910 gene and PCR amplification was performed using RIN4-FW/RIN4-RV as primers. Amplification was performed using FastPfu PCR SuperMix.
其中，引物RIN4-FW/RIN4-RV的核苷酸序列如下(5’-3’)：The nucleotide sequence of primers RIN4-FW/RIN4-RV is as follows (5'-3'):
RIN4-FW：ATGGCGGAGGAATCAGGCC；RIN4-FW: ATGGCGGAGGAATCAGGCC;
RIN4-RV：TCAGGATTGCGTAGGGCTGG。RIN4-RV: TCAGGATTGCGTAGGGCTGG.
PCR反应体系如下：The PCR reaction system is as follows:
PCR反应程序如下：The PCR reaction procedure is as follows:
94℃5min；94℃30s，62℃30s，72℃1min，30-35个循环；72℃10min。94℃5min; 94℃30s, 62℃30s, 72℃1min, 30-35 cycles; 72℃10min.
扩增产物经琼脂糖凝胶电泳检测后，切下目的条带，并按照Maker电泳条带亮度来估算DNA总量，加入适量的洗脱液使最终DNA浓度符合后续实验，使用天根DNA回收试剂盒进行目的片段回收。After the amplified product was detected by agarose gel electrophoresis, the target band was cut out and the total DNA amount was estimated according to the brightness of the Maker electrophoresis band. An appropriate amount of eluent was added to make the final DNA concentration meet the requirements of subsequent experiments, and the target fragment was recovered using the Tiangen DNA recovery kit.
(2)构建中间载体(2) Construction of intermediate vector
中间载体使用带有ubi启动子的9zf载体，采用同源重组的方法进行中间载体的构建。先使用EcoRV酶切将9zf载体线性化，使用引物9zf-RIN4-FW/9zf-RIN4-RV扩增用于同源重组的目的片段(步骤(1)制备)，在引物的5’端分别加上载体插入位点两侧的同源序列。利用同源重组的原理将目的基因连接到9zf载体上，构建中间载体9zf-ZmRIN4-Tnos。随后进行大肠杆菌转化及阳性重组子的鉴定，首先取5μL连接产物至50μL在冰上融化的Trans1-T1大肠感受态细胞中，轻轻混匀，冰浴30min。42℃水浴热激42s，立即置于冰上静置2min。加入500μL不含抗生素的液体LB培养基，37℃，200rpm振荡培养1h。3,000g离心2min，弃掉部分上清，用移液器轻吹菌体至充分悬浮，取全部菌液涂布于含氨苄抗生素的固体LB培养基平板上，37℃过夜培养。挑取大小中等的克隆单菌落至5mL含抗生素的LB液体培养基中37℃，200rpm振荡培养10-16h左右，提取质粒进行酶切鉴定、PCR鉴定及测序鉴定。测序结果表明：中间载体9zf-ZmRIN4-Tnos的结构为：将核苷酸序列是SEQ ID No.2的DNA分子替换9zf载体的两个EcoRV酶切识别位点之间的片段，保持9zf载体的其它核苷酸序列不变。测序正确的克隆用于后续过表达载体的构建。The intermediate vector uses the 9zf vector with the ubi promoter, and the intermediate vector is constructed by homologous recombination. First, the 9zf vector is linearized by EcoRV digestion, and the target fragment for homologous recombination (prepared in step (1)) is amplified using primers 9zf-RIN4-FW/9zf-RIN4-RV, and the homologous sequences on both sides of the vector insertion site are added to the 5' end of the primer. The target gene is connected to the 9zf vector using the principle of homologous recombination to construct the intermediate vector 9zf-ZmRIN4-Tnos. Then, Escherichia coli transformation and positive recombinant identification are carried out. First, 5 μL of the connection product is added to 50 μL of Trans1-T1 colon competent cells melted on ice, gently mixed, and ice bathed for 30 minutes. Heat shock in a 42°C water bath for 42 seconds, and immediately place on ice for 2 minutes. Add 500 μL of liquid LB medium without antibiotics, and culture at 37°C, 200rpm shaking for 1 hour. Centrifuge at 3,000g for 2min, discard part of the supernatant, blow the bacteria gently with a pipette until they are fully suspended, take all the bacterial liquid and spread it on a solid LB medium plate containing ampicillin antibiotics, and culture it at 37℃ overnight. Pick a single colony of medium size and put it into 5mL LB liquid medium containing antibiotics at 37℃, shake and culture it at 200rpm for about 10-16h, extract the plasmid for enzyme identification, PCR identification and sequencing identification. The sequencing results show that the structure of the intermediate vector 9zf-ZmRIN4-Tnos is: replace the fragment between the two EcoRV enzyme recognition sites of the 9zf vector with a DNA molecule with a nucleotide sequence of SEQ ID No.2, and keep the other nucleotide sequences of the 9zf vector unchanged. The clones with correct sequencing are used for the subsequent construction of overexpression vectors.
其中，引物9zf-RIN4-FW/9zf-RIN4-RV的核苷酸序列如下(5’-3’)：The nucleotide sequence of primers 9zf-RIN4-FW/9zf-RIN4-RV is as follows (5'-3'):
9zf-RIN4-FW：GTTACTTCTGCAGGAATTCGATATCATGGCGGAGGAATCAGGC(下划线为同源臂)；9zf-RIN4-FW:GTTACTTCTGCAGGAATTCGATATC ATGGCGGAGGAATCAGGC (homology arms are underlined);
9zf-RIN4-RV：TAACGAGCTCTAGAAGCTTGATATCTCAGGATTGCGTAGGGCTGG(下划线为同源臂)。9zf-RIN4-RV:TAACGAGCTCTAGAAGCTTGATATC TCAGGATTGCGTAGGGCTGG (homology arms are underlined).
(3)构建过表达载体(3) Construction of overexpression vector
过表达载体的构建使用pCAMBIA3301，采用酶连的方法进行载体构建。根据载体上的多克隆位点，选用限制性内切酶BamHI和AflII双酶切pCAMBIA3301载体和中间载体9zf-ZmRIN4-Tnos，回收pCAMBIA3301的大片段和中间载体9zf-ZmRIN4-Tnos的小片段，利用T4DNA连接酶将两片段连接起来，构建过表达载体pCAMBIA3301-ZmRIN4-Tnos。The overexpression vector was constructed by using pCAMBIA3301 and enzyme ligation. According to the multiple cloning site on the vector, the restriction endonucleases BamHI and AflII were selected to double-digest the pCAMBIA3301 vector and the intermediate vector 9zf-ZmRIN4-Tnos, and the large fragment of pCAMBIA3301 and the small fragment of the intermediate vector 9zf-ZmRIN4-Tnos were recovered. The two fragments were connected by T4 DNA ligase to construct the overexpression vector pCAMBIA3301-ZmRIN4-Tnos.
载体线性化酶切体系(20μL体系)：Vector linearization enzyme digestion system (20μL system):
酶切反应的条件：37℃水浴酶切(24h及以上)。Enzyme digestion reaction conditions: 37°C water bath digestion (24h or more).
1％琼脂糖凝胶电泳检测酶切产物并按照天根DNA回收试剂盒说明书回收线性化载体。根据中美泰和生物技术公司的Seamless Assembly Cloning Kit设计重组反应体系，线性化载体使用20-60ng，DNA片段按照与载体摩尔比3:1使用。1% agarose gel electrophoresis was used to detect the digestion product and the linearized vector was recovered according to the instructions of Tiangen DNA recovery kit. The recombination reaction system was designed according to the Seamless Assembly Cloning Kit of Sino-US Taihe Biotechnology Co., Ltd., 20-60ng of linearized vector was used, and the DNA fragment was used at a molar ratio of 3:1 to the vector.
重组反应体系如下(10μL)：The recombinant reaction system is as follows (10 μL):
重组反应的条件：50℃反应15min。Recombination reaction conditions: 50°C for 15 min.
随后进行大肠杆菌转化及阳性重组子的鉴定，取5μL连接产物至50μL在冰上融化的Trans1-T1大肠感受态细胞中，轻轻混匀，冰浴30min；42℃水浴热激42s；立即置于冰上静置2min；加入500μL不含抗生素的液体LB培养基，37℃，200rpm振荡培养1h；3,000g离心2min，弃掉部分上清，用移液器轻吹菌体至充分悬浮，取全部菌液涂布于含卡纳抗生素的固体LB培养基平板上，37℃过夜培养；挑取大小中等的克隆单菌落至5mL含抗生素的LB液体培养基中37℃，200rpm振荡培养10-16h左右，提取质粒进行酶切鉴定、PCR鉴定及测序鉴定。测序正确的克隆用于后续的遗传转化。图1所示为ZmRIN4基因过表达载体pCAMBIA3301-ZmRIN4的图谱，该载体大小为10.749Kb。ZmRIN4基因来源于玉米，由Ubi启动子驱动(Ubipromoter)，由根癌农杆菌NOS终止子(NOS-Ter)终止。bar基因由CaMV 35S启动子驱动，由CaMV 35S终止子终止。Then, E. coli transformation and positive recombinant identification were performed. 5 μL of the ligation product was added to 50 μL of Trans1-T1 competent cells melted on ice, gently mixed, and placed on ice for 30 minutes; heat-shocked in a 42°C water bath for 42 seconds; immediately placed on ice for 2 minutes; added 500 μL of liquid LB medium without antibiotics, and cultured at 37°C, 200 rpm for 1 hour; centrifuged at 3,000 g for 2 minutes, discarded part of the supernatant, and gently blew the bacteria with a pipette until they were fully suspended. All the bacterial liquid was spread on a solid LB medium plate containing kanamycin, and cultured at 37°C overnight; a single colony of medium size was picked and placed in 5 mL of LB liquid medium containing antibiotics, and cultured at 37°C, 200 rpm for about 10-16 hours, and the plasmid was extracted for enzyme digestion identification, PCR identification, and sequencing identification. The clones with correct sequencing were used for subsequent genetic transformation. Figure 1 shows the map of the ZmRIN4 gene overexpression vector pCAMBIA3301-ZmRIN4, which is 10.749 Kb in size. The ZmRIN4 gene is derived from corn, driven by the Ubi promoter (Ubipromoter), and terminated by the Agrobacterium tumefaciens NOS terminator (NOS-Ter). The bar gene is driven by the CaMV 35S promoter and terminated by the CaMV 35S terminator.
2.2、CRISPR-Cas9载体的构建2.2 Construction of CRISPR-Cas9 vector
(1)设计sgRNA(1) Design of sgRNA
利用sgRNA设计网站：http://www.rgenome.net/cas-designer/设计Zm00001eb280910基因的sgRNA，最终筛选获得sgRNA的靶标的核苷酸序列如下：The sgRNA of the Zm00001eb280910 gene was designed using the sgRNA design website: http://www.rgenome.net/cas-designer/. The nucleotide sequence of the target of the sgRNA was finally screened as follows:
sgRNA：GGCCGTCCCTTGCCAAAGTTTGG(下划线标记为PAM序列，其余为靶标序列，靶标序列也即SEQ ID No.2第16-35位/SEQ ID No.3第2759-2778位)。sgRNA: GGCCGTCCCTTGCCAAAGTTTGG (the underlined part is the PAM sequence, and the rest is the target sequence, which is also the 16th to 35th position of SEQ ID No.2/the 2759th to 2778th position of SEQ ID No.3).
(2)扩增U6启动子(2) Amplification of U6 promoter
以含有U6启动子的质粒为模板扩增U6启动子(核苷酸序列是SEQ ID No.4第1-500位)，扩增引物为MU61-3F/MU61-1R。The U6 promoter (nucleotide sequence is SEQ ID No. 4, position 1-500) was amplified using the plasmid containing the U6 promoter as a template, and the amplification primers were MU61-3F/MU61-1R.
PCR反应体系如下：The PCR reaction system is as follows:
其中引物MU61-3F/MU61-1R的核苷酸序列如下(5’-3’)：The nucleotide sequence of primers MU61-3F/MU61-1R is as follows (5'-3'):
MU61-3F：TGCACTGCACAAMU61-3F:TGCACTGCACAA
MU61-1R：AATTCGGTGCTTGCGGCTCMU61-1R:AATTCGGTGCTTGCGGCTC
PCR反应程序如下：The PCR reaction procedure is as follows:
94℃5min；94℃30s，60℃20s，72℃1min，30-35cycles；72℃10min。94℃5min; 94℃30s, 60℃20s, 72℃1min, 30-35cycles; 72℃10min.
(3)融合U6和sgRNA(3) Fusion of U6 and sgRNA
利用上一步的PCR产物和合成的sgRNA为模板，使用引物MUsgR-1F/MUsgR-2R将U6和sgRNA通过overlapPCR融合在一起获得含有sgRNA表达盒的DNA片段，PCR体系和程序参照步骤(2)。Using the PCR product from the previous step and the synthesized sgRNA as templates, primers MUsgR-1F/MUsgR-2R were used to fuse U6 and sgRNA together through overlap PCR to obtain a DNA fragment containing the sgRNA expression cassette. The PCR system and procedure refer to step (2).
其中引物MU61-3F/MUsgR-2R的核苷酸序列如下(5’-3’)：The nucleotide sequence of primer MU61-3F/MUsgR-2R is as follows (5'-3'):
MUsgR-1F：GAGCCGCAAGCACCGAATTGGCCGTCCCTTGCCAAAGTTMUsgR-1F:GAGCCGCAAGCACCGAATTGGCCGTCCCTTGCCAAAGTT
MUsgR-2R：GGCCAGTGCCAAGCTTAAAAAAAGCACCGACTCGMUsgR-2R: GGCCAGTGCCAAGCTTAAAAAAAGCACCGACTCG
sgRNA表达盒的DNA片段的核苷酸序列是SEQ ID No.4，其中SEQ ID No.4第1-500位为U6启动子，第501-603位为sgRNA基因，第501-520位为sgRNA的spacer序列，第521-603位为sgRNA的骨架序列。The nucleotide sequence of the DNA fragment of the sgRNA expression cassette is SEQ ID No. 4, wherein positions 1-500 of SEQ ID No. 4 are U6 promoter, positions 501-603 are sgRNA gene, positions 501-520 are spacer sequence of sgRNA, and positions 521-603 are backbone sequence of sgRNA.
(4)最后利用同源重组的原理将含有sgRNA表达盒的DNA片段融合到CRISPR-cas9载体上，构建CRISPR-ZmRIN4载体。随后进行连接转化及阳性重组子的鉴定，取5μL连接产物至50μL在冰上融化的Trans1-T1大肠感受态细胞中，轻轻混匀，冰浴30min；42℃水浴热激42s；立即置于冰上静置2min；加入500μL不含抗生素的液体LB培养基，37℃，200rpm振荡培养1h；3,000g离心2min，弃掉部分上清，用移液器轻吹菌体至充分悬浮，取全部菌液涂布于含卡纳抗生素的固体LB培养基平板上，37℃过夜培养；挑取大小中等的克隆单菌落至5mL含抗生素的LB液体培养基中37℃，200rpm振荡培养10-16h左右，提取质粒进行酶切鉴定、PCR鉴定及测序鉴定。测序正确的克隆用于后续的遗传转化。(4) Finally, the DNA fragment containing the sgRNA expression cassette was fused to the CRISPR-cas9 vector using the principle of homologous recombination to construct the CRISPR-ZmRIN4 vector. Then, the ligation transformation and positive recombinant identification were performed. 5 μL of the ligation product was added to 50 μL of Trans1-T1 colon competent cells melted on ice, gently mixed, and placed in an ice bath for 30 minutes; heat shock in a 42°C water bath for 42 seconds; immediately placed on ice for 2 minutes; 500 μL of liquid LB medium without antibiotics was added, and cultured at 37°C, 200 rpm for 1 hour; centrifuged at 3,000 g for 2 minutes, discarded part of the supernatant, and the bacteria were gently blown with a pipette until fully suspended, and all the bacterial liquid was spread on a solid LB medium plate containing kanamycin, and cultured at 37°C overnight; a single colony of medium size was picked and placed in 5 mL of LB liquid medium containing antibiotics and cultured at 37°C, 200 rpm for about 10-16 hours, and the plasmid was extracted for enzyme digestion identification, PCR identification, and sequencing identification. The clones with correct sequencing were used for subsequent genetic transformation.
实施例3、过表达植株的构建及检测Example 3: Construction and detection of overexpression plants
3.1、过表达植株的构建3.1 Construction of overexpression plants
玉米遗传转化委托玉米遗传转化由中国种子集团有限公司生命科学技术中心(武汉)负责，过表达转化受体为玉米自交系KN5585。转基因T0代幼苗经除草剂抗性筛选结合PCR鉴定阳性转基因植株，连续自交，辅以基因型鉴定获得纯合转基因T3用于性状测定。T0转基因玉米的制备方法如下：Maize genetic transformation was entrusted to China National Seed Group Corporation Life Science and Technology Center (Wuhan) for maize genetic transformation. The overexpression transformation receptor was maize inbred line KN5585. TransgenicT0 seedlings were screened for herbicide resistance and positive transgenic plants were identified by PCR. They were self-pollinated continuously and supplemented with genotype identification to obtain homozygous transgenicT3 for trait determination. The preparation method ofT0 transgenic maize is as follows:
1.愈伤组织准备1. Callus Preparation
生长速度较快、质地松软、松散易碎、颜色较为鲜艳的胚性愈伤组织。这类愈伤组织可以长期继代，并且可以较长时间保持其胚胎发生能力。Embryogenic callus tissue with fast growth rate, soft texture, loose and fragile, and bright color. This type of callus tissue can be subcultured for a long time and can maintain its embryogenic ability for a long time.
2.农杆菌准备2. Agrobacterium Preparation
1)侵染前两天将农杆菌在LB培养基上划线，28℃暗培养，或者挑单克隆使用液体LB培养基摇菌；1) Two days before infection, streak Agrobacterium on LB medium and culture in the dark at 28°C, or select a single clone and shake the culture in liquid LB medium;
2)收集菌体，转移在50mL离心管中，使用侵染培养基重悬菌体，使得侵染液浓度在OD＝0.8-1.5之间；2) Collect the bacteria, transfer them to a 50 mL centrifuge tube, and resuspend them in infection medium so that the concentration of the infection medium is between OD = 0.8-1.5;
3)将配置好的侵染液在28℃摇床，200rpm摇菌2小时活化农杆菌，用于侵染。3) The prepared infection solution was shaken in a 28°C incubator at 200 rpm for 2 hours to activate the Agrobacterium for infection.
3.侵染3. Infection
1)选取预处理过的愈伤组织，转入侵染培养基；1) Select pretreated callus tissue and transfer it into infection medium;
2)将愈伤组织加入侵染液中浸泡10-30min，打散愈伤组织团块，摇匀使得愈伤组织充分接触农杆菌；2) Soak the callus in the invading dye solution for 10-30 minutes, break up the callus clumps, and shake well to allow the callus to fully contact the Agrobacterium;
3)吸出剩余的侵染液，将侵染后愈伤组织转入共培养培养基，19℃暗培养3天；3) Aspirate the remaining infection solution, transfer the infected callus into co-cultivation medium, and culture in the dark at 19°C for 3 days;
4.恢复培养4. Resume Culture
使用含有抗生素的无菌水冲洗愈伤组织表面3-5次，待加入的水不再浑浊时倒掉液体，将愈伤组织转入铺有滤纸的平皿中，在超净台中吹干愈伤表面水分。转入恢复培养基，28℃暗培养7-10天，转入筛选培养基。Use sterile water containing antibiotics to rinse the surface of the callus 3-5 times, and when the added water is no longer turbid, pour out the liquid, transfer the callus to a plate covered with filter paper, and blow dry the surface water of the callus in a clean bench. Transfer to recovery medium, culture in the dark at 28℃ for 7-10 days, and then transfer to screening medium.
5.筛选5. Screening
1)恢复培养后将转化愈伤组织转入添加抗生素的筛选培养基中，28℃暗培养20-30天；1) After the culture is resumed, the transformed callus is transferred to a screening medium supplemented with antibiotics and cultured in the dark at 28°C for 20-30 days;
2)将愈伤组织转入无菌平皿中，打散后压薄；2) Transfer the callus tissue into a sterile plate, break it up and press it thinly;
3)在紫外操作台上挑出较亮的愈伤颗粒，3-5个颗粒堆成一团，转入新的筛选培养基中；3) Pick out the brighter callus particles on the UV operating table, pile 3-5 particles into a ball, and transfer them to a new screening medium;
4)28℃暗培养20天，转入新的筛选培养基中继代扩繁；4) Cultivate in the dark at 28°C for 20 days, then transfer to a new screening medium for subculture and propagation;
5)继代两轮(40天)后再次挑荧光(不再将愈伤组织打散)。5) After two rounds of subculture (40 days), the fluorescent cells were picked again (without breaking up the callus tissue).
6.分化6. Differentiation
1)将热激后愈伤组织转入预分化培养基，28℃暗培养10天，转入28℃，光照培养10天；1) The heat-shocked callus was transferred to the pre-differentiation medium, cultured in the dark at 28°C for 10 days, and then transferred to the light culture at 28°C for 10 days;
2)转入分化培养基中，再生芽长至3-5cm时转入生根培养基；2) Transfer to differentiation medium, and transfer to rooting medium when the regenerated buds grow to 3-5 cm;
3)待长出大量健壮根后，炼苗移栽3) After a large number of strong roots have grown, harden the seedlings and transplant them
8.炼苗移栽8. Hardening and transplanting
1)生根后揭去生根瓶上的封口膜，在培养基中炼苗2-3天；1) After rooting, remove the sealing film on the rooting bottle and harden the seedlings in the culture medium for 2-3 days;
2)洗净根部培养基移栽在灭菌的营养土中，室内炼苗7天；2) Wash the root culture medium and transplant it into sterilized nutrient soil, and harden the seedlings indoors for 7 days;
3)移栽在温室或是大田。3) Transplant in a greenhouse or field.
3.2、目的基因ZmRIN4的PCR检测3.2. PCR detection of target gene ZmRIN4
3.2.1、方法：3.2.1 Methods:
根据基因序列设计PCR引物Ubi-Fw/Tnos-Rv，预期扩增片段大小为445bp。PCR primers Ubi-Fw/Tnos-Rv were designed according to the gene sequence, and the expected amplified fragment size was 445 bp.
引物Ubi-Fw/Tnos-Rv的核苷酸序列如下(5’-3’)：The nucleotide sequence of primer Ubi-Fw/Tnos-Rv is as follows (5'-3'):
Ubi-Fw：AGCCCTGCCTTCATACGCTA；Ubi-Fw: AGCCCTGCCTTCATACGCTA;
Tnos-Rv：AATCATCGCAAGACCGGCAA；Tnos-Rv:AATCATCGCAAGACCGGCAA;
提取基因组DNA，按照以下PCR参数进行扩增：Extract genomic DNA and amplify according to the following PCR parameters:
反应体系：30ng模板DNA、2.0μL 10×buffer、1.0μL 2mM dNTPs、1.5μL 25mMMgCl2、0.4μl 10μM引物、1.5U Taq酶。Reaction system: 30 ng template DNA, 2.0 μL 10× buffer, 1.0 μL 2 mM dNTPs, 1.5 μL 25 mM MgCl2 , 0.4 μl 10 μM primer, and 1.5 U Taq enzyme.
反应程序如下：The reaction procedure is as follows:
94℃变性5min；94℃变性30sec、60℃退火30sec、72℃延伸45sec，共35个循环；72℃延伸5min。Denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 45 sec, for a total of 35 cycles; extension at 72°C for 5 min.
3.2.2、结果：3.2.2 Results:
对转化体植株进行PCR检测，结果如图2所示：转化体T1代的目的基因扩增片段大小与阳性质粒对照PCR的扩增片段大小一致，约为445bp。图2的结果表明：ZmRIN4基因过表达表达盒已整合至玉米基因组中。The transformant plants were tested by PCR, and the results are shown in Figure 2: the size of the target gene amplified fragment of the transformantT1 generation was consistent with the size of the amplified fragment of the positive plasmid control PCR, which was about 445 bp. The results in Figure 2 show that the ZmRIN4 gene overexpression cassette has been integrated into the corn genome.
实施例4、基因敲除纯合株系的制备及基因型检测Example 4: Preparation of homozygous knockout strains and genotype detection
构建好的CRISPR-ZmRIN4载体，交由中国种子集团有限公司生命科学技术中心(武汉)负责开展遗传转化，转化受体为玉米自交系C01。CRISPR/Cas9基因编辑株系自交筛选纯合株系，测序确定编辑位点及类型。基因敲除纯合株系的制备方法参照实施例3，其区别仅在于将过表达质粒替换为敲除质粒，将受体玉米替换为玉米自交系C01。The constructed CRISPR-ZmRIN4 vector was handed over to the Life Science and Technology Center of China National Seed Group Co., Ltd. (Wuhan) for genetic transformation, and the transformation recipient was the maize inbred line C01. The CRISPR/Cas9 gene-edited strain was self-pollinated to screen homozygous strains, and the editing sites and types were determined by sequencing. The preparation method of the gene knockout homozygous strain was referred to Example 3, the only difference being that the overexpression plasmid was replaced by the knockout plasmid, and the recipient maize was replaced by the maize inbred line C01.
4.1、方法4.1 Methods
根据基因序列设计PCR引物RIN4-Crispr-F/RIN4-Crispr-R，预期扩增片段大小为663bp。PCR primers RIN4-Crispr-F/RIN4-Crispr-R were designed according to the gene sequence, and the expected amplified fragment size was 663 bp.
引物RIN4-Crispr-F/RIN4-Crispr-R的核苷酸序列如下(5’-3’)：The nucleotide sequences of primers RIN4-Crispr-F/RIN4-Crispr-R are as follows (5'-3'):
RIN4-Crispr-F：ATCTGCTTGCAGAAGCCTGTRIN4-Crispr-F: ATCTGCTTGCAGAAGCCTGT
RIN4-Crispr-R：GCTGGCTGTCACACAACAAARIN4-Crispr-R:GCTGGCTGTCACACAACAAA
提取基因组DNA，按照以下PCR参数进行扩增：Extract genomic DNA and amplify according to the following PCR parameters:
反应体系：30ng模板DNA、2.0μL 10×buffer、1.0μL 2mM dNTPs、1.5μL 25mMMgCl2、0.4μl 10μM引物、1.5U Taq酶。Reaction system: 30 ng template DNA, 2.0 μL 10× buffer, 1.0 μL 2 mM dNTPs, 1.5 μL 25 mM MgCl2 , 0.4 μl 10 μM primer, and 1.5 U Taq enzyme.
反应程序如下：The reaction procedure is as follows:
94℃变性5min；94℃变性30sec、60℃退火30sec、72℃延伸45sec，共35个循环；72℃延伸5min。Denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 45 sec, for a total of 35 cycles; extension at 72°C for 5 min.
4.2、结果4.2 Results
经Sanger测序，确定基因敲除纯合株系，获得两种纯合突变体KO-2和KO-21。敲除纯合株系测序结果如图3。纯合突变体KO-2和KO-21的突变如下：The gene knockout homozygous strains were determined by Sanger sequencing, and two homozygous mutants KO-2 and KO-21 were obtained. The sequencing results of the knockout homozygous strains are shown in Figure 3. The mutations of the homozygous mutants KO-2 and KO-21 are as follows:
纯合突变植株KO-2：相比于野生型玉米C01，突变株KO-2的两条同源染色体中的目的基因发生如下变化：SEQ ID No.3的第2773-2774位的核苷酸AA缺失，导致目的基因发生移码突变，进而导致目的蛋白无法正常表达，从而实现了目基因的敲除。Homozygous mutant plant KO-2: Compared with the wild-type corn C01, the target genes in the two homologous chromosomes of the mutant KO-2 underwent the following changes: the nucleotide AA at positions 2773-2774 of SEQ ID No.3 was deleted, resulting in a frameshift mutation in the target gene, which in turn caused the target protein to be unable to be expressed normally, thereby achieving the knockout of the target gene.
纯合突变植株KO-21：相比于野生型玉米C01，突变株KO-21的两条同源染色体中的目的基因发生如下变化：SEQ ID No.3的第2776-2796位的核苷酸AAGTTTGGTGAATGGGATGTC缺失，导致目的基因发生突变，从而实现了目基因的敲除。Homozygous mutant plant KO-21: Compared with the wild-type corn C01, the target genes in the two homologous chromosomes of the mutant KO-21 underwent the following changes: the nucleotides AAGTTTGGTGAATGGGATGTC at positions 2776-2796 of SEQ ID No.3 were deleted, resulting in a mutation in the target gene, thereby achieving the knockout of the target gene.
实施例5、ZmRIN4调控玉米株高、穗位高Example 5: ZmRIN4 regulates maize plant height and ear height
实施例3制备的过表达T3纯合株系、实施例4制备的敲除纯合株系(KO-2和KO-21)、以及野生型对照KN5585和C01于开花期在新疆农业科学院安宁渠试验站进行干旱胁迫。根据有效积温(GDD)进行干旱处理，旱区至开花前7天时土壤水势达到150cBar，干旱胁迫持续至吐丝后14天，期间土壤水势维持150-180cBar，对照区正常灌溉。开花期干旱胁迫(WS)及正常灌溉(WW)条件下，分别调查株高、吐丝期、散粉期等农艺性状；收获时去掉头尾两株，统计收获株数和有效穗数，测定小区产量，以非转基因株系为对照，比较耐旱性差异。于新疆(2021，2022夏)开展田间耐旱性鉴定，2022年冬于海南进一步调查了正常条件下株高、穗位高。The overexpressionT3 homozygous strain prepared in Example 3, the knockout homozygous strain (KO-2 and KO-21) prepared in Example 4, and the wild-type controls KN5585 and C01 were subjected to drought stress at the Anningqu Experimental Station of Xinjiang Academy of Agricultural Sciences during the flowering period. Drought treatment was carried out according to the effective accumulated temperature (GDD). The soil water potential in the drought area reached 150 cBar 7 days before flowering, and the drought stress lasted until 14 days after silking, during which the soil water potential was maintained at 150-180 cBar, and the control area was irrigated normally. Under drought stress (WS) during the flowering period and normal irrigation (WW) conditions, agronomic traits such as plant height, silking period, and powder shedding period were investigated respectively; the head and tail plants were removed at harvest, the number of harvested plants and the number of effective ears were counted, the plot yield was determined, and the non-transgenic strains were used as controls to compare the differences in drought resistance. Field drought tolerance identification was carried out in Xinjiang (summer 2021, 2022), and plant height and ear height under normal conditions were further investigated in Hainan in the winter of 2022.
测定方法：Determination method:
株高：玉米开花期时雄穗顶部到地面的距离。为排除边际行效应，测量时选取每行中间8-10个单株，并记录，统计分析。Plant height: The distance from the top of the tassel to the ground during the flowering period of corn. To eliminate the marginal row effect, 8-10 plants in the middle of each row were selected for measurement, recorded, and statistically analyzed.
穗位高：上穗穗柄着生处到地面的高度。为排除边际行效应，测量时选取每行中间8-10个单株，并记录，统计分析。Ear height: the height from the place where the upper ear stalk is attached to the ground. In order to eliminate the marginal row effect, 8-10 plants in the middle of each row were selected for measurement, recorded, and statistically analyzed.
常规表型数据的统计方法，利用Excel(Excel 2013，Microsoft)中Student's t-test方法进行统计分析(P<0.05)。*表示具有显著性差异(P＜0.05)，**表示具有极显著性差异(P＜0.01)，***表示具有极显著性差异(P＜0.001)。The statistical method of conventional phenotypic data was performed using the Student's t-test method in Excel (Excel 2013, Microsoft) for statistical analysis (P<0.05). * indicates a significant difference (P<0.05), ** indicates an extremely significant difference (P<0.01), and *** indicates an extremely significant difference (P<0.001).
5.1、2021年新疆实验设计及结果5.1. 2021 Xinjiang Experiment Design and Results
过表达T3纯合株系(OE-1、OE-2、OE-3)、敲除纯合株系(KO-2和KO-21)、野生型对照KN5585和C01植株设置正常灌溉和开花期干旱胁迫两种水分条件，分别种植三个重复，每个重复种植2行区，每行12株。OverexpressionT3 homozygous lines (OE-1, OE-2, OE-3), knockout homozygous lines (KO-2 and KO-21), wild-type control KN5585 and C01 plants were set up with two water conditions: normal irrigation and drought stress during flowering period. Three replicates were planted, each replicate was planted with two rows, and each row had 12 plants.
试验结果如图4，结果表明：The test results are shown in Figure 4, which show that:
正常灌溉条件下，ZmRIN4基因过表达玉米(OE-1、OE-2、OE-3)株高为189.60cm-196.10cm，较对照KN5585(201.20cm)降低2％-6％；穗位高为95.67-108.10cm，较对照KN5585(113.40)降低5％-16％。基因编辑植株(KO-2和KO-21)株高为204.56cm-209.10cm，较对照C01株高(196.47cm)可提高4％-6％；穗位高为102.64cm-102.93cmcm，较对照C01(101.13)提高1.5％-1.8％无显著差异。Under normal irrigation conditions, the plant height of ZmRIN4 gene overexpressing corn (OE-1, OE-2, OE-3) was 189.60cm-196.10cm, which was 2%-6% lower than the control KN5585 (201.20cm); the ear height was 95.67-108.10cm, which was 5%-16% lower than the control KN5585 (113.40). The plant height of gene-edited plants (KO-2 and KO-21) was 204.56cm-209.10cm, which was 4%-6% higher than the control C01 (196.47cm); the ear height was 102.64cm-102.93cmcm, which was 1.5%-1.8% higher than the control C01 (101.13), with no significant difference.
开花期干旱胁迫下，ZmRIN4过表达玉米株高155.53cm-165.20cm，较对照KN5585(174.00cm)降低6％-11％；穗位高为76.73cm-85.60cm，较对照KN5585(87.70cm)降低2％-13％。基因编辑植株玉米株高182.72cm-197.08cm，较对照C01株高(172.07cm)提高6％-15％；穗位高为91.13cm-92.84cm，较对照C01(86.13cm)提高6％-8％。Under drought stress during the flowering period, the plant height of ZmRIN4 overexpressing corn was 155.53cm-165.20cm, which was 6%-11% lower than the control KN5585 (174.00cm); the ear height was 76.73cm-85.60cm, which was 2%-13% lower than the control KN5585 (87.70cm). The plant height of gene-edited corn plants was 182.72cm-197.08cm, which was 6%-15% higher than the control C01 plant height (172.07cm); the ear height was 91.13cm-92.84cm, which was 6%-8% higher than the control C01 (86.13cm).
5.2、2022年新疆实验设计及结果5.2. 2022 Xinjiang Experiment Design and Results
在2021年田间鉴定基础上，选择过表达植株OE-3和OE-6，基因编辑植株选择KO-21进一步鉴定。分别设置正常灌溉和开花期干旱胁迫两种水分条件，分别种植三个重复，每个重复种植4行区，每行15株。Based on the field identification in 2021, the overexpression plants OE-3 and OE-6 and the gene-edited plant KO-21 were selected for further identification. Two water conditions, normal irrigation and drought stress during flowering, were set, and three replicates were planted, with 4 rows in each replicate and 15 plants in each row.
试验结果如图5，结果表明：The test results are shown in Figure 5. The results show that:
正常条件下，RIN4过表达(OE-3和OE-6)株高152.1-152.7，较对照KN5585(178.0cm)降低14.2-14.6％；RIN4过表达穗位高69.0-72.6cm较对照KN5585(88.5cm)降低17.9-22.0％。基因编辑植株KO-21株高205.2cm，较对照C01(179.6cm)提高14.2％；KO-21穗位高100.4cm较对照C01(87.7cm)提高14.5％。Under normal conditions, the plant height of RIN4 overexpression (OE-3 and OE-6) was 152.1-152.7, which was 14.2-14.6% lower than the control KN5585 (178.0 cm); the ear height of RIN4 overexpression was 69.0-72.6 cm, which was 17.9-22.0% lower than the control KN5585 (88.5 cm). The plant height of gene-edited plant KO-21 was 205.2 cm, which was 14.2% higher than the control C01 (179.6 cm); the ear height of KO-21 was 100.4 cm, which was 14.5% higher than the control C01 (87.7 cm).
开花期干旱胁迫下，RIN4过表达株高93.8-100.2cm较对照KN5585(88.5cm)降低11.3-17.0％；RIN4过表达穗位高43.8-45.7cm较对照KN5585(88.5cm)降低18.3-21.8％。KO-21株高130.2cm较对照C01(120.5cm)提高8.1％；KO-21穗位高66.1cm较对照C01(57.9cm)提高14.1％。Under drought stress during flowering, the plant height of RIN4 overexpression was 93.8-100.2 cm, which was 11.3-17.0% lower than the control KN5585 (88.5 cm); the ear height of RIN4 overexpression was 43.8-45.7 cm, which was 18.3-21.8% lower than the control KN5585 (88.5 cm). The plant height of KO-21 was 130.2 cm, which was 8.1% higher than the control C01 (120.5 cm); the ear height of KO-21 was 66.1 cm, which was 14.1% higher than the control C01 (57.9 cm).
5.3、2022年海南实验设计及结果5.3. Hainan Experiment Design and Results in 2022
选择过表达植株OE-3和OE-6、基因编辑植株KO-2和KO-21、野生型对照KN5585和C01，每种转基因纯合植株设置正常灌溉一种条件，种植一个重复，每个重复种植4行区，每行12株，此次试验只调查了株高性状。Overexpression plants OE-3 and OE-6, gene-edited plants KO-2 and KO-21, and wild-type controls KN5585 and C01 were selected. Normal irrigation conditions were set for each transgenic homozygous plant, and one replicate was planted. Each replicate was planted in 4 rows, with 12 plants in each row. Only plant height traits were investigated in this experiment.
试验结果如图6，结果表明：The test results are shown in Figure 6, which show that:
正常条件下，RIN4过表达株高120.1cm-133.1cm较对照KN5585(160.0cm)降低17.7％-22.1％，KO-2和KO-21株高175.2cm和174.0cm较对照C01(160.1cm)提高8.8％-9.5％。进一步调查发现，ZmRIN4调控玉米株高主要是由于茎节数的变化，其中过表达植株，茎节数减少1-2节，基因敲除植株茎节数可增加1-2节。Under normal conditions, the height of RIN4 overexpression plants was 120.1cm-133.1cm, which was 17.7%-22.1% lower than the control KN5585 (160.0cm), and the height of KO-2 and KO-21 plants was 175.2cm and 174.0cm, which was 8.8%-9.5% higher than the control C01 (160.1cm). Further investigation found that ZmRIN4 regulated maize plant height mainly due to changes in the number of stem nodes, among which the number of stem nodes of overexpression plants decreased by 1-2 nodes, and the number of stem nodes of gene knockout plants increased by 1-2 nodes.
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。按以下附带的权利要求的范围，可以进行一些基本特征的应用。The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.
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