CN115011631A - Proteins regulating drought resistance in maize seedlings and their encoding genes and applications - Google Patents

Abstract

The invention discloses a protein for regulating and controlling drought resistance of a corn seedling stage, and a coding gene and application thereof, belonging to the technical field of genetic engineering breeding. The invention provides an application of protein or a substance for regulating the expression of a protein coding gene or a substance for regulating the activity or the content of the protein in regulating and controlling the drought resistance of plants, wherein the protein is a protein with an amino acid sequence of SEQ ID No. 3. The invention also provides a method for regulating and controlling the drought resistance of plants, which comprises regulating and controlling the expression of the protein coding gene or regulating and controlling the activity or the content of the protein to regulate and control the drought resistance of plants. The method provided by the invention can obtain the target corn with improved drought resistance, and provides a solution for cultivating the drought-resistant corn.

Description

Translated fromChinese

调控玉米苗期抗旱性的蛋白及其编码基因和应用Proteins regulating drought resistance in maize seedlings and their encoding genes and applications

技术领域technical field

本发明属于基因工程育种技术领域，具体涉及调控玉米苗期抗旱性的蛋白及其编码基因和应用。The invention belongs to the technical field of genetic engineering breeding, and in particular relates to a protein for regulating and controlling the drought resistance of maize seedling stage and its encoding gene and application.

背景技术Background technique

玉米是我国重要的工业原料、饲料和粮食作物，在我国广泛种植，而干旱是制约玉米生产的主要非生物胁迫因素之一。在我国大部分玉米主产区，由于降雨量不足或分布不均，或没有灌溉条件，导致玉米产量提高受到干旱的极大制约。培育耐旱玉米品种是减少干旱损失的有效途径。玉米的抗旱性是一个复杂的数量性状，目前通过QTL、GWAS等已经挖掘到了大量抗旱性遗传位点，玉米抗旱基因克隆与分子机制解析取得重要进展，但植物抗旱性是一个涉及多基因多信号途径的复杂过程，在玉米抗旱分子育种中得到实际利用的基因很少，有待进一步挖掘玉米抗旱相关的基因。Maize is an important industrial raw material, fodder and food crop in my country, and it is widely grown in China. Drought is one of the main abiotic stress factors restricting maize production. In most major maize producing areas in my country, the increase in maize yield is greatly restricted by drought due to insufficient or uneven distribution of rainfall, or lack of irrigation conditions. Breeding drought-tolerant maize varieties is an effective way to reduce drought losses. The drought resistance of maize is a complex quantitative trait. At present, a large number of drought resistance genetic loci have been excavated through QTL, GWAS, etc., and important progress has been made in the cloning and molecular mechanism analysis of maize drought resistance genes. However, plant drought resistance is a multi-gene and multi-signal. Due to the complex process of the pathway, few genes have actually been used in the molecular breeding of maize drought resistance, and further exploration of the genes related to maize drought resistance is needed.

发明内容SUMMARY OF THE INVENTION

本发明要解决的技术问题是如何调控植物的抗逆性，例如如何提高玉米的耐旱性。The technical problem to be solved by the present invention is how to regulate the stress resistance of plants, for example, how to improve the drought resistance of maize.

为解决上述技术问题，第一个方面，本发明提供蛋白质或调控所述蛋白质编码基因表达的物质或调控所述蛋白质活性或含量的物质的应用，其特征在于：所述应用可为下述任一种：In order to solve the above technical problems, in the first aspect, the present invention provides the application of a protein or a substance that regulates the expression of the protein-encoding gene or a substance that regulates the activity or content of the protein, characterized in that the application can be any of the following: A sort of:

A1)、蛋白质或调控所述蛋白质编码基因表达的物质或调控所述蛋白质活性或含量的物质在调控植物抗逆性中的应用；A1), the application of a protein or a substance regulating the expression of the protein-encoding gene or a substance regulating the activity or content of the protein in regulating plant stress resistance;

A2)、蛋白质或调控所述蛋白质编码基因表达的物质或调控所述蛋白质活性或含量的物质在制备调控植物抗逆性的产品中的应用；A2), the application of a protein or a substance that regulates the expression of the protein-encoding gene or a substance that regulates the activity or content of the protein in the preparation of a product for regulating plant stress resistance;

A3)、蛋白质或调控所述蛋白质编码基因表达的物质或调控所述蛋白质活性或含量的物质在培育耐旱植物中的应用；A3), the application of protein or a substance that regulates the expression of the protein-encoding gene or a substance that regulates the activity or content of the protein in cultivating drought-tolerant plants;

A4)、蛋白质或调控所述蛋白质编码基因表达的物质或调控所述蛋白质活性或含量的物质在制备培育耐旱植物的产品中的应用；A4), the application of a protein or a substance that regulates the expression of the protein-encoding gene or a substance that regulates the activity or content of the protein in the preparation of a product for cultivating drought-tolerant plants;

A5)、蛋白质或调控所述蛋白质编码基因表达的物质或调控所述蛋白质活性或含量的物质在植物育种中的应用；A5), the application of protein or the substance that regulates the expression of the protein-encoding gene or the substance that regulates the activity or content of the protein in plant breeding;

所述蛋白质可为如下D1)、D2)或D3)：The protein may be D1), D2) or D3) as follows:

D1)、氨基酸序列是SEQ ID No.3的蛋白质；D1), the amino acid sequence is the protein of SEQ ID No.3;

D2)、SEQ ID No.3所示的氨基酸序列经过一个以上氨基酸残基的取代和/或缺失和/或添加得到的与D1)所示的蛋白质具有80％以上的同一性且与植物抗逆性相关的蛋白质；The amino acid sequence shown in D2) and SEQ ID No.3 has more than 80% identity with the protein shown in D1) and is resistant to plant stress, obtained by substitution and/or deletion and/or addition of one or more amino acid residues sex-related proteins;

D3)、在D1)或D2)的N端和/或C端连接标签得到的融合蛋白质。D3), a fusion protein obtained by linking a tag at the N-terminus and/or C-terminus of D1) or D2).

进一步地，上述的应用中，所述蛋白质来源于玉米。Further, in the above application, the protein is derived from corn.

其中，SEQ ID No.3由304个氨基酸残基组成。Among them, SEQ ID No.3 consists of 304 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 obtained by first synthesizing their coding genes and then carrying out biological expression.

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 a target protein by using DNA recombination technology in vitro, so as to facilitate the expression, detection, tracking and/or purification of the target protein. The protein tag can be a Flag protein tag, His protein tag, MBP protein tag, HA protein tag, myc protein tag, GST protein tag and/or SUMO protein tag and the like.

进一步地，A5)中所述植物育种的目的可为培育耐旱性植物。Further, the purpose of plant breeding described in A5) may be to cultivate drought-tolerant plants.

进一步地，上述的应用中，所述调控所述蛋白质编码基因表达的物质或调控所述蛋白质活性或含量的物质可为提高或上调所述蛋白质编码基因表达的物质或提高或上调所述蛋白质活性或含量的物质，所述调控植物抗逆性可为提高所述植物的抗逆性。Further, in the above application, the substance that regulates the expression of the protein-coding gene or the substance that regulates the activity or content of the protein can be a substance that improves or up-regulates the expression of the protein-coding gene or increases or up-regulates the protein activity. Or the content of the substance, the regulation of plant stress resistance can be to improve the stress resistance of the plant.

进一步地，上述的应用中，所述调控所述蛋白质编码基因表达的物质或调控所述蛋白质活性或含量的物质可为生物材料，所述生物材料可为下述B1)至B7)中的任一种：Further, in the above application, the substance that regulates the expression of the protein-coding gene or the substance that regulates the activity or content of the protein can be a biological material, and the biological material can be any of the following B1) to B7). A sort of:

B1)、编码上述蛋白质的核酸分子；B1), a nucleic acid molecule encoding the above-mentioned protein;

B2)、含有B1)所述核酸分子的表达盒；B2), the 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；B5), a transgenic plant cell line containing the nucleic acid molecule of B1), or a transgenic plant cell line containing the expression cassette of B2);

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；B6), a transgenic plant tissue containing the nucleic acid molecule of B1), or a transgenic plant tissue containing the expression cassette of B2);

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。B7), a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2).

进一步地，上述的应用中，B1)所述核酸分子可为如下b1)至b3)任一项所示的DNA分子：Further, in the above-mentioned application, the nucleic acid molecule of B1) can be the DNA molecule shown in any one of the following b1) to b3):

b1)、编码链的编码序列是SEQ ID No.2所示的DNA分子；b1), the coding sequence of the coding strand is the DNA molecule shown in SEQ ID No.2;

b2)、编码链的核苷酸序列是SEQ ID No.1所示的DNA分子；b2), the nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No.1;

b3)、与b1)或b2)限定的核苷酸序列具有80％或80％以上同一性，且编码上述蛋白质的DNA分子。b3), a DNA molecule that has 80% or more identity with the nucleotide sequence defined in b1) or b2) and encodes the above-mentioned protein.

进一步地，上述的应用中，所述植物为下述任一种：Further, in the above-mentioned application, the plant is any of the following:

P1)、单子叶植物；P1), monocotyledonous plants;

P2)、禾本目植物；P2), Gramineae;

P3)、禾本科植物；P3), Poaceae;

P4)、玉蜀黍属植物；P4), Zea mays;

P5)、玉米；P5), corn;

P6)、双子叶植物；P6), dicotyledonous plants;

P7)、罂粟目植物；P7), Poppy plants;

P8)、十字花科植物；P8), cruciferous plants;

P9)、鼠耳芥属植物P9), Arabidopsis

P10)、拟南芥。P10), Arabidopsis.

本发明中，B4)所述重组微生物具体可为酵母、细菌、藻类或真菌。所述细菌可为革兰氏阳性细菌或革兰氏阴性细菌。所述革兰氏阴性细菌可为根癌农杆菌(Agrobacteriumtumefaciens)。所述根癌农杆菌(Agrobacterium tumefaciens)具体可为根癌农杆菌EHA105。In the present invention, the recombinant microorganism in B4) can specifically be yeast, bacteria, algae or fungi. The bacteria can be Gram-positive bacteria or Gram-negative bacteria. The gram-negative bacteria may be Agrobacterium tumefaciens. The Agrobacterium tumefaciens can specifically be Agrobacterium tumefaciens EHA105.

本发明中，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。In the present invention, the plant tissue of B6) can be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

本发明中，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。In the present invention, the transgenic plant organ of B7) can be the root, stem, leaf, flower, fruit and seed of the transgenic plant.

本发明中，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。In the present invention, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs may or may not include propagating materials.

为解决上述技术问题，第二个方面，本发明提供上述应用中的所述蛋白质或上述应用中的所述生物材料。In order to solve the above technical problem, in a second aspect, the present invention provides the protein in the above application or the biological material in the above application.

为解决上述技术问题，第三个方面，本发明提供一种调控植物抗逆性的方法，所述方法包括通过调控上述蛋白编码基因的表达或调控所述蛋白的活性或含量，来调控植物植物抗逆性。In order to solve the above-mentioned technical problems, in the third aspect, the present invention provides a method for regulating plant stress resistance, the method comprising regulating the expression of the above-mentioned protein-encoding gene or regulating the activity or content of the protein to regulate the plant plant. Stress resistance.

进一步地，所述调控为正向调控或上调。Further, the regulation is positive regulation or up regulation.

进一步地，上述的方法包括向受体植物中导入上述应用中B1)所述的编码基因，促进或提高上述蛋白编码基因的表达或上述蛋白活性或含量，得到植物抗逆性高于所述受体植物的目的植物。Further, the above-mentioned method comprises introducing the coding gene described in B1) in the above-mentioned application into the recipient plant, promoting or improving the expression of the above-mentioned protein-coding gene or the above-mentioned protein activity or content, so that the plant stress resistance is higher than that of the recipient plant. The target plant of the body plant.

本发明中，所述抗逆性可为抗旱性。In the present invention, the stress resistance may be drought resistance.

所述抗旱性提高具体可表现为：干旱胁迫条件下存活率高于野生型；复水后能够更快的恢复正常生长水平。The improvement of the drought resistance can be embodied as follows: the survival rate is higher than that of the wild type under drought stress conditions; and the normal growth level can be recovered more quickly after rehydration.

为解决上述技术问题，第四个方面，本发明提供一种培育抗旱性植物的方法，包括上调或促进或提高目的植物中上述蛋白质的编码基因的表达量，得到抗旱性植物，所述抗旱性植物的耐旱性高于所述目的植物。In order to solve the above-mentioned technical problem, in the fourth aspect, the present invention provides a method for cultivating drought-resistant plants, comprising up-regulating or promoting or improving the expression level of the above-mentioned protein-encoding genes in the target plant to obtain drought-resistant plants, the drought-resistant plants. The drought tolerance of the plant is higher than that of the target plant.

进一步地，上述的方法中，所述植物为下述任一种：Further, in the above-mentioned method, the plant is any of the following:

P1)、单子叶植物；P1), monocotyledonous plants;

P2)、禾本目植物；P2), Gramineae;

P3)、禾本科植物；P3), Poaceae;

P4)、玉蜀黍属植物；P4), Zea mays;

P5)、玉米；P5), corn;

P6)、双子叶植物；P6), dicotyledonous plants;

P7)、罂粟目植物；P7), Poppy plants;

P8)、十字花科植物；P8), cruciferous plants;

P9)、鼠耳芥属植物P9), Arabidopsis

P10)、拟南芥。P10), Arabidopsis.

本发明中，所述玉米可包括玉米自交系。In the present invention, the maize may include maize inbred lines.

本发明中，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 present invention, the identity refers to the identity of amino acid sequences or nucleotide sequences. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page of the NCBI homepage website. For example, in advanced BLAST2.1, by using 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 Lambdaratio to 11 respectively , 1 and 0.85 (default value) and search for the identity of a pair of amino acid sequences to calculate the identity value (%).

本发明中，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 present invention, the identity of more than 80% 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.

本发明中，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 present invention, the substance that regulates gene expression may be a substance that performs at least one of the following six types of regulation: 1) regulation at the level of gene transcription; 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 mRNA transport of the gene from the nucleus to the cytoplasm) 4) regulation of the translation of the gene; 5) regulation of the mRNA degradation of the gene; 6) post-translation regulation of the gene (that is, the activity of the protein translated by the gene control).

本发明中，所述调控基因表达可为促进或提高所述基因表达，所述促进或提高所述基因表达可通过基因过表达实现。In the present invention, the regulation of gene expression may be to promote or increase the gene expression, and the promotion or increase of the gene expression may be achieved by gene overexpression.

本发明取得的有益技术效果：The beneficial technical effect obtained by the present invention:

(1)对本发明Zm00001d005920抗旱蛋白编码的基因进行基因过表达后，玉米抗旱性显著提高。(1) After the gene encoding the Zm00001d005920 drought resistance protein of the present invention is overexpressed, the drought resistance of maize is significantly improved.

(2)本发明为创制玉米过表达植株和/或玉米育种提供了更精准、高效、安全的技术方法，实现了对玉米抗旱性的精准改良，可以促进商业化玉米育种进程，克服传统育种的短板，缩短育种周期的同时不影响其他性状。(2) The present invention provides a more precise, efficient and safe technical method for creating maize overexpression plants and/or maize breeding, realizes precise improvement of the drought resistance of maize, can promote the process of commercial maize breeding, and overcomes the problems of traditional breeding. Short board, shorten the breeding cycle without affecting other traits.

附图说明Description of drawings

图1为玉米自交系地上部生物量计算获得的抗旱指数(DTI)全基因组关联分析的结果。Figure 1 shows the results of the genome-wide association analysis of the drought resistance index (DTI) obtained by calculating the shoot biomass of maize inbred lines.

图2为qDR2位点的区段的候选基因分析结果。Figure 2 shows the results of candidate gene analysis of segments of the qDR2 locus.

图3为重组载体pCAMBIA3301-Zm00001d005920的构建示意图及重组载体的载体图谱。Figure 3 is a schematic diagram of the construction of the recombinant vector pCAMBIA3301-Zm00001d005920 and the vector map of the recombinant vector.

图4为过表达Zm00001d005920的拟南芥抗旱性测定结果。Figure 4 shows the results of drought resistance assay in Arabidopsis thaliana overexpressing Zm00001d005920.

图5为过表达Zm00001d005920基因的拟南芥根系生长测定结果。Figure 5 shows the results of root growth assay of Arabidopsis thaliana overexpressing Zm00001d005920 gene.

图6为过表达Zm00001d005920基因的玉米抗旱性测定结果。Figure 6 shows the results of the drought resistance assay of maize overexpressing the Zm00001d005920 gene.

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 further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting 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 are not intended to 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 conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product specification. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

玉米自交系B104来源于U.S.National Plant Germplasm System(https://npgsweb.ars-grin.gov/gringlobal/search)。The maize inbred lineB104 was derived from the US National Plant Germplasm System (https://npgsweb.ars-grin.gov/gringlobal/search ).

拟南芥Col-0，哥伦比亚生态型拟南芥为Arabidopsis Biological ResourceCenter的产品，详见网址http://abrc.osu.edu/。Arabidopsis Col-0, the Columbia ecotype Arabidopsis thaliana is a product of the Arabidopsis Biological Resource Center, see http://abrc.osu.edu/ for details.

载体pCAMBIA3301购自北京庄盟国际生物基因科技有限公司，货号为ZK868。The vector pCAMBIA3301 was purchased from Beijing Zhuangmeng International Bio-Gene Technology Co., Ltd., the product number is ZK868.

CPB载体由中国农业科学院作物科学研究所谢传晓课题组提供，在文献“Zhao,Y.,Zhang,C.,Liu,W.et al.An alternative strategy for targeted gene replacement inplants using a dual-sgRNA/Cas9 design.Sci Rep 6,23890(2016).”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 CPB vector was provided by the research group of Xie Chuanxiao, Institute of Crop Science, Chinese Academy of Agricultural Sciences, in the document "Zhao, Y., Zhang, C., Liu, W. et al. An alternative strategy for targeted gene replacement inplants using a dual-sgRNA/Cas9Design.Sci Rep 6, 23890 (2016).", the public can obtain the above-mentioned biological materials from the applicant, and the obtained above-mentioned biological materials are only used for repeating the experiments of the present invention and cannot be used for other purposes.

pLB载体购自天根生化科技(北京)有限公司，货号为VT205。The pLB vector was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., the product number is VT205.

Fast-T1大肠杆菌感受态细胞购自南京诺唯赞生物科技股份有限公司，货号为C505-03。Fast-T1 Escherichia coli competent cells were purchased from Nanjing Novizan Biotechnology Co., Ltd., the product number is C505-03.

EHA105农杆菌感受态细胞购自北京博迈德基因技术有限公司，货号为BC303。EHA105 Agrobacterium competent cells were purchased from Beijing Bomed Gene Technology Co., Ltd., the product number is BC303.

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，P＜0.001(***)表示具有极显著性差异。The following examples use SPSS11.5 statistical software to process the data, the experimental results are expressed as the mean ± standard deviation, and the One-way ANOVA test is used, P<0.05 (*) indicates a significant difference, P<0.01 (** ) indicates a very significant difference, and P<0.001 (***) indicates a very significant difference.

实施例1、目的基因Zm00001d005920的确定和获得Example 1. Determination and acquisition of target gene Zm00001d005920

1.1、目的基因Zm00001d005920的确定1.1. Determination of the target gene Zm00001d005920

1.1.1、抗旱表型收集和全基因组关联分析1.1.1. Drought resistance phenotype collection and genome-wide association analysis

本实验在温室种植374份优良玉米种质资源自交系，种植温度维持在白天28℃(14h)-夜间24℃(10h)，利用PVC管和黑色塑料薄膜桶套用的方法，每个PVC管(60cm高×25cm直径)中放置3个黑色塑料薄膜桶(60cm高×13cm直径)，每个塑料薄膜桶使用混合土壤(草炭土：蛭石＝1:1)，每个黑色塑料薄膜桶中种植玉米XXX株。在设置有正常水分处理(WW)和干旱胁迫(WS)两种环境的土壤中播种籽粒饱满程度一致、大小一致的种子，待出苗35天后，对地上部植株进行烘干称重，计算抗旱指数(Drought Tolerance Index,DTI)。In this experiment, 374 inbred lines of high-quality corn germplasm resources were planted in the greenhouse. The planting temperature was maintained at 28°C (14h) during the day and 24°C (10h) at night. Using the method of applying PVC pipes and black plastic film buckets, each PVC pipe (60cm high x 25cm diameter) placed 3 black plastic film buckets (60cm high x 13cm diameter), each plastic film bucket used mixed soil (peat soil: vermiculite = 1:1), each black plastic film bucket Plant corn XXX strains. Seeds with the same grain filling and the same size were sown in the soil with normal water treatment (WW) and drought stress (WS) environments. After 35 days of emergence, the aboveground plants were dried and weighed to calculate the drought resistance index. (Drought Tolerance Index, DTI).

基于重测序得到覆盖全基因组的11,800,000个高质量SNP，根据WW和WS处理下得到的关联群体表型值，利用GEMMA的分析模型，进行全基因组关联分析，设阈值为P＝1×10⁶。Based on resequencing, 11,800,000 high-quality SNPs covering the whole genome were obtained. According to the associated population phenotype values obtained under the WW and WS treatments, the GEMMA analysis model was used to conduct genome-wide association analysis, and the threshold was set to P=1×10⁶ .

1.1.2、控制玉米苗期抗旱性基因Zm00001d0059520的鉴定1.1.2. Identification of Zm00001d0059520, a gene controlling drought resistance in maize seedling stage

通过对地上部的生物量计算获得的抗旱指数(DTI)进行全基因组关联分析结果中，发现在2号染色体上有与抗旱性相关的重要位点qDR2(图1)。在qDR2位点的区段内共发现了3个候选基因(图2中a)，其中最显著关联的SNP位于基因Ⅲ内，且该SNP形成的两种单倍型具有极显著的抗旱性表型差异(图2中b)。为确定候选基因，利用课题组前期用10份玉米自交系苗期在不同水分处理下的RNA-Seq结果，发现基因型I和基因型II表达量较低，基因型III表达量高，且干旱下的表达量与正常水分呈显著差异(图2中c)。因此初步确定基因Ⅲ为抗旱性的候选基因。为了进一步确定候选基因，在关联群体中随机挑选出10份抗旱材料和10份干旱敏感材料，在不同处理下进行表达量分析，结果发现基因Ⅲ在正常水分处理下抗旱材料和干旱敏感材料之间相对表达量没有显著差异，而在干旱胁迫下该基因在抗旱材料中的相对表达量显著高于干旱敏感材料(图2中d)。因此，确定Zm00001d005920为本项目所研究的抗旱基因。在此基础上，开展了该基因的功能验证。Based on the results of genome-wide association analysis of the drought resistance index (DTI) calculated from the above-ground biomass, it was found that there is an important locus qDR2 associated with drought resistance on chromosome 2 (Fig. 1). A total of 3 candidate genes were found in the segment of the qDR2 locus (a in Figure 2), of which the most significantly associated SNP was located in gene III, and the two haplotypes formed by this SNP had extremely significant drought resistance. type difference (b in Figure 2). In order to determine the candidate genes, using the RNA-Seq results of 10 maize inbred lines under different water treatments in the early stage of the research group, it was found that the expression levels of genotype I and genotype II were low, the expression level of genotype III was high, and The expression under drought was significantly different from that under normal moisture (c in Figure 2). Therefore, gene Ⅲ was preliminarily identified as a candidate gene for drought resistance. In order to further identify candidate genes, 10 drought-resistant materials and 10 drought-sensitive materials were randomly selected from the associated population, and their expression levels were analyzed under different treatments. It was found that gene III was between drought-resistant materials and drought-sensitive materials under normal water treatment. There was no significant difference in relative expression, while the relative expression of this gene in drought-resistant materials was significantly higher than that in drought-sensitive materials under drought stress (d in Figure 2). Therefore, Zm00001d005920 was identified as the drought resistance gene studied in this project. On this basis, the functional verification of the gene was carried out.

1.2、Zm00001d005920基因组及编码基因的扩增及回收测序1.2. Amplification, recovery and sequencing of Zm00001d005920 genome and coding genes

选取籽粒饱满的玉米自交系PHR36种子，将种子种植在装有营养土的花盆中，每个盆种植3株，置于光照培养箱中培养(28℃，光照)。待幼苗长至六叶一心期时采集叶片，一部分用于提取基因组DNA；另一部分叶片，分别取植株地上部分和地下部分提取RNA，-80℃保存。The seeds of the maize inbred line PHR36 with plump grains were selected, and the seeds were planted in flowerpots with nutrient soil, 3 plants per pot, and cultivated in a light incubator (28°C, light). When the seedlings grow to the six-leaf and one-heart stage, the leaves are collected, and one part is used to extract genomic DNA; the other part of the leaves is taken from the aerial part and the underground part of the plant to extract RNA, and stored at -80°C.

1.2.1、玉米材料基因组DNA的提取1.2.1. Extraction of genomic DNA from maize material

(1)将叶片迅速放入到已灭菌的研钵中，倒液氮充分研磨，期间不断加入液氮，研磨充分后加入2.0ml离心管中，加到离心管1/3处；(1) Put the blade into the sterilized mortar quickly, pour the liquid nitrogen into the mortar and grind it, continuously add liquid nitrogen during the period, add it into the 2.0ml centrifuge tube after the grinding is sufficient, and add it to 1/3 of the centrifuge tube;

(2)将提前配置好的CTAB提取液事先放入65℃水浴锅中预热，然后在上一步中的离心管中加入800μl预热的CTAB缓冲液，用力摇晃，使之充分混匀；(2) Put the CTAB extract prepared in advance into a 65°C water bath to preheat, then add 800 μl of preheated CTAB buffer to the centrifuge tube in the previous step, and shake vigorously to mix it well;

(3)将混合好的提取液置于65℃恒温水浴30min，期间每隔10min晃动一次，使之充分发生反应；(3) place the mixed extract in a constant temperature water bath at 65°C for 30min, and shake it every 10min to make it fully react;

(4)取出经过充分反应的离心管，加入等体积氯仿/异戊醇(体积比24:1)，缓慢混匀15min，静止10min；(4) Take out the fully reacted centrifuge tube, add an equal volume of chloroform/isoamyl alcohol (volume ratio 24:1), slowly mix for 15min, and stand still for 10min;

(5)将离心管放入到离心机中，离心(12000rpm，15-20min)；(5) put the centrifuge tube into the centrifuge, centrifuge (12000rpm, 15-20min);

(6)小心吸取上清液于另一洁净的2.0ml离心管中，加入等体积经过预冷(-20℃)的异丙醇，轻混摇匀，将离心管放置到-20℃的冰箱中冷30min,至大量白色沉淀析出；(6) Carefully pipette the supernatant into another clean 2.0ml centrifuge tube, add an equal volume of pre-cooled (-20°C) isopropanol, mix gently, and place the centrifuge tube in a -20°C refrigerator Cool in the middle for 30min until a large amount of white precipitate separates out;

(7)用灭菌的枪头将白色沉淀勾出，放入另一洁净的离心管中，加入500μl 75％的乙醇冲洗2-3次；(7) Tick off the white precipitate with a sterilized pipette tip, put it into another clean centrifuge tube, and add 500 μl of 75% ethanol to rinse it for 2-3 times;

(8)离心，倒掉75％的乙醇，用枪头吸走多余的乙醇后于室温干燥，用1×TE溶解DNA；(8) Centrifuge, pour off 75% ethanol, suck off excess ethanol with a pipette tip, dry at room temperature, and dissolve DNA with 1×TE;

(9)加入RNase进行纯化(终浓度为10ug/μl)，37℃保温箱恒温放置1h；(9) Add RNase for purification (final concentration is 10ug/μl), and place it in a 37°C incubator at a constant temperature for 1h;

(10)加入等体积的苯酚/氯仿/异戊醇(体积比25:24:1)抽提一次，高速离心(12000rpm)10min；(10) Add equal volume of phenol/chloroform/isoamyl alcohol (volume ratio 25:24:1) to extract once, and centrifuge at high speed (12000rpm) for 10min;

(11)小心吸取上清，再用等体积氯仿/异戊醇(体积比24:1)抽提一次，高速离心(12000rpm)10min；(11) Carefully aspirate the supernatant, then extract once with an equal volume of chloroform/isoamyl alcohol (volume ratio 24:1), and centrifuge at high speed (12000 rpm) for 10 min;

(12)用预冷的无水乙醇沉淀DNA，离心(12000rpm)10min；(12) Precipitate DNA with pre-cooled absolute ethanol and centrifuge (12000rpm) for 10min;

(13)倒掉乙醇，彻底晾干，以免抑制下游的PCR反应，加入适量TE溶解，于-20℃冰箱保存备用。(13) Pour off the ethanol, dry it thoroughly to avoid inhibiting the downstream PCR reaction, add an appropriate amount of TE to dissolve, and store it in a -20°C refrigerator for later use.

1.2.2、玉米材料总RNA的提取和纯化1.2.2. Extraction and purification of total RNA from corn material

主要采用无苯酚、氯仿RNA快速提取技术提取植物总RNA：Mainly use phenol-free, chloroform RNA rapid extraction technology to extract total plant RNA:

具体方法参考gene-better公司多糖多酚植物总RNA提取试剂盒(带gDNA过滤器)试剂说明书，以下所用实验器皿都经过高温灭菌处理，以除掉RNase。For the specific method, please refer to the instruction manual of the polysaccharide and polyphenol plant total RNA extraction kit (with gDNA filter) from gene-better company.

(1)在经过180℃高温处理过的无RNase的研钵中倒入液氮，之后从-76℃超低温冰箱中取出冻存的叶片，加入液氮充分研磨，期间不断补充液氮，研磨充分；(1) Pour liquid nitrogen into an RNase-free mortar that has been treated at a high temperature of 180 °C, then take out the frozen leaves from the -76 °C ultra-low temperature refrigerator, add liquid nitrogen to fully grind, and continuously replenish liquid nitrogen during the period to fully grind ;

(2)在向离心管中加入样品之前，先将无RNase的1.5ml的离心管完全浸于液氮中速冻，之后后迅速加入研磨的样品，样品不宜加入过多一般加到1/3处(约50mg)，向离心管中加入500μl裂解液和50μl PLANTaid旋涡震荡20s,使其充分裂解。(2) Before adding the sample to the centrifuge tube, firstly immerse the 1.5ml centrifuge tube without RNase in liquid nitrogen for quick freezing, and then quickly add the ground sample. (about 50 mg), add 500 μl lysis buffer and 50 μl PLANTaid to the centrifuge tube and vortex for 20s to fully lyse.

(3)将裂解物13000rpm离心10分钟，沉淀不能裂解的碎片和结合有多糖多酚的PLANTaid。(3) Centrifuge the lysate at 13,000 rpm for 10 minutes to precipitate unlysable fragments and PLANTaid bound to polysaccharides and polyphenols.

(4)取裂解物上清转到一个新的离心管。加入上清体积一般的无水乙醇，立即吹打混匀，不要离心。(4) Transfer the lysate supernatant to a new centrifuge tube. Add the normal volume of absolute ethanol to the supernatant, and immediately mix by pipetting without centrifugation.

(5)将混合物(每次小于720μl，多可以分两次加入)加入一个基因组DNA过滤器中，13000rpm离心2min，弃掉废液。(5) Add the mixture (less than 720 μl each time, it can be added twice at most) into a genomic DNA filter, centrifuge at 13000 rpm for 2 min, and discard the waste liquid.

(6)将基因组DNA过滤器放在一个干净的2ml离心管内，在基因组内过滤器内加入500μl裂解液RLT PLUS，13000rpm离心30s，收集滤液，用移液器较精确估计过滤体积，加入0.5倍体积的无水乙醇，此时可能出现沉淀，但是不影响提取过程，立即吹打混匀，不要离心。(6) Put the genomic DNA filter in a clean 2ml centrifuge tube, add 500μl of lysate RLT PLUS to the genomic DNA filter, centrifuge at 13000rpm for 30s, collect the filtrate, use a pipette to accurately estimate the filtration volume, add 0.5 times volume of anhydrous ethanol, precipitation may occur at this time, but it will not affect the extraction process, immediately mix by pipetting, do not centrifuge.

(7)立刻将混合物加入一个吸附柱RA中，13000rpm离心2min，弃掉废液。(7) Immediately add the mixture to an adsorption column RA, centrifuge at 13000 rpm for 2 min, and discard the waste liquid.

(8)加700μl去蛋白液RW1，室温放置1min，13000rpm离心30s，弃掉废液。(8) Add 700 μl of deproteinized solution RW1, place at room temperature for 1 min, centrifuge at 13000 rpm for 30 s, and discard the waste liquid.

(9)加入500μl漂洗液RW，13000rpm离心30s弃掉废液。加入500μl漂洗液RW，重复一遍。将吸附柱RA放回空收集管中，13000rpm离心2min，尽量除去漂洗液，以免漂洗液中残留乙醇抑制下游反应。(9) Add 500 μl of rinse solution RW, and centrifuge at 13000 rpm for 30 s to discard the waste liquid. Add 500 μl of rinse solution RW and repeat. Put the adsorption column RA back into the empty collection tube, centrifuge at 13,000 rpm for 2 min, and remove the rinsing solution as much as possible, so as to avoid residual ethanol in the rinsing solution to inhibit the downstream reaction.

(10)取出RA吸附柱，放入一个RNase free离心管中，在吸附膜中间加入30-50μlRNase free water，室温放置1min，13000rpm离心1min。(10) Take out the RA adsorption column, put it into an RNase free centrifuge tube, add 30-50 μl RNase free water in the middle of the adsorption membrane, place it at room temperature for 1 min, and centrifuge at 13000 rpm for 1 min.

(11)1％琼脂糖凝胶电泳(18min左右)鉴定所提取RNA的质量，并用分光光度计测定所提取RNA的浓度，吸取适量的RNA用于纯化和反转，剩余的RNA放置于-76℃长期保存。(11) 1% agarose gel electrophoresis (about 18 minutes) to identify the quality of the extracted RNA, and use a spectrophotometer to measure the concentration of the extracted RNA, draw an appropriate amount of RNA for purification and inversion, and place the remaining RNA at -76 ℃ for long-term storage.

(11)反转录合成第一链cDNA(11) Synthesis of first-strand cDNA by reverse transcription

以(11)获得的RNA为模板，参考Transgen公司One-Step gDNA Removal and CdnaSynthesis SuperMix说明书，反转录合成第一链cDNA，20μl反转录反应体系(置于0.2ml无RNase离心管)如表1。50℃孵育30min，85℃加热5s使酶失活，迅速转至冰上，得到cDNA。将cDNA原液稀释5倍，用内参基因GADPH进行PCR扩增，检测反转效果，-20℃保存备用。Using the RNA obtained in (11) as a template, referring to the instructions of Transgen's One-Step gDNA Removal and CdnaSynthesis SuperMix, reverse transcription to synthesize first-strand cDNA, 20 μl reverse transcription reaction system (placed in a 0.2 ml RNase-free centrifuge tube) as shown in the table 1. Incubate at 50°C for 30min, heat at 85°C for 5s to inactivate the enzyme, and quickly transfer to ice to obtain cDNA. The cDNA stock solution was diluted 5 times, and the internal reference gene GADPH was used for PCR amplification to detect the inversion effect, and stored at -20°C for future use.

表1：20μl反转录反应体系Table 1: 20 μl reverse transcription reaction system

组分component体积volumeTotal RNA(总量50ng-5ug)Total RNA (total 50ng-5ug)4μl4μlAnchored Oligo(dT)₂₀Primer(0.5ug/μl)Anchored Oligo(dT)₂₀Primer(0.5ug/μl)1μl1μl2×TS-Uni Reaction Mix2×TS-Uni Reaction Mix10μl10μlUni RT/RI Enzyme MixUni RT/RI Enzyme Mix1μl1μlgDNA RemovergDNA Remover1μl1μlRNase-free ddH₂ORNase-free ddH₂O3μl3μl总计total20μl20μl

1.2.3、候选基因组DNA的扩增1.2.3. Amplification of candidate genomic DNA

从玉米Maize GDB数据库(https://www.maizegdb.org/)下载参考基因组B73对应的候选基因序列，在基因组5’UTR和3’UTR区域利用Primer5软件设计引物，以上述1.2.1获得的玉米自交系PHR36幼苗基因组DNA为模板扩增候选基因组全长DNA，扩增体系如表2。Download the candidate gene sequence corresponding to the reference genome B73 from the maize Maize GDB database (https://www.maizegdb.org/), use Primer5 software to design primers in the 5'UTR and 3'UTR regions of the genome, and use the primers obtained in 1.2.1 above. The genomic DNA of the maize inbred line PHR36 seedlings was used as the template to amplify the full-length DNA of the candidate genome, and the amplification system was shown in Table 2.

表2：PCR扩增体系Table 2: PCR amplification system

组分component体积volumeDNA模板DNA template1μl1μlForward PrimerForward Primer1μl1μlReverse PrimerReverse Primer1μl1μlMCLAB SuperMixMCLAB SuperMix1 0μl10μlddH2OddH2O7μl7μl总计total20μl20μl

由于候选基因的基因较长，因此设计引物分段扩增基因的全长。引物序列如下：Since the gene of the candidate gene is long, primers are designed to amplify the full length of the gene in sections. The primer sequences are as follows:

Forward Primer1：5'-CCCAAACAGTCCAATCACGG-3'；Forward Primer1: 5'-CCCAAACAGTCCAATCACGG-3';

Reverse Primer1：5'-CAAGCTAAGGTACATAGTAAGCCACC-3'；Reverse Primer1: 5'-CAAGCTAAGGTACATAGTAAGCCACC-3';

Forward Primer2：5'-GTCTGTCTTCACGATTCCCTT-3'；Forward Primer2: 5'-GTCTGTCTTCACGATTCCCTT-3';

Reverse Primer2：5'-TCTGATCCCTCATGCTGGT-3'；Reverse Primer2: 5'-TCTGATCCCTCATGCTGGT-3';

Forward Primer3：5'-GTCTGCCCTACCGCTGTTT-3'；Forward Primer3: 5'-GTCTGCCCTACCGCTGTTT-3';

Reverse Primer3：5'-CCATTGTGGCTTTGTGCTT-3'；Reverse Primer3: 5'-CCATTGTGGCTTTGTGCTT-3';

PCR扩增程序如下：The PCR amplification procedure is as follows:

1.2.4、候选基因全长CDS的扩增1.2.4. Amplification of candidate gene full-length CDS

根据扩增的基因组DNA序列设计引物，将上述1.2.2获得的玉米自交系PHR36幼苗cDNA分别作为基因扩增的模板，设计引物进行基因全长CDS序列的扩增，扩增体系如表3。The primers were designed according to the amplified genomic DNA sequence, and the cDNA of the maize inbred line PHR36 seedling obtained in the above 1.2.2 was used as a template for gene amplification, and the primers were designed to amplify the full-length CDS sequence of the gene. The amplification system is shown in Table 3 .

表3：PCR扩增体系Table 3: PCR amplification system

组分component体积volumecDNA模板cDNA template1μl1μlForward PrimeForward Prime1μl1μlReverse PrimerReverse Primer1μl1μlMCLAB SuperMixMCLAB SuperMix1 0μl10μlddH2OddH2O7μl7μl总计total20μl20μl

其中，引物序列如下：The primer sequences are as follows:

Forward Primer：5'-ATGGCTAGCGGCGTCCTCC-3'Forward Primer: 5'-ATGGCTAGCGGCGTCCTCC-3'

Reverse Primer：5'-TCAAGACCAGGTGGACCTATTCT-3'Reverse Primer: 5'-TCAAGACCAGGTGGACCTATTCT-3'

PCR扩增程序如下：The PCR amplification procedure is as follows:

1.2.5、候选基因组DNA以及CDS片段回收与测序1.2.5. Recovery and sequencing of candidate genomic DNA and CDS fragments

A、候选基因组DNA以及CDS片段回收A. Recovery of candidate genomic DNA and CDS fragments

片段回收主要参考北京诺唯赞生物技术股份有限公司的FastPure Gel DNAExtraction Mini Kit说明书For fragment recovery, please refer to the FastPure Gel DNAExtraction Mini Kit instruction manual of Beijing Novizan Biotechnology Co., Ltd.

(1)向100ml TAE溶液中加入1g的琼脂糖，在微波炉中加热煮沸，之后加入10μl的10000×GelStain染料，倒入插有梳子的平板中，制备琼脂1％琼脂糖凝胶，将加入Loadingbuffer的PCR产物进行琼脂糖凝胶电泳(125v)25min，在长波紫外灯下切取目的条带放入1.5ml离心管中；(1) Add 1 g of agarose to 100 ml of TAE solution, heat and boil in a microwave oven, then add 10 μl of 10000×GelStain dye, pour it into a plate with a comb, prepareagar 1% agarose gel, and add Loadingbuffer The PCR product was subjected to agarose gel electrophoresis (125v) for 25min, and the target band was cut under a long-wave ultraviolet lamp and placed in a 1.5ml centrifuge tube;

(2)向离心管中加入400μl的Buffer GDP，用于溶胶；(2) Add 400 μl of Buffer GDP to the centrifuge tube for sol;

(3)将离心管放入到55℃水浴锅中，水浴，每5min上下颠倒混匀一次，直到凝胶完全融化，溶液呈现为淡黄色，室温下静置待其温度降至室温后进行下一步反应；(3) Put the centrifuge tube into a 55°C water bath, invert the water bath, and mix it upside down every 5 minutes until the gel is completely melted and the solution is pale yellow. one-step reaction;

(4)将上一步离心管中的溶液转入吸附柱之中，将离心管放入离心机中，12000rpm离心30s，弃废液，再将吸附柱放回空的收集管中；(4) transfer the solution in the centrifuge tube of the previous step into the adsorption column, put the centrifuge tube into the centrifuge, centrifuge at 12000rpm for 30s, discard the waste liquid, and put the adsorption column back into the empty collection tube;

(5)向吸附柱中加入300μl的Buffer GDP，静置1min，将离心管加入到离心机中，12000rpm离心30s，弃废液；(5) Add 300 μl of Buffer GDP to the adsorption column, let it stand for 1 min, add the centrifuge tube to the centrifuge, centrifuge at 12000 rpm for 30 s, and discard the waste liquid;

(6)把吸附柱置于收集管中，加入700μl Buffer GW至吸附柱中，12000rpm离心30s；弃滤液。洗两遍。(6) Put the adsorption column in the collection tube, add 700 μl Buffer GW to the adsorption column, centrifuge at 12000 rpm for 30 s; discard the filtrate. Wash twice.

(7)将吸附柱放回空的收集管中，12000rpm离心2min；(7) Put the adsorption column back into the empty collection tube and centrifuge at 12000rpm for 2min;

(8)取出吸附柱，放入干净的1.5ml无菌离心管中，室温下静置5min，在吸附膜的中间部位加入25μl的洗脱液，室温下静置5min，12000rpm离心2min；(8) Take out the adsorption column, put it into a clean 1.5ml sterile centrifuge tube, let stand for 5 minutes at room temperature, add 25 μl of eluent to the middle part of the adsorption membrane, let stand for 5 minutes at room temperature, and centrifuge at 12000 rpm for 2 minutes;

(9)重复步骤(8)。(9) Repeat step (8).

B、候选基因组DNA以及CDS片段测序B. Sequencing of candidate genomic DNA and CDS fragments

参考天根生物的pLB零背景快速克隆试剂盒说明书，操作步骤如下：Refer to the instructions of Tiangen Biotech's pLB zero background rapid cloning kit, the operation steps are as follows:

(1)将上述的DNA回收的片段连接到pLB载体，连接体系(10μl)如表4，轻弹离心管以混匀反应液。(1) The above DNA recovered fragment was ligated to the pLB vector. The ligation system (10 μl) was as shown in Table 4, and the centrifuge tube was flicked to mix the reaction solution.

表4：Table 4:

组分component体积volume候选基因回收片段Candidate gene recovery fragment3μl3μlpLB载体pLB vector1μl1μl2×Reaction Solution2×Reaction Solution5μl5μlT4 DNA连接酶T4 DNA ligase1μl1μl总计total10μl10μl

(2)将混合反应液放入22℃恒温金属浴中反应15min。反应结束后，将离心管置于冰上，进行后续的转化实验。(2) Put the mixed reaction solution into a constant temperature metal bath at 22°C for 15 minutes. After the reaction, place the centrifuge tube on ice for subsequent transformation experiments.

(3)制备含有氨苄青霉素终浓度为100ug/ml的LB琼脂糖平板。将平板放置在37℃，预热20min。(3) Prepare an LB agarose plate containing ampicillin with a final concentration of 100ug/ml. The plate was placed at 37°C and preheated for 20 min.

(4)将10μl的连接产物加入到100μl的TOP10感受态细胞中(感受态细胞从-80℃冰箱取出放置于冰上，待刚刚解冻时加入连接产物)，用手轻弹混匀，冰浴30min。(4) Add 10 μl of the ligation product to 100 μl of TOP10 competent cells (the competent cells are taken out from the -80°C refrigerator and placed on ice, and the ligation product will be added when it is just thawed), mix by hand, and ice bath for 30 minutes .

(5)之后将离心管放置于42℃水浴锅中90s，取出后立即冰浴5min。(5) After that, place the centrifuge tube in a water bath at 42°C for 90s, and immediately take it out in an ice bath for 5min.

(6)向离心管中加入500μl的LB(不含抗生素)培养基，150rpm、37℃震荡培养60min，使菌体复苏。(6) 500 μl of LB (antibiotic-free) medium was added to the centrifuge tube, and the cells were cultured with shaking at 150 rpm and 37° C. for 60 min to recover the cells.

(7)将离心管中的菌液混匀，取100μl加到含氨苄青霉素的LB固体琼脂培养基上，用无菌的弯头玻璃棒轻轻地将菌液涂开。待平板表面干燥后，倒置平板，37℃恒温培养12h。(7) Mix the bacterial liquid in the centrifuge tube, add 100 μl to the LB solid agar medium containing ampicillin, and gently spread the bacterial liquid with a sterile curved glass rod. After the surface of the plate was dry, the plate was inverted and incubated at a constant temperature of 37°C for 12 h.

(8)取出平板，挑取乳白色的阳性单克隆至600μl的LBA液体培养基(加入抗生素)上，37℃震荡培养8h，pLB载体通用引物进行菌液PCR，经琼脂糖凝胶电泳鉴定为阳性的克隆进行测序验证，提取质粒用于后续实验。(8) Take out the plate, pick the milky white positive monoclone onto 600 μl of LBA liquid medium (add antibiotics), shake and culture at 37°C for 8 hours, carry out bacterial liquid PCR with the universal primer of pLB vector, and identify it as positive by agarose gel electrophoresis The clones were verified by sequencing, and the plasmids were extracted for subsequent experiments.

pLB载体通用引物序列如下：The sequences of the universal primers for the pLB vector are as follows:

Forward Primer：5'-CGACTCACTATAGGGAGAGCGGC-3'；Forward Primer: 5'-CGACTCACTATAGGGAGAGCGGC-3';

Reverse Primer：5'-AAGAACATCGATTTTCCATGGCAG-3'；Reverse Primer: 5'-AAGAACATCGATTTTCCATGGCAG-3';

结果如下：The result is as follows:

Zm00001d005920基因的基因组DNA的核苷酸序列为序列1；Zm00001d005920的CDS核苷酸序列为序列2；编码的蛋白命名为Zm00001d005920蛋白，该蛋白的氨基酸序列为序列3。The nucleotide sequence of the genomic DNA of Zm00001d005920 gene issequence 1; the CDS nucleotide sequence of Zm00001d005920 issequence 2; the encoded protein is named Zm00001d005920 protein, and the amino acid sequence of this protein issequence 3.

实施例2、Zm00001d005920基因转化拟南芥的功能验证Example 2. Functional verification of Zm00001d005920 gene transforming Arabidopsis thaliana

2.1、Zm00001d005920基因转化拟南芥Col-02.1. Transforming Arabidopsis Col-0 with Zm00001d005920 gene

2.1.1、候选基因植物表达载体的构建2.1.1. Construction of candidate gene plant expression vector

构建pCAMBIA3301-Zm00001d005920载体的步骤如下：The steps for constructing the pCAMBIA3301-Zm00001d005920 vector are as follows:

1)PCAMBIA 3301载体线性化1) Linearization ofPCAMBIA 3301 vector

用Nco1和BstE2内切酶37℃水浴切割载体PCAMBIA 3301，得到线性化PCAMBIA3301载体。反应体系如表5。Thevector PCAMBIA 3301 was cleaved with Nco1 and BstE2 endonucleases in a water bath at 37°C to obtain a linearized PCAMBIA3301 vector. The reaction system is shown in Table 5.

表5：载体线性化反应体系Table 5: Vector linearization reaction system

37℃水浴3h，之后切胶回收。37 ℃ water bath for 3h, then the glue was cut and recovered.

2)Zm00001d005920片段制备2) Zm00001d005920 fragment preparation

引物：包括目的片段特异性引物和载体重叠序列Primers: including target fragment-specific primers and vector overlapping sequences

LF：5'-ACGGGGGACTCTTGACATGGCTAGCGGCGTCCTCC-3'(下划线部分为载体重叠序列)；LF: 5'-ACGGGGGACTCTTGAC ATGGCTAGCGGCGTCCTCC-3' (the underlined part is the overlapping sequence of the vector);

LR：5'-ATTCGAGCTGGTCACTCAAGACCAGGTGGACCTATTCT-3'(下划线部分为载体重叠序列)；LR: 5'-ATTCGAGCTGGTCAC TCAAGACCAGGTGGACCTATTCT-3' (the underlined part is the overlapping sequence of the vector);

以PHR36的cDNA为模板，用上述引物进行PCR扩增，得到扩增产物，即为Zm00001d005920片段(两端包含重叠序列的序列2)。Using the cDNA of PHR36 as a template, PCR amplification was performed with the above-mentioned primers to obtain an amplified product, which is the Zm00001d005920 fragment (sequence 2 containing overlapping sequences at both ends).

上述PCR反应体系(由南京诺唯赞生物科技有限公司提供，目录号是P505-d1/d2/d3)如表6。The above PCR reaction system (provided by Nanjing Novizan Biotechnology Co., Ltd., catalog number is P505-d1/d2/d3) is shown in Table 6.

表6：PCR扩增体系Table 6: PCR amplification system

组分component体积volumecDNA模板cDNA template1μl1μlForward PrimeForward Prime1μl1μlReverse PrimerReverse Primer1μl1μl2×Phanta Max Buffer a2 x Phanta Max Buffer a25μl25μldNTP MixdNTP Mix1μl1μlPhanta Max Super-Fidelity DNA PolymerasePhanta Max Super-Fidelity DNA Polymerase1μl1μlddH₂OddH₂O20μl20μl总计total50μl50μl

PCR扩增程序如下：The PCR amplification procedure is as follows:

扩增产物进行电泳和切胶回收。The amplified products were subjected to electrophoresis and gel cutting recovery.

3)连接载体线性片段和目的基因片段，连接体系如表7，轻轻混合，50℃反应15分钟。3) Connect the linear fragment of the vector and the target gene fragment, the ligation system is shown in Table 7, mix gently, and react at 50°C for 15 minutes.

表7：连接体系Table 7: Connection System

4)转化4) Conversion

在冰上融化Fast-T1感受态细胞，在100μl感受态细胞中加入10μl的重组产物，轻轻弹动离心管混匀，冰上放置30分钟，之后42℃水浴中热激30秒，之后马上转移至冰上冷却2分钟，加入450μl常温的LB培养基，之后37℃摇床中200rpm培养1小时，之后取100μl细胞均匀涂在具有卡那霉素抗性的LB平板上，在37℃培养箱中过夜培养。Thaw Fast-T1 competent cells on ice, add 10 μl of recombinant product to 100 μl of competent cells, gently shake the centrifuge tube to mix, place on ice for 30 minutes, heat shock in a 42°C water bath for 30 seconds, and then immediately Transfer to ice for 2 minutes, add 450 μl of LB medium at room temperature, and then culture at 200 rpm in a shaker at 37 °C for 1 hour, then take 100 μl of cells and spread them evenly on LB plates with kanamycin resistance and culture at 37 °C Incubate overnight.

5)阳性克隆筛选5) Positive clone screening

利用引物LF/LR进行阳性克隆筛选，测序正确的菌液提取质粒待用。Use primers LF/LR to screen positive clones, and extract plasmids from bacterial liquids with correct sequencing.

LF：5'-ACGGGGGACTCTTGACATGGCTAGCGGCGTCCT-3'；LF: 5'-ACGGGGGACTCTTGACATGGCTAGCGGCGTCCT-3';

LR：5'-ATTCGAGCTGGTCACTCAAGACCAGGTGGACC-3'；LR: 5'-ATTCGAGCTGGTCACTCAAGACCAGGTGGACC-3';

测序结果表明：重组载体pCAMBIA3301-Zm00001d005920是将pCAMBIA3301载体中的5'-ACGGGGGACTCTTGAC-3'和5'-GTGACCAGCTCGAAT-3'之间的小片段替换为序列2所示的Zm00001d005920的编码区序列，保持pCAMBIA3301的其它核苷酸不变，得到的表达Zm00001d005920基因的重组表达载体。该载体中启动Zm00001d005920基因表达的启动子为CaMV 35S promoter。The sequencing results show that the recombinant vector pCAMBIA3301-Zm00001d005920 is a small fragment between 5'-ACGGGGGACTCTTGAC -3' and 5'-GTGACCAGCTCGAAT -3' in the pCAMBIA3301 vector is replaced with the coding region sequence of Zm00001d005920 shown insequence 2, and the pCAMBIA3301 The other nucleotides remain unchanged, and the recombinant expression vector expressing Zm00001d005920 gene is obtained. The promoter for initiating the expression of Zm00001d005920 gene in this vector is CaMV 35S promoter.

重组载体pCAMBIA3301-Zm00001d005920的构建示意图及重组载体的载体图谱如图3所示。The schematic diagram of the construction of the recombinant vector pCAMBIA3301-Zm00001d005920 and the vector map of the recombinant vector are shown in Figure 3 .

2.1.2、农杆菌侵染液制备2.1.2. Preparation of Agrobacterium infection solution

1)取-70℃保存的农杆菌(EHA105农杆菌感受态细胞购自北京博迈德基因技术有限公司，货号为BC303)感受态细胞于冰上融化；1) Take Agrobacterium (EHA105 Agrobacterium competent cells purchased from Beijing Bomed Gene Technology Co., Ltd., item number BC303) stored at -70°C and thaw on ice;

2)向感受态细胞中加入1ug上述1制备的重组载体pCAMBIA3301-Zm00001d005920，轻轻混匀，冰浴静置5min；2) Add 1 ug of the recombinant vector pCAMBIA3301-Zm00001d005920 prepared in the above 1 to the competent cells, mix gently, and let stand in an ice bath for 5 minutes;

3)将离心管置于液氮中速冻5min，快速将离心管置于37℃水浴中保持5min,不要晃动水面；再将离心管放回冰上，冰浴5min；3) Quick-freeze the centrifuge tube in liquid nitrogen for 5 minutes, quickly place the centrifuge tube in a 37°C water bath for 5 minutes, do not shake the water surface; then put the centrifuge tube back on ice for 5 minutes;

4)无菌条件下加入800μl无抗生素的LB液体培养基，于28℃震荡培养基2-3小时，菌体复苏。4) Add 800 μl of antibiotic-free LB liquid medium under sterile conditions, shake the medium at 28° C. for 2-3 hours, and recover the bacteria.

5)5000rpm离心1分钟收菌，留100μl上清，轻轻吹打重悬菌体，涂布于相应抗生素的LB平板上，于28℃培养箱中倒置培养72小时，得到重组菌。5) Collect bacteria by centrifugation at 5000 rpm for 1 minute, leave 100 μl of supernatant, gently pipette to resuspend the bacteria, spread on LB plates with corresponding antibiotics, and invert in a 28°C incubator for 72 hours to obtain recombinant bacteria.

2.1.3、农杆菌侵染拟南芥2.1.3. Agrobacterium infection of Arabidopsis

1)用接种针挑取重组菌的单菌落在含相应抗生素的50ml LB液体培养基培养，28℃培养2天。1) Pick a single colony of recombinant bacteria with an inoculating needle and culture it in 50 ml of LB liquid medium containing the corresponding antibiotics, and culture at 28°C for 2 days.

2)5000rpm离心8min，收集菌体。2) Centrifuge at 5000 rpm for 8 min to collect bacterial cells.

3)重悬菌体；先配置菌体重悬液(100ml ddH₂O,蔗糖5g,Silwet L-77 10μl),使用重悬液将菌体重悬，使最终OD₆₀₀值为0.8左右。3) Resuspend the bacteria; firstly prepare the bacteria suspension (100ml ddH₂ O, 5 g of sucrose, 10 μl of Silwet L-77), and use the suspension to resuspend the bacteria so that the final OD₆₀₀ value is about 0.8.

4)选取生长4周左右，发育正常刚开花的野生型拟南芥植株。首先去除植株上的果荚，将准备好的菌液重悬液倒入培养皿中，将整个花序浸入菌液10s。4) Select wild-type Arabidopsis thaliana plants that have grown for about 4 weeks and have just bloomed normally. First remove the fruit pods on the plant, pour the prepared bacterial suspension into a petri dish, and immerse the entire inflorescence in the bacterial solution for 10s.

5)将浸好的拟南芥植株在黑暗环境下处理24h后恢复正常光照。5) The soaked Arabidopsis plants were treated in a dark environment for 24 hours and returned to normal light.

6)拟南芥成熟后收取拟南芥种子，烘干后，用6％的次氯酸钠消毒，种植于含有50ug/ml的草铵膦除草剂的MS培养基上，培养拟南芥直到得到T2代转化植株。6) Arabidopsis thaliana seeds are collected after maturity, dried, sterilized with 6% sodium hypochlorite, planted on the MS medium containing 50ug/ml of glufosinate-ammonium herbicide, and cultivated Arabidopsis thaliana until the T2 generation is obtained Transformed plants.

2.1.4、分子鉴定2.1.4. Molecular identification

取生长了一个月的到T2代转化植株叶片，提取到T2代转化植株和野生型拟南芥(col-0)叶片的DNA，用如下引物扩增：Take the leaves of the transformed plants in the T2 generation that have grown for one month, extract the DNA from the T2 generation transformed plants and the leaves of wild-type Arabidopsis thaliana (col-0), and use the following primers to amplify:

其中，目标基因Zm00001d005920引物序列如下：Among them, the primer sequence of the target gene Zm00001d005920 is as follows:

Forward Primer1：5'-CAGCTGGAAATTTGAGTTTTGA-3'；Forward Primer1: 5'-CAGCTGGAAATTTGAGTTTTGA-3';

Reverse Primer1：5'-AAGTTGTAGTAGCCTCCGTGGA-3'；Reverse Primer1: 5'-AAGTTGTAGTAGCCTCCGTGGA-3';

拟南芥内参引物序列如下：The Arabidopsis internal reference primer sequences are as follows:

Tublin-F：5'-ATCCGTGAAGAGTACCCAGAT-3'；Tublin-F: 5'-ATCCGTGAAGAGTACCCAGAT-3';

Tublin-R 5'-AAGAACCATGCACTCATCAGC-3'；Tublin-R 5'-AAGAACCATGCACTCATCAGC-3';

结果如图4中a所示，可以看出，与野生型拟南芥(WT)相比，基因Zm00001d005920的引物扩增结果说明已获得到三个纯合转Zm00001d005920基因拟南芥T2代OE6、OE10和OE18过表达株系。The results are shown in a in Figure 4. It can be seen that, compared with the wild-type Arabidopsis (WT), the primer amplification results of the gene Zm00001d005920 indicate that three homozygous Zm00001d005920 transgenic Arabidopsis T2 generation OE6, OE10 and OE18 overexpressing lines.

上述扩增体系如表8。The above amplification system is shown in Table 8.

表8：扩增体系Table 8: Amplification system

PCR扩增程序如下：The PCR amplification procedure is as follows:

2.2、过表达Zm00001d005920导致拟南芥抗旱性增强2.2. Overexpression of Zm00001d005920 leads to enhanced drought resistance in Arabidopsis

对纯合转Zm00001d005920的拟南芥T2株系OE6、OE10和OE18种子播种，收获纯合转Zm00001d005920的拟南芥OE6、OE10和OE18的T3代种子。对获得的T3代转基因拟南芥株系OE6、OE10和OE18和野生型col-0(WT)进行土壤干旱胁迫下的存活率鉴定。具体如下：The seeds of the homozygous Zm00001d005920 Arabidopsis thaliana T2 lines OE6, OE10 and OE18 were sown, and the T3 seeds of the homozygous Zm00001d005920 Arabidopsis thaliana OE6, OE10 and OE18 were harvested. The obtained T3 generation transgenic Arabidopsis lines OE6, OE10 and OE18 and wild-type col-0 (WT) were subjected to the identification of survival rate under soil drought stress. details as follows:

将纯合转Zm00001d005920的拟南芥OE6、OE10和OE18的T3代种子利用6％的次氯酸钠消毒后，再利用蒸馏水清洗5遍，洗掉种子表面的次氯酸钠，然后播种在MS培养基上培养7d，移栽4株长势相同的幼苗到提前称重并吸饱水的小钵中，生长在22℃(光照16h/黑暗8h)的温室，设置三个重复，每个重复中包含野生型拟南芥和3个OE6、OE10和OE18的T3代株系，野生型和过表达株系分别种植5个小钵。待拟南芥幼苗生长15d后停止浇水，干旱处理23d，复水7d后；统计野生型拟南芥和OE6、OE10和OE18的T2代株系的存活率，试验结果取平均值。The T3 seeds of Arabidopsis OE6, OE10 and OE18, which were homozygous for Zm00001d005920, were sterilized with 6% sodium hypochlorite, then washed with distilled water for 5 times to remove the sodium hypochlorite on the surface of the seeds, and then sown on MS medium for 7 days. Four seedlings with the same growth vigor were transplanted into small pots weighed in advance and saturated with water, grown in a greenhouse at 22°C (light 16h/dark 8h), and three replicates were set, each replicate containing wild-type Arabidopsis thaliana And 3 T3 generation lines of OE6, OE10 and OE18, wild type and overexpression lines were planted in 5 small pots respectively. After Arabidopsis seedlings grew for 15 days, watering was stopped, and after 23 days of drought treatment and 7 days of rehydration, the survival rates of wild-type Arabidopsis and T2 generation lines of OE6, OE10 and OE18 were counted, and the test results were averaged.

结果如图4中b和图4中c所示，图4中b为表型观测，c为存活率统计；可以看出，与野生型拟南芥相比，纯合转Zm00001d005920的拟南芥复水后生存状态优于野生型(图4中b)；统计存活率，与野生型拟南芥相比，纯合转Zm00001d005920的拟南芥存活率显著提高(图4中c)，说明Zm00001d005920基因提高拟南芥抗旱性。The results are shown in b in Figure 4 and c in Figure 4. In Figure 4, b is the phenotype observation, and c is the survival rate statistics; it can be seen that compared with the wild-type Arabidopsis, the homozygous Zm00001d005920 Arabidopsis The survival state after rehydration was better than that of the wild type (b in Figure 4); according to the survival rate, compared with the wild type Arabidopsis, the survival rate of the homozygous Zm00001d005920 Arabidopsis was significantly improved (c in Figure 4), indicating that Zm00001d005920 Genes improve drought resistance in Arabidopsis thaliana.

2.3、过表达Zm00001d005920基因促进拟南芥根系生长2.3. Overexpression of Zm00001d005920 gene promotes root growth of Arabidopsis thaliana

对Zm00001d005920转拟南芥株系OE6、OE10和OE18的T3代植株的根系表型进行调查，具体如下：The root phenotypes of the T3 generation plants of Zm00001d005920 transfected Arabidopsis lines OE6, OE10 and OE18 were investigated, as follows:

将获得的T3代转基因拟南芥株系OE6、OE10和OE18、野生型col-0(WT)种子种植在用于模拟正常水分处理环境的MS培养基上，生长在22℃(光照16h/黑暗8h)的温室7天后，选择生长状况一致的幼苗重新移栽在模拟干旱的含有250mM甘露醇的MS培养基上，再生长7天，统计转基因拟南芥和野生型col-0(WT)在正常水分处理和模拟干旱处理下的根系总长(cm)，试验共设置三个重复，用于统计数据的植株每个株系有20株，结果取平均值。The obtained T3 generation transgenic Arabidopsis lines OE6, OE10 and OE18, and wild-type col-0 (WT) seeds were planted on MS medium used to simulate a normal water treatment environment and grown at 22°C (light 16h/dark). After 7 days in the greenhouse of 8h), the seedlings with the same growth status were selected and re-transplanted on MS medium containing 250mM mannitol to simulate drought for another 7 days. For the total root length (cm) under normal water treatment and simulated drought treatment, three replicates were set up in the experiment, and there were 20 plants in each line used for statistical data, and the results were averaged.

结果如图5所示，图5中a为根系表型观测，图5中b为根系总长度统计；发现三个T3代转基因株系OE6、OE10和OE18在正常水分处理条件下的根系总长度无显著差异，三个T3代转基因株系OE6、OE10和OE18在模拟干旱处理条件下的根系总长度显著高于野生型，说明Zm00001d005920可以促进拟南芥根系的生长发育。The results are shown in Figure 5. In Figure 5, a is the root phenotype observation, and Figure 5 in b is the statistics of the total root length. It was found that the total root length of the three T3 transgenic lines OE6, OE10 and OE18 under normal water treatment conditions There was no significant difference. The total root length of the three T3 transgenic lines OE6, OE10 and OE18 was significantly higher than that of the wild type under simulated drought conditions, indicating that Zm00001d005920 can promote the growth and development of Arabidopsis roots.

实施例3、Zm00001d005920基因转化玉米的功能验证Example 3. Functional verification of Zm00001d005920 gene transformation in maize

3.1、Zm00001d005920基因转化玉米B1043.1. Transformation of Zm00001d005920 gene into maize B104

3.1.1、候选基因植物表达载体的构建3.1.1. Construction of candidate gene plant expression vector

采用实施例2构建的pCAMBIA3301-Zm00001d005920载体。The pCAMBIA3301-Zm00001d005920 vector constructed in Example 2 was used.

3.1.2、重组农杆菌制备3.1.2. Preparation of recombinant Agrobacterium

参照实施例2。Refer to Example 2.

3.1.3、农杆菌侵染玉米3.1.3. Agrobacterium infection of corn

采用农杆菌侵染玉米幼胚的转化方法(王宏伟,梁业红,史振声,等.农杆菌介导玉米幼胚遗传转化体系的研究.玉米科学,2011,19(3):3.)，将上述2制备的重组载体pCAMBIA3301-Zm00001d005920通过上述2制备的重组农杆菌转至野生型玉米B104中，获得T0代转基因玉米。Using the transformation method of Agrobacterium-infected maize young embryos (Wang Hongwei, Liang Yehong, Shi Zhensheng, et al. Research on Agrobacterium-mediated genetic transformation system of maize young embryos. Maize Science, 2011, 19(3): 3.), the above 2 The prepared recombinant vector pCAMBIA3301-Zm00001d005920 was transformed into wild-type maize B104 by the recombinant Agrobacterium prepared in the above 2 to obtain the TO generation transgenic maize.

在T0代拟转基因玉米的叶片上涂抹Basta，叶片能够正常生长的植株(抗性苗)即为T0代转基因植株。转基因T0植株自交，成熟后收获，直至获得T3代转基因种子。Apply Basta on the leaves of the pseudo-transgenic maize of the T0 generation, and the plants (resistant seedlings) whose leaves can grow normally are the transgenic plants of the T0 generation. The transgenic T0 plants were selfed and harvested after maturity until the T3 generation transgenic seeds were obtained.

3.1.4、分子鉴定3.1.4. Molecular identification

在温室种植收获的T3代转基因株系和野生型玉米B104，取叶片，并提取RNA，利用实时荧光定量(qRT-PCR)分析Zm00001d005920基因在转基因过表达株系和B104中的相对表达量，筛选出纯合并且表达量显著高于B104的转基因过表达株系(图6中a)。The harvested T3 generation transgenic lines and wild-type maize B104 were grown in the greenhouse, the leaves were taken, and RNA was extracted, and the relative expression levels of Zm00001d005920 gene in the transgenic overexpression lines and B104 were analyzed by real-time fluorescence quantitative (qRT-PCR), and the screening was carried out. A transgenic overexpression line that was homozygous and whose expression level was significantly higher than that of B104 was obtained (a in Figure 6).

首先提取叶片RNA，再反转成cDNA，然后利用南京诺唯赞生物技术股份有限公司的Taq Pro Universal SYBR qPCR Master Mix进行野生型(B104)和过表达Zm00001d005920株系的相对表达量分析，反应的体系为表9。First, leaf RNA was extracted, and then reversed into cDNA. Then, the relative expression levels of wild-type (B104) and overexpressing Zm00001d005920 lines were analyzed using Taq Pro Universal SYBR qPCR Master Mix from Nanjing Novizan Biotechnology Co., Ltd. The system is in Table 9.

表9：反应体系Table 9: Reaction system

上述反应用到的引物序列如下：The primer sequences used in the above reaction are as follows:

用于扩增内参基因：Used to amplify the reference gene:

GAPGH-F：5'-CCCTTCATCACCACGGACTAC-3'；GAPGH-F: 5'-CCCTTCATCACCACGGACTAC-3';

GAPDH-R：5'-AACCTTCTTGGCACCACCCT-3'；GAPDH-R: 5'-AACCTTCTTGGCACCACCCT-3';

用于扩增Zm00001d005920：Used to amplify Zm00001d005920:

Zm00001d005920-F：5'-CAGCTGGAAATTTGAGTTTTGA-3'；Zm00001d005920-F:5'-CAGCTGGAAATTTGAGTTTTGA-3';

Zm00001d005920-R：5'-AAGTTGTAGTAGCCTCCGTGGA-3'；Zm00001d005920-R: 5'-AAGTTGTAGTAGCCTCCGTGGA-3';

配制好体系后，按照表10的程序进行qPCR反应。After the system was prepared, the qPCR reaction was performed according to the procedure in Table 10.

表10：qPCR反应程序Table 10: qPCR reaction program

反应完成后观察扩增曲线和溶解曲线，剔除掉一些不符合实验标准的数据，符合误差范围内的数据可利用2^-ΔΔCt算法计算相对表达量。本次实验中每个样品均设置有3个生物学重复。After the reaction was completed, the amplification curve and the dissolution curve were observed, and some data that did not meet the experimental standards were eliminated. The data within the error range could be calculated using the 2-^ΔΔCt algorithm. Three biological replicates were set for each sample in this experiment.

结果如图6中a所示，可以看出，与野生型玉米(B104)相比，T3代转基因株系OE2、OE4和OE16的表达量显著提高。The results are shown in a in Figure 6. It can be seen that the expression levels of T3 transgenic lines OE2, OE4 and OE16 are significantly increased compared with wild-type maize (B104).

T3代转基因纯合株系OE2、OE4和OE16命名为T3代转Zm00001d005920玉米OE2、OE4和OE16。T3 generation of transgenic homozygous lines OE2, OE4 and OE16 were named as T3 generation transgenic Zm00001d005920 maize OE2, OE4 and OE16.

3.2、过表达Zm00001d005920导致玉米抗旱性增强3.2. Overexpression of Zm00001d005920 leads to enhanced drought resistance in maize

在土壤干旱胁迫条件下，开展转基因株系和野生型B104的存活率鉴定。首先将T3代转Zm00001d005920玉米OE2、OE4和OE16和野生型玉米(B104)分别播种在穴盘中，待其生长至三叶一心，选取生长状况一致的12株幼苗转移至提前称重一致且为正常水分处理的小屉中，后期不再进行浇水(干旱处理)，干旱处理31d，复水7d后；统计野生型玉米B104(WT)和T3代转Zm00001d005920玉米OE2、OE4和OE16的存活率。试验重复3次，结果取平均值。Under soil drought stress conditions, the survival rate identification of transgenic lines and wild-type B104 was carried out. First, the T3 generation transgenic Zm00001d005920 maize OE2, OE4 and OE16 and wild-type maize (B104) were sown in plug trays, and when they grew to three leaves and one heart, 12 seedlings with the same growth status were selected and transferred to the same weight in advance. In the small trays treated with normal water, no watering was performed in the later stage (drought treatment), after 31 days of drought treatment and 7 days of rehydration; the survival rates of wild-type maize B104 (WT) and T3 generation transgenic Zm00001d005920 maize OE2, OE4 and OE16 were counted . The experiment was repeated 3 times, and the results were averaged.

结果如图6所示，发现在正常水分条件下过表达株系和WT之间没有明显的差别(图6中b)，干旱胁迫31天后，WT和OE2、OE4和OE16恢复浇水，发现过表达株系T3代转Zm00001d005920玉米OE2、OE4和OE16相较于WT有更好的恢复能力且更容易使叶片恢复到正常形态特征(图6中c)。The results are shown in Fig. 6. It was found that there was no obvious difference between the overexpression lines and WT under normal water conditions (b in Fig. 6). Compared with WT, the Zm00001d005920 maize OE2, OE4 and OE16 of the expression line T3 had better recovery ability and easier to restore leaves to normal morphological characteristics (Fig. 6 c).

统计复水7d后T3代转Zm00001d005920玉米OE2、OE4和OE16和WT玉米的存活率(图6中d)，WT在复水后的存活率只有33％，而OE2、OE4和OE16的存活率分别达到了70％、88％、83％，均显著高于WT，进而说明在玉米中过表达Zm00001d005920可积极响应干旱胁迫，并提高玉米的抗旱性。The survival rates of OE2, OE4 and OE16 maize and WT maize in T3 generation after rehydration for 7 days were counted (d in Figure 6). The survival rate of WT after rehydration was only 33%, while the survival rates of OE2, OE4 and OE16 were respectively It reached 70%, 88%, and 83%, which were significantly higher than those of WT, indicating that overexpression of Zm00001d005920 in maize could positively respond to drought stress and improve the drought resistance of maize.

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, without departing from the spirit and scope of the present invention, and without unnecessary experimentation, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. While the invention has been given particular embodiments, it should be understood that the invention can be further modified. In conclusion, in accordance with the principles of the present invention, this application is intended to cover any alterations, uses or improvements of the invention, including changes made using conventional techniques known in the art, departing from the scope disclosed in this application.

序列表sequence listing

<110> 中国农业科学院作物科学研究所<110> Institute of Crop Science, Chinese Academy of Agricultural Sciences

<120> 调控玉米苗期抗旱性的蛋白及其编码基因和应用<120> Proteins regulating drought resistance in maize seedlings and their encoding genes and applications

<160> 3<160> 3

<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0

<210> 1<210> 1

<211> 3232<211> 3232

<212> DNA<212> DNA

<213> 玉米(Zea mays L.)<213> Corn (Zea mays L.)

<400> 1<400> 1

cccaaacagt ccaatcacgg taactaatca ctttgcacga aaacagatta aaacaataaa 60cccaaacagt ccaatcacgg taactaatca ctttgcacga aaacagatta aaacaataaa 60

accaccgcca ccgccacctc gacctcgact cctccgcccc atcgccgtcg tccccgtctc 120accaccgcca ccgccacctc gacctcgact cctccgcccc atcgccgtcg tccccgtctc 120

ctccgccttg ctgtctgtcc gctgctcacc tcccctaaaa ggaggcaccc ccggccattg 180ctccgccttg ctgtctgtcc gctgctcacc tcccctaaaa ggaggcaccc ccggccattg 180

ccgcaccaag ctccccttcc tcgaatgccc caggcccgga caccacgacg ccctgccccg 240ccgcaccaag ctccccttcc tcgaatgccc caggcccgga caccacgacg ccctgccccg 240

tgctcacatg gctagcggcg tcctccaccg ctccgcctcc cgatgtctcc gccctgccgc 300tgctcacatg gctagcggcg tcctccaccg ctccgcctcc cgatgtctcc gccctgccgc 300

cgcgccaacc cccgactccg gcaggggcca cggcgcggtt atggtgagct tctatccgcg 360cgcgccaacc cccgactccg gcaggggcca cggcgcggtt atggtgagct tctatccgcg 360

ctcgctttcg ccgtctcttg gggtttccga ctcgctggga ctccctcttt gcttatctct 420ctcgctttcg ccgtctcttg gggtttccga ctcgctggga ctccctcttt gcttatctct 420

gggttttctc gggtgccgca gatcggtgat tcggggacgc gcggtgttgc tctgcggctg 480gggttttctc gggtgccgca gatcggtgat tcggggacgc gcggtgttgc tctgcggctg 480

ggaggatcta gccggtgagt cgagtcgatc tcctctgtaa tttctgcagc ttctaagttt 540ggaggatcta gccggtgagt cgagtcgatc tcctctgtaa tttctgcagc ttctaagttt 540

ggatcagcga tcaggtcgcg caactgtctg tcttcacgat tcccttgctt ccatcgtgtc 600ggatcagcga tcaggtcgcg caactgtctg tcttcacgat tcccttgctt ccatcgtgtc 600

cggcgcaacc tcccctggtt ccttgagtat ctaaactact tatctagctt ctccgtgcaa 660cggcgcaacc tcccctggtt ccttgagtat ctaaactact tatctagctt ctccgtgcaa 660

taactccaat tgcctcctcg gtggcttact atgtacctta gcttggtgaa ttctgcagcg 720taactccaat tgcctcctcg gtggcttact atgtacctta gcttggtgaa ttctgcagcg 720

caagaaatga gatccaaatt gagatttgag aatttgtatt tctgggggcc gtttagctta 780caagaaatga gatccaaatt gagatttgag aatttgtatt tctgggggcc gtttagctta 780

gtcaactact tggttcaaca gtttagatca acgttccact ttcttgcgat ttgaatttgc 840gtcaactact tggttcaaca gtttagatca acgttccact ttcttgcgat ttgaatttgc 840

agtagcctgt agtacactgt tgtcggatgg aaacactaat ggtagatgga tatatttgtt 900agtagcctgt agtacactgt tgtcggatgg aaacactaat ggtagatgga tatatttgtt 900

ttcctctgtg tggattgaca tattgtaagt gctttaatgt actcacgaac ggttccaatt 960ttcctctgtg tggattgaca tattgtaagt gctttaatgt actcacgaac ggttccaatt 960

taagcctatc ctaaaacatc gctttcagat aactctatat ttattattat cagctaatta 1020taagcctatc ctaaaacatc gctttcagat aactctatat ttattattat cagctaatta 1020

gtggttttca gataagtttt tttttttggg gggggggggg gggggtcgca tccaatttac 1080gtggttttca gataagtttt tttttttggg gggggggggg gggggtcgca tccaatttac 1080

accaattatc tgtagaccta tatagtgttg tgatctccgg acattacata aactcacatg 1140accaattatc tgtagaccta tatagtgttg tgatctccgg acattacata aactcacatg 1140

aattttgcca gccaggattt tgcattttct tgtaccacaa aaacatcgtt gtgatccttc 1200aattttgcca gccaggattt tgcattttct tgtaccacaa aaacatcgtt gtgatccttc 1200

agagatcacc ctggcctcca atccacccag ttcttaatat gtttgggcag aaaattattt 1260agagatcacc ctggcctcca atccacccag ttcttaatat gtttgggcag aaaattattt 1260

aataagatcg tcttctgcag gagagagtta ttatctgtca caatggcctc cagagaccac 1320aataagatcg tcttctgcag gagagagtta ttatctgtca caatggcctc cagagaccac 1320

actggcttga cccgacaact tttggatttt caacatgata caatagatga ggtaggtgca 1380actggcttga cccgacaact tttggatttt caacatgata caatagatga ggtaggtgca 1380

gaacacggtc cattcatgga tttgaaagcg agattcatgg acttcaaaca gagaaactat 1440gaacacggtc cattcatgga tttgaaagcg agattcatgg acttcaaaca gagaaactat 1440

gtgtaagtgg atcatgaagt acaacacata tcattcacct taacgtttca cagttttttt 1500gtgtaagtgg atcatgaagt acaacacata tcattcacct taacgtttca cagttttttt 1500

gacattgctg tatctggaac tatggacagg gagaattttt caaattacca aaatcttgct 1560gacattgctg tatctggaac tatggacagg gagaattttt caaattacca aaatcttgct 1560

gaacagcaaa caccaaaggt acacgataat gactttgata caaaataatt ttaatattgg 1620gaacagcaaa caccaaaggt acacgataat gactttgata caaaataatt ttaatattgg 1620

cattatgagc cttacgggat tacttttggt tttctgttct tgtgtgcagt tcatggtgat 1680cattatgagc cttacgggat tacttttggt tttctgttct tgtgtgcagt tcatggtgat 1680

tgcttgtgct gactccagag tctgccctac cgctgttttg gggtttcagc ctggcgaagc 1740tgcttgtgct gactccagag tctgccctac cgctgttttg gggtttcagc ctggcgaagc 1740

atttacggtt cgtaatgtgg cgaatttggt accaccatat gaggtattac aatttttttg 1800atttacggtt cgtaatgtgg cgaatttggt accaccatat gaggtattac aatttttttg 1800

catctttagg ctgtgattga actctcaagc aatgcattcc tttgtggaag tactgacagt 1860catctttagg ctgtgattga actctcaagc aatgcattcc tttgtggaag tactgacagt 1860

aaacaaaatg ttgcaatacc agcatgaggg atcagagact agtgcagcac tagagtttgc 1920aaacaaaatg ttgcaatacc agcatgaggg atcagagact agtgcagcac tagagtttgc 1920

tatcaataca ctcgaggtat aagtttactg tgtgtttcat taatgatttg tttcttttat 1980tatcaataca ctcgaggtat aagtttactg tgtgtttcat taatgatttg tttcttttat 1980

cctatttatt ccttttttgc tgaagtaaca taaaagcatt cttgagactg gcacatttgc 2040cctatttatt ccttttttgc tgaagtaaca taaaagcatt cttgagactg gcacatttgc 2040

cttcatttgc tcaggtagag aatgtactgg tggtaggcca cagtaggtgt gggggcatcc 2100cttcatttgc tcaggtagag aatgtactgg tggtaggcca cagtaggtgt gggggcatcc 2100

aggcactaat gagcatgaaa gatgattcaa cgtccgggta aaagttctag ccatttcttc 2160aggcactaat gagcatgaaa gatgattcaa cgtccgggta aaagttctag ccatttcttc 2160

attacatgtt cttttgttgt ccgtttagac tttagactag tctctgaatt tctgatttca 2220attacatgtt cttttgttgt ccgtttagac tttagactag tctctgaatt tctgatttca 2220

tttattttat cgacacagaa gcttcattaa aaactgggtt tcaattggga agagcgcaag 2280tttattttat cgacacagaa gcttcattaa aaactgggtt tcaattggga agagcgcaag 2280

gttaagcaca agagctgcag ctggaaattt gagttttgac atgcagtgca aacattgtga 2340gttaagcaca agagctgcag ctggaaattt gagttttgac atgcagtgca aacattgtga 2340

aaaggtgatt cttgggtgat gtgtgatcac gagccttaac ttttacaaaa atattgccta 2400aaaggtgatt cttgggtgat gtgtgatcac gagccttaac ttttacaaaa atattgccta 2400

atgtagtgca caggatcaat caattccatg gctttcacta attcagattc atctcaagat 2460atgtagtgca caggatcaat caattccatg gctttcacta attcagattc atctcaagat 2460

cttgcctgct catcctattg ttcttccctg ttcatagtct ttatctatac tagggttaga 2520cttgcctgct catcctattg ttcttccctg ttcatagtct ttatctatac tagggttaga 2520

tatggatgtc tcgtactagt tatacaattc aaatttatct tggcaaatat gttggttttc 2580tatggatgtc tcgtactagt tatacaattc aaatttatct tggcaaatat gttggttttc 2580

ttttgttcaa ggtttatgta gtcatagtat gatattacta agcacaacca ctcacggtgt 2640ttttgttcaa ggtttatgta gtcatagtat gatattacta agcacaacca ctcacggtgt 2640

ctgacatgta ttgatttctt ttttttgtta caggagtcaa taaatagctc ggtgtctgac 2700ctgacatgta ttgatttctt ttttttgtta caggagtcaa taaatagctc ggtgtctgac 2700

atgtattgat ttcttccttt tgttacagga atcaataaat agctctctgt tgaacttgtt 2760atgtattgat ttcttccttt tgttacagga atcaataaat agctctctgt tgaacttgtt 2760

aacataccca tggatagaga agagggtgaa tgaaggtact ttgaatctcc acggaggcta 2820aacataccca tggatagaga agagggtgaa tgaaggtact ttgaatctcc acggaggcta 2820

ctacaacttc gttgattgca catttgagaa atggacatta ttgtaccgtg aagggttgga 2880ctacaacttc gttgattgca catttgagaa atggacatta ttgtaccgtg aagggttgga 2880

aggtggaagc aagtacgcta taaagaatag gtccacctgg tcttgaccaa tgaaggcctc 2940aggtggaagc aagtacgcta taaagaatag gtccacctgg tcttgaccaa tgaaggcctc 2940

atttacttgg gtaaatttca ctctgcgccc tgcagttaag catggttttg ctttgccact 3000atttacttgg gtaaatttca ctctgcgccc tgcagttaag catggttttg ctttgccact 3000

acgctgtgca tttccgtcgc acctttgctc cattgtggta ttgatgtcct gcagaagcga 3060acgctgtgca tttccgtcgc acctttgctc cattgtggta ttgatgtcct gcagaagcga 3060

gtccctgtat caaataactg tgtccattgc gatgcatggc actatggcac tagcatgtca 3120gtccctgtat caaataactg tgtccattgc gatgcatggc actatggcac tagcatgtca 3120

gcttgctgta gtattgcata ctcacgggca ggtgcatcgg cattgtagtt gtagcagcta 3180gcttgctgta gtattgcata ctcacgggca ggtgcatcgg cattgtagtt gtagcagcta 3180

ttgtttgata cgtacaggaa ctctccactg tggaagcaca aagccacaat gg 3232ttgtttgata cgtacaggaa ctctccactg tggaagcaca aagccacaat gg 3232

<210> 2<210> 2

<211> 915<211> 915

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 2<400> 2

atggctagcg gcgtcctcca ccgttccgcc tcccgatgtc tccgccctgc cgccgcgcca 60atggctagcg gcgtcctcca ccgttccgcc tcccgatgtc tccgccctgc cgccgcgcca 60

aaccccgact ccggccgggg ccacggcgcg gttatgatcg gtgattcggg gacgcgcggt 120aaccccgact ccggccgggg ccacggcgcg gttatgatcg gtgattcggg gacgcgcggt 120

gttgctctgc ggatgggagg atctagccgg agagagttat tatctgtcac aatggcctcc 180gttgctctgc ggatgggagg atctagccgg agagagttat tatctgtcac aatggcctcc 180

agagatcaca ctggcttgac ccgacaactt ttggattttc aacatgatac aatagatgag 240agagatcaca ctggcttgac ccgacaactt ttggattttc aacatgatac aatagatgag 240

gtaggtgcag aacacggtcc attcatggat ttgaaagcga gattcatgga cttcaaacag 300gtaggtgcag aacacggtcc attcatggat ttgaaagcga gattcatgga cttcaaacag 300

agaaactatg tggagaattt ttcaaattac caaaatcttg ctgaacagca aacacccaag 360agaaactatg tggagaattt ttcaaattac caaaatcttg ctgaacagca aacacccaag 360

ttcatggtga ttgcttgtgc tgactccaga gtctgcccta ccgctgtttt ggggtttcag 420ttcatggtga ttgcttgtgc tgactccaga gtctgcccta ccgctgtttt ggggtttcag 420

cctggcgaag catttacggt tcgtaatgtg gcgaatttgg taccaccata tgagcatgag 480cctggcgaag catttacggt tcgtaatgtg gcgaatttgg taccaccata tgagcatgag 480

ggatcagaga ctagtgcagc actagagttt gctatcaata cactcgaggt agagaatgta 540ggatcagaga ctagtgcagc actagagttt gctatcaata cactcgaggt agagaatgta 540

ctggtggtag gccacagtag gtgtggaggc atccaggcac taatgagcat gaaagatgat 600ctggtggtag gccacagtag gtgtggaggc atccaggcac taatgagcat gaaagatgat 600

tcaacgtccg gaagcttcat taaaaactgg gtttcaattg ggaagagcgc aaggttaagc 660tcaacgtccg gaagcttcat taaaaactgg gtttcaattg ggaagagcgc aaggttaagc 660

acaagagctg cagctggaaa tttgagtttt gacatgcagt gcaaacattg tgaaaaggaa 720acaagagctg cagctggaaa tttgagtttt gacatgcagt gcaaacattg tgaaaaggaa 720

tcaataaata gctctctgtt gaacttgtta acatacccat ggatagagaa gagggtgaat 780tcaataaata gctctctgtt gaacttgtta acatacccat ggatagagaa gagggtgaat 780

gaaggtactt tgaatctcca cggaggctac tacaacttta ttgattgcac atttgagaaa 840gaaggtactt tgaatctcca cggaggctac tacaacttta ttgattgcac atttgagaaa 840

tggacattat tgtaccgtga ggggttggaa ggtggaagca agtacgctat aaagaatagg 900tggacattat tgtaccgtga ggggttggaa ggtggaagca agtacgctat aaagaatagg 900

tccacctggt cttga 915tccacctggt cttga 915

<210> 3<210> 3

<211> 304<211> 304

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 3<400> 3

Met Ala Ser Gly Val Leu His Arg Ser Ala Ser Arg Cys Leu Arg ProMet Ala Ser Gly Val Leu His Arg Ser Ala Ser Arg Cys Leu Arg Pro

1 5 10 151 5 10 15

Ala Ala Ala Pro Asn Pro Asp Ser Gly Arg Gly His Gly Ala Val MetAla Ala Ala Pro Asn Pro Asp Ser Gly Arg Gly His Gly Ala Val Met

20 25 30 20 25 30

Ile Gly Asp Ser Gly Thr Arg Gly Val Ala Leu Arg Met Gly Gly SerIle Gly Asp Ser Gly Thr Arg Gly Val Ala Leu Arg Met Gly Gly Ser

35 40 45 35 40 45

Ser Arg Arg Glu Leu Leu Ser Val Thr Met Ala Ser Arg Asp His ThrSer Arg Arg Glu Leu Leu Ser Val Thr Met Ala Ser Arg Asp His Thr

50 55 60 50 55 60

Gly Leu Thr Arg Gln Leu Leu Asp Phe Gln His Asp Thr Ile Asp GluGly Leu Thr Arg Gln Leu Leu Asp Phe Gln His Asp Thr Ile Asp Glu

65 70 75 8065 70 75 80

Val Gly Ala Glu His Gly Pro Phe Met Asp Leu Lys Ala Arg Phe MetVal Gly Ala Glu His Gly Pro Phe Met Asp Leu Lys Ala Arg Phe Met

85 90 95 85 90 95

Asp Phe Lys Gln Arg Asn Tyr Val Glu Asn Phe Ser Asn Tyr Gln AsnAsp Phe Lys Gln Arg Asn Tyr Val Glu Asn Phe Ser Asn Tyr Gln Asn

100 105 110 100 105 110

Leu Ala Glu Gln Gln Thr Pro Lys Phe Met Val Ile Ala Cys Ala AspLeu Ala Glu Gln Gln Thr Pro Lys Phe Met Val Ile Ala Cys Ala Asp

115 120 125 115 120 125

Ser Arg Val Cys Pro Thr Ala Val Leu Gly Phe Gln Pro Gly Glu AlaSer Arg Val Cys Pro Thr Ala Val Leu Gly Phe Gln Pro Gly Glu Ala

130 135 140 130 135 140

Phe Thr Val Arg Asn Val Ala Asn Leu Val Pro Pro Tyr Glu His GluPhe Thr Val Arg Asn Val Ala Asn Leu Val Pro Pro Tyr Glu His Glu

145 150 155 160145 150 155 160

Gly Ser Glu Thr Ser Ala Ala Leu Glu Phe Ala Ile Asn Thr Leu GluGly Ser Glu Thr Ser Ala Ala Leu Glu Phe Ala Ile Asn Thr Leu Glu

165 170 175 165 170 175

Val Glu Asn Val Leu Val Val Gly His Ser Arg Cys Gly Gly Ile GlnVal Glu Asn Val Leu Val Val Gly His Ser Arg Cys Gly Gly Ile Gln

180 185 190 180 185 190

Ala Leu Met Ser Met Lys Asp Asp Ser Thr Ser Gly Ser Phe Ile LysAla Leu Met Ser Met Lys Asp Asp Ser Thr Ser Gly Ser Phe Ile Lys

195 200 205 195 200 205

Asn Trp Val Ser Ile Gly Lys Ser Ala Arg Leu Ser Thr Arg Ala AlaAsn Trp Val Ser Ile Gly Lys Ser Ala Arg Leu Ser Thr Arg Ala Ala

210 215 220 210 215 220

Ala Gly Asn Leu Ser Phe Asp Met Gln Cys Lys His Cys Glu Lys GluAla Gly Asn Leu Ser Phe Asp Met Gln Cys Lys His Cys Glu Lys Glu

225 230 235 240225 230 235 240

Ser Ile Asn Ser Ser Leu Leu Asn Leu Leu Thr Tyr Pro Trp Ile GluSer Ile Asn Ser Ser Leu Leu Asn Leu Leu Thr Tyr Pro Trp Ile Glu

245 250 255 245 250 255

Lys Arg Val Asn Glu Gly Thr Leu Asn Leu His Gly Gly Tyr Tyr AsnLys Arg Val Asn Glu Gly Thr Leu Asn Leu His Gly Gly Tyr Tyr Asn

260 265 270 260 265 270

Phe Ile Asp Cys Thr Phe Glu Lys Trp Thr Leu Leu Tyr Arg Glu GlyPhe Ile Asp Cys Thr Phe Glu Lys Trp Thr Leu Leu Tyr Arg Glu Gly

275 280 285 275 280 285

Leu Glu Gly Gly Ser Lys Tyr Ala Ile Lys Asn Arg Ser Thr Trp SerLeu Glu Gly Gly Ser Lys Tyr Ala Ile Lys Asn Arg Ser Thr Trp Ser

290 295 300 290 295 300

Claims (10)

Translated fromChinese

1.蛋白质或调控所述蛋白质编码基因表达的物质或调控所述蛋白质活性或含量的物质的应用，其特征在于：所述应用为下述任一种：1. the application of protein or the material that regulates the expression of the protein-encoding gene or the material that regulates the activity or content of the protein, characterized in that: the application is any of the following:A1)、蛋白质或调控所述蛋白质编码基因表达的物质或调控所述蛋白质活性或含量的物质在调控植物抗逆性中的应用；A1), the application of a protein or a substance regulating the expression of the protein-encoding gene or a substance regulating the activity or content of the protein in regulating plant stress resistance;A2)、蛋白质或调控所述蛋白质编码基因表达的物质或调控所述蛋白质活性或含量的物质在制备调控植物抗逆性的产品中的应用；A2), the application of a protein or a substance that regulates the expression of the protein-encoding gene or a substance that regulates the activity or content of the protein in the preparation of a product for regulating plant stress resistance;A3)、蛋白质或调控所述蛋白质编码基因表达的物质或调控所述蛋白质活性或含量的物质在培育耐旱植物中的应用；A3), the application of protein or a substance that regulates the expression of the protein-encoding gene or a substance that regulates the activity or content of the protein in cultivating drought-tolerant plants;A4)、蛋白质或调控所述蛋白质编码基因表达的物质或调控所述蛋白质活性或含量的物质在制备培育耐旱植物的产品中的应用；A4), the application of a protein or a substance that regulates the expression of the protein-encoding gene or a substance that regulates the activity or content of the protein in the preparation of a product for cultivating drought-tolerant plants;A5)、蛋白质或调控所述蛋白质编码基因表达的物质或调控所述蛋白质活性或含量的物质在植物育种中的应用；A5), the application of protein or the substance that regulates the expression of the protein-encoding gene or the substance that regulates the activity or content of the protein in plant breeding;所述蛋白质为如下D1)、D2)或D3)：Said protein is D1), D2) or D3) as follows:D1)、氨基酸序列是SEQ ID No.3的蛋白质；D1), the amino acid sequence is the protein of SEQ ID No.3;D2)、SEQ ID No.3所示的氨基酸序列经过一个以上氨基酸残基的取代和/或缺失和/或添加得到的与D1)所示的蛋白质具有80％以上的同一性且与植物抗逆性相关的蛋白质；The amino acid sequence shown in D2) and SEQ ID No.3 has more than 80% identity with the protein shown in D1) and is resistant to plant stress, obtained by substitution and/or deletion and/or addition of one or more amino acid residues sex-related proteins;D3)、在D1)或D2)的N端和/或C端连接标签得到的融合蛋白质。D3), a fusion protein obtained by linking a tag at the N-terminus and/or C-terminus of D1) or D2).2.根据权利要求1所述的应用，其特征在于：所述蛋白质来源于玉米。2. The application according to claim 1, wherein the protein is derived from corn.3.根据权利要求1或2所述的应用，其特征在于：所述调控所述蛋白质编码基因表达的物质或调控所述蛋白质活性或含量的物质为提高或上调所述蛋白质编码基因表达的物质或提高或上调所述蛋白质活性或含量的物质，所述调控植物抗逆性为提高所述植物的抗逆性。3. The application according to claim 1 or 2, wherein: the substance that regulates the expression of the protein-coding gene or the substance that regulates the activity or content of the protein is a substance that improves or up-regulates the expression of the protein-coding gene Or a substance that increases or up-regulates the activity or content of the protein, and the regulation of plant stress resistance is to increase the stress resistance of the plant.4.根据权利要求1-3中任一项所述的应用，其特征在于：所述调控所述蛋白质编码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B1)至B7)中的任一种：4. The application according to any one of claims 1-3, characterized in that: the substance that regulates the expression of the protein-coding gene or the substance that regulates the activity or content of the protein is a biological material, and the biological material is any of the following B1) to B7):B1)、编码权利要求1中所述蛋白质的核酸分子；B1), a nucleic acid molecule encoding the protein described in claim 1;B2)、含有B1)所述核酸分子的表达盒；B2), the 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；B5), a transgenic plant cell line containing the nucleic acid molecule of B1), or a transgenic plant cell line containing the expression cassette of B2);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；B6), a transgenic plant tissue containing the nucleic acid molecule of B1), or a transgenic plant tissue containing the expression cassette of B2);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。B7), a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2).5.根据权利要求4所述的应用，其特征在于：B1)所述核酸分子为如下b1)至b3)任一项所示的DNA分子：5. application according to claim 4 is characterized in that: B1) described nucleic acid molecule is the DNA molecule shown in any one of following b1) to b3):b1)、编码链的编码序列是SEQ ID No.2所示的DNA分子；b1), the coding sequence of the coding strand is the DNA molecule shown in SEQ ID No.2;b2)、编码链的核苷酸序列是SEQ ID No.1所示的DNA分子；b2), the nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No.1;b3)、与b1)或b2)限定的核苷酸序列具有80％或80％以上同一性，且编码权利要求1中所述蛋白质的DNA分子。b3), a DNA molecule having 80% or more identity with the nucleotide sequence defined in b1) or b2) and encoding the protein of claim 1.6.根据权利要求1-5中任一项所述的应用，其特征在于：所述植物为下述任一种：6. according to the application described in any one of claim 1-5, it is characterized in that: described plant is following any one:P1)、单子叶植物；P1), monocotyledonous plants;P2)、禾本目植物；P2), Gramineae;P3)、禾本科植物；P3), Poaceae;P4)、玉蜀黍属植物；P4), Zea mays;P5)、玉米；P5), corn;P6)、双子叶植物；P6), dicotyledonous plants;P7)、罂粟目植物；P7), Poppy plants;P8)、十字花科植物；P8), cruciferous plants;P9)、鼠耳芥属植物P9), ArabidopsisP10)、拟南芥。P10), Arabidopsis.7.权利要求1或2所述应用中的所述蛋白质或权利要求4或5所述应用中的所述生物材料。7. The protein in the use of claim 1 or 2 or the biomaterial in the use of claim 4 or 5.8.一种调控植物抗逆性的方法，其特征在于：所述方法包括通过调控权利要求1中所述蛋白编码基因的表达或调控所述蛋白的活性或含量，来调控植物植物抗逆性。8. A method for regulating plant stress resistance, characterized in that: the method comprises regulating the expression of the protein-encoding gene described in claim 1 or regulating the activity or content of the protein to regulate plant stress resistance .9.根据权利要求8所述的方法，其特征在于：所述方法包括向受体植物中导入权利要求4或5所述应用中B1)所述的编码基因，促进或提高权利要求1中所述蛋白编码基因的表达或权利要求1中所述蛋白活性或含量，得到植物抗逆性高于所述受体植物的目的植物。9. The method according to claim 8, characterized in that: the method comprises introducing the coding gene described in B1) in the application of claim 4 or 5 into the recipient plant, promoting or improving the coding in claim 1. The expression of the protein-encoding gene or the activity or content of the protein in claim 1 can obtain a target plant whose plant stress resistance is higher than that of the recipient plant.10.一种培育抗旱性植物的方法，包括上调或促进或提高目的植物中权利要求1或2所述蛋白质的编码基因的表达量，得到抗旱性植物，所述抗旱性植物的耐旱性高于所述目的植物。10. A method for cultivating drought-resistant plants, comprising up-regulating or promoting or improving the expression level of the gene encoding the protein of claim 1 or 2 in the plant of interest, to obtain drought-resistant plants, the drought-resistant plants of which are high. in the plant of interest.

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