阅读说明：本技术 大豆sHSP26基因及其应用 (Soybean sHSP26 gene and application thereof ) 是由 刘思言 关淑艳 曲静 姚丹 刘慧婧 江源 李广隆 鲁中爽 王蕊 刘金凤 胡绍旺 于 2019-10-31 设计创作，主要内容包括：本发明提供一种大豆sHSP26基因及其应用,属于植物基因工程领域。该基因的核苷酸序列如SEQ ID NO.1所示。本发明利用RT-PCR技术克隆了大豆sHSP26,并对该基因的基本理化性质和结构域等进行了生物信息学分析,并同时首次成功构建了sHSP26基因的植物超表达载体和CRISPR载体,经遗传转化获得转基因后代株系,对转基因后代的生理生化指标和农艺性状进行统计和分析,结果显示转超表达载体的大豆株系的SOD和POD活性、PRO含量均高于对照植株,MDA含量低于对照植株,尤其在干旱胁迫后趋势更加明显,说明该基因的超量表达可以提高转基因大豆的抗旱能力。(the invention provides a soybean sHSP26 gene and application thereof, belonging to the field of plant genetic engineering. The nucleotide sequence of the gene is shown in SEQ ID NO. 1. According to the invention, the soybean sHSP26 is cloned by using an RT-PCR technology, bioinformatics analysis is carried out on the basic physicochemical property, the structural domain and the like of the gene, a plant over-expression vector and a CRISPR vector of the sHSP26 gene are successfully constructed for the first time, a transgenic progeny plant is obtained through genetic transformation, statistics and analysis are carried out on physiological and biochemical indexes and agronomic characters of the transgenic progeny, the result shows that the SOD activity, POD activity and PRO content of the soybean plant transformed with the over-expression vector are higher than those of a control plant, the MDA content is lower than that of the control plant, particularly, the trend is more obvious after drought stress, and the over-expression of the gene can improve the drought resistance of the transgenic soybean.)

1. A soybean sHSP26 gene is characterized in that the nucleotide sequence is shown in SEQ ID NO. 1.

2. An amino acid sequence encoding the soybean sHSP26 gene of claim 1, which is represented by SEQ ID No. 2.

3. The plant overexpression vector containing the soybean sHSP26 gene of claim 1, wherein the overexpression vector is named as pCAMBIA3301-sHSP 26.

4. A plant gene editing vector containing the soybean sHSP26 gene of claim 1, wherein the gene editing vector is named CRISPR-sHSP26 gene.

5. The use of the soybean sHSP26 gene of claim 1 in drought resistance of plants.

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to a soybean sHSP26 gene and application thereof.

Background

soybean (Glycine max (L.) Merr.) is an important crop used in both oil and high-protein food and feed in the world, and has wide application in the fields of food, medicine, industry, animal husbandry and the like. The soybean is extremely sensitive to drought, the yield is closely related to the number of pods, the number of grains per pod, the weight of hundreds of grains and the like, and the soybean seeds are formed by ovary development, so the development stage of the soybean ovary is a key period for determining the yield of the soybean, and the yield of the soybean can be seriously influenced if the soybean suffers from stress such as drought and the like in the period, so that the method has very important significance for finding the gene which is related to the development of the soybean ovary and can improve the drought resistance of the soybean and obtaining a new drought-resistant transgenic soybean strain. The experiment clones the screened differential expression gene sHSP26 and further researches the gene function through the overexpression and CRISPR/Cas9 technology on the basis of sequencing of a soybean drought-resistant mutant (TB18) seven-leaf stage unpolished ovary transcriptome, and lays a foundation for creating a new strain of drought-resistant transgenic soybean through a genetic engineering technology.

The generation of drought seriously affects the yield and quality of crops, and the cultivation of high-quality drought-resistant crop varieties is very important. The molecular biology approach is applied to the research of crop drought resistance mechanism, the drought resistance related gene is cloned, and the cultivation of a new drought-resistant transgenic crop variety becomes an important target in the research of crop breeding.

In 2007, wheat protein phosphatase 2A catalytic subunit genes were cloned in xu heavily yi, and bioinformatics analysis and function research were carried out on the genes. Researches show that the protein phosphatase 2A catalytic subunit gene is mainly expressed in cell nucleus and cytoplasm, and the transgenic tobacco over-expressing the protein phosphatase 2A catalytic subunit gene has higher drought resistance than a control plant under drought stress. In 2009, the national security research finds that the drought resistance and salt tolerance of transgenic plants over expressing GmUBC2 and GmPK genes are obviously higher than those of control plants. In 2010, Penghui research finds that the expression of CarNAC3 gene can improve the drought tolerance of Arabidopsis plants. In 2010, a heat shock transcription factor CarHSFB2 is separated from chickpea by a comet, and the drought resistance and heat resistance of an arabidopsis plant transformed with the gene are obviously improved. In 2015, GhCBF3 gene is separated from cotton by Mallotus philippinarum, and researches show that the drought resistance and salt tolerance of Arabidopsis with over-expression of the gene are higher than those of control plants.

Disclosure of Invention

The invention aims to provide a soybean sHSP26 gene and application thereof, and the expression of the sHSP26 gene can improve the drought resistance of soybeans.

In order to realize the purpose, the invention adopts the following technical scheme:

the invention firstly provides a soybean sHSP26 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.

The invention also provides an amino acid sequence of the soybean sHSP26 gene, which is shown in SEQ ID NO. 2.

The invention also provides a plant overexpression vector containing the soybean sHSP26 gene, and the overexpression vector is named as pCAMBIA3301-sHSP 26.

The invention also provides a plant gene editing vector containing the soybean sHSP26 gene, wherein the gene editing vector is named as a CRISPR-sHSP26 gene.

the invention also provides application of the soybean sHSP26 gene in drought resistance of plants.

The invention has the advantages of

The invention firstly provides a soybean sHSP26 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1. According to the invention, the soybean sHSP26 is cloned by using an RT-PCR technology, bioinformatics analysis is carried out on the basic physicochemical property, the structural domain and the like of the gene, a plant over-expression vector and a CRISPR vector of the sHSP26 gene are successfully constructed for the first time, a transgenic progeny plant is obtained through genetic transformation, statistics and analysis are carried out on physiological and biochemical indexes and agronomic characters of the transgenic progeny, the result shows that the SOD activity, POD activity and PRO content of the soybean plant transformed with the over-expression vector are higher than those of a control plant, the MDA content is lower than that of the control plant, particularly, the trend is more obvious after drought stress, and the over-expression of the gene can improve the drought resistance of the transgenic soybean.

Drawings

FIG. 1 is a schematic diagram of the T-DNA structure of plant over-expression vector pCAMBIA3301-sHSP 26;

FIG. 2 is an RT-PCR electrophoresis picture of sHSP26 gene. M: DL2000 molecular weight standards 1-4: RT-PCR products;

FIG. 3 is a PCR identification electrophoresis of cloning vectors, M: DL2000 molecular weight standard 1: negative controls 2-7: PCR product a: bacterial liquid PCR b: carrying out plasmid PCR;

FIG. 4 is a double restriction enzyme digestion verification detection map of the cloning vector; m: DL2000 molecular weight standard 1: cloning vector plasmid 2-3: performing enzyme digestion on the product;

FIG. 5 is a comparison chart of sequencing results;

FIG. 6 is a diagram of analysis of protein conserved domains;

FIG. 7 is a graph of hydropathic and hydrophobic analysis;

FIG. 8 is a signal peptide prediction graph;

FIG. 9 is a coiled coil prediction graph;

FIG. 10 is a model of the tertiary structure of sHSP26 protein;

FIG. 11 shows the PCR identification of the overexpression vector pCAMBIA3301-sHSP26, M: DL2000 molecular weight standard 1: negative controls 2-7: PCR products;

FIG. 12 shows the restriction enzyme identification of the overexpression vector pCAMBIA3301-sHSP26, M: DL2000 molecular weight standard 1: overexpression vector plasmids 2-3: performing enzyme digestion on the product;

FIG. 13 is a comparison of sequencing results of overexpression vectors;

Fig. 14 is an electrophoresis diagram of CRISPR-sHSP26 vector PCR identification, M: DL2000 molecular weight standard 1: negative controls 2-7: PCR products;

FIG. 15 is a comparison graph of sequencing results of CRISPR-sHSP26 vector;

FIG. 16 is a PCR identification chart of Agrobacterium tumefaciens liquid, M: DL2000 molecular weight standard 1: negative controls 2-6: PCR product, a: the overexpression vector pCAMBIA3301-sHSP26 bacterial liquid PCR b: PCR of CRISPR-sHSP26 carrier bacterial liquid;

FIG. 17 is a PCR assay of transgenic overexpression vector pCAMBIA3301-sHSP26 plants, M: DL2000 molecular weight standard 1: positive control 2: negative controls 3-12: PCR product, a: t is₀Generation detection result b: t is₁and (c) generation detection result: t is₂generating a detection result;

Fig. 18 is a PCR detection map of a CRISPR-sHSP26 transformed vector plant, M: DL2000 molecular weight standard 1: positive control 2: negative controls 3-15: PCR product, a: t is₀generation detection result b: t is₁and (c) generation detection result: t is₂generating a detection result;

FIG. 19 is a Southern blot assay of transgenic plants, M: southern standard molecular weight +: positive control-: negative controls 1-4: t is₂Transforming plants;

FIG. 20 is a graph showing the relative expression amounts of sHSP26 gene;

FIG. 21 is a graph showing detection of superoxide dismutase activity;

FIG. 22 is a peroxidase activity assay;

FIG. 23 is a graph showing the results of detecting the malonaldehyde content;

FIG. 24 is a graph showing the results of measurement of proline content

Detailed Description

The soybean materials used were Jinong 18(JN18) and Jinong 18 drought-resistant mutant (TB18), both provided by the plant Biotechnology center of Jilin agriculture university.

the used molecular biological reagent is a RNAiso plus kit and T₄Ligase, pMD-18T Vector, DL2000 Marker and the like are purchased from Bao bioengineering (Dalian) Co., Ltd., All-in-One qPCR Kit is purchased from GeneCopoeia Co., Ltd., plasmid minium extraction Kit is purchased from Kangji century Biotech Co., Ltd., southern blot digoxin Kit is purchased from Roche, CRISPR/Cas Vector construction Kit is purchased from Baige Gene technology (Jiangsu) Co., Ltd., and detection Kit of superoxide dismutase and the like is purchased from Suzhou Keming Biotechnology Ltd. Consumables such as centrifuge tube and pipette tip are supplied by Changchun Feikai biological Co Ltd, and biochemical reagents such as isopropanol are purchased from Changchun Ci Jintai Co Ltd