CN109536515B

CN109536515B - Glycine protein-rich gene of soybean plant and application thereof

Info

Publication number: CN109536515B
Application number: CN201910001346.6A
Authority: CN
Inventors: 王丕武; 王楠; 王鑫雨; 曲静; 吴楠; 薄晓雪; 关淑艳
Original assignee: Jilin Agricultural University
Current assignee: Jilin Agricultural University
Priority date: 2019-01-02
Filing date: 2019-01-02
Publication date: 2022-04-08
Anticipated expiration: 2039-01-02
Also published as: CN109536515A

Abstract

The invention relates to the field of biotechnology, in particular to a glycine-rich protein gene of a soybean plant and application thereof, wherein the nucleotide sequence of the glycine-rich protein gene of the soybean plant is shown as SEQ ID No.1, the invention lays a foundation for further understanding the molecular mechanism of GRP gene participating in adverse reactions such as drought and the like, a GRP overexpression vector plant has stronger drought resistance compared with CK, the significance of overground fresh weight and overground dry weight is more obvious, and the drought resistance of the soybean is obviously improved by the change of 5 indexes of plant height, overground fresh weight, underground fresh weight, overground dry weight and underground dry weight.

Description

Glycine protein-rich gene of soybean plant and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a glycine-rich protein gene of a soybean plant and application thereof.

Background

The soybean is a very important food crop and oil crop in China, and the planting area is second to that of rice, corn and wheat and is the fourth place.

Plant glycine-rich proteins (GRPs) are a class of proteins that are composed of highly repetitive sequences of glycine in their constituent structure. In 1986, 150 kinds of plants rich in glycine protein are separated and identified from different plants such as corn, rice, arabidopsis, tobacco and the like, and in 1986, Codit and Meagher separate a gene PtGRP.1 from petunia. In 1987, Reddy and Poovaah extracted GRP from strawberry. The research shows that almost all organisms contain the gene protein, the expression of the plant glycine-rich protein gene GRP has strict development regulation characteristics, and the expression quantity is induced to generate different changes by various environmental factors. The glycine-rich protein gene GRP protein of plants in soybean maps to all lignified cells such as phloem fiber, primary xylem and secondary xylem cell walls. The existing research shows that the factors of drought, cold damage, mechanical damage, abscisic acid (ABA) and the like can induce the increase of the expression level of GRPs genes.

Therefore, the research on glycine-rich protein (GRP) genes of soybean plants by using a molecular biology method, the cloning of the GRP genes in soybeans, the establishment of plant expression vectors and the continuous research on the change of expression levels in different tissues under the drought stress condition have important significance.

The soybean has weak high temperature resistance and drought resistance, and is subjected to abiotic adversity stresses such as drought, high temperature, salt and alkali and the like in the growth period, which is an important influence factor causing yield reduction of the soybean. Drought is one of the most major environmental factors that cause a decrease in soybean yield, and can cause 10% -15% yield loss in the general year and 30% -50% in severe years. Therefore, the method is very important for improving the drought resistance of soybean varieties and cultivating high-yield soybean new varieties with strong drought resistance.

Disclosure of Invention

The invention aims to provide a glycine-rich protein gene of a soybean plant and application thereof, so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme:

a soybean plant rich in glycine protein gene has a nucleotide sequence shown in SEQ ID No. 1.

As a further scheme of the invention: the primers used in the offspring detection of transgenic plants are herbicide resistance (bar),35s promoter, nos terminator, and target Gene (GRP).

As a further scheme of the invention: the nucleotide sequence of the herbicide (bar) resistance is shown in SEQ ID NO.2 and SEQ ID NO. 3.

As a further scheme of the invention: the nucleotide sequence of the 35s promoter is shown in SEQ ID NO.4 and SEQ ID NO. 5.

As a further scheme of the invention: the nucleotide sequence of the noss terminator is shown as SEQ ID NO.6 and SEQ ID NO. 7.

As a further scheme of the invention: the nucleotide sequence of the target Gene (GRP) is shown as SEQ ID NO.8 and SEQ ID NO. 9.

An application of glycine-rich protein gene in soybean plant in improving soybean drought resistance is disclosed.

Compared with the prior art, the invention has the beneficial effects that: the invention lays a foundation for further understanding the molecular mechanism of GRP gene participating in adverse reactions such as drought, the plant of the GRP overexpression vector has stronger drought resistance compared with CK, the significance of the overground fresh weight and the overground dry weight is more obvious, and the drought resistance of the soybean is obviously improved by the change of 5 indexes of plant height, the overground fresh weight, the underground fresh weight, the overground dry weight and the underground dry weight.

Drawings

FIG. 1 is a GRP gene phylogenetic tree;

FIG. 2 is a graph showing the results of detecting the hydrophobicity of a protein of a GRP gene;

FIG. 3 is a diagram showing the results of analysis of the transmembrane region of a GRP gene protein;

FIG. 4 is a diagram of the structure of a plant over-expression vector pCAMBIA-3301-GRP-over expression;

FIG. 5 shows the structure of plant interference expression vector pCAMBIA-3301-GRP-RNAi;

FIG. 6 is a graph showing the results of electrophoresis of a glycine-rich protein Gene (GRP) fragment of a soybean plant (A: pcr product of GRP gene B: PMD18-T-GRP pcr product);

FIG. 7 shows the verification of recombinant plasmid (A: pCAMBIA-3301-35 s-GRP-nos; B: pcr product);

FIG. 8 shows the verification of the PCR product of recombinant plasmid (A: 1-2: pCAMBIA3301-GRP-RNAi cleavage product; B:1,4,7 water; 2-3 sense fragment; 5-6 antisense fragment; 8-9 intron);

FIG. 9 is a PCR map of the GRP-transgenic overexpression vector gene (A:35s promoter; B: terminator Nos; C: screening marker bar);

FIG. 10 is a PCR map of a GRP-transfected interference expression vector gene (A:35s promoter; B: terminator Nos; C: screening marker bar);

FIG. 11 is a Southern blot analysis of GRP overexpression vector transgenic plants;

FIG. 12 is a Southern blot analysis of GRP interference expression vector transgenic plants;

FIG. 13 is a qRT-PCR identification of GRP over-expression vector gene;

FIG. 14 is a qRT-PCR identification chart of the gene of the GRP-transfected interference expression vector.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

1. Bioinformatics analysis

1.1 construction of a plant evolutionary tree rich in glycine protein genes: sequence homology between NCBI network and phytozome12 is checked by blast, homologous sequences containing GRP gene in soybean and GRP gene in Arabidopsis, maize, tobacco, etc. are selected for downloading, and MEGA7.0 software is used to construct phylogenetic evolution tree, as shown in FIG. 1. Analysis shows that the cloned GRP gene has close relationship with Arabidopsis thaliana and far relationship with other Arabidopsis thaliana.

1.2 prediction and analysis of physicochemical Properties of GRP Gene protein

The basic protein physicochemical property analysis of the GRP gene was performed using the ProtParam tool on the ExPASy server. The results show that: the molecular formula of the GRP gene is C₉₃₃H₁₅₃₀N₂₅₄O₄₁₆S₁₀The relative molecular mass was 23282.46, the total number of atoms was 3143, the theoretical isoelectric point (theactical pI) was 9.06, the number of amino acids was 236, of which 4 positively charged amino acids (Arg + Lys), 1 negatively charged amino acid (Asp + Glu), the instability coefficient was 63.85, and this wasAn unstable protein. The half-life period is 30 h. Analysis of the composition of amino acids showed that: the content of serine is 36.0%, the content of threonine is 31.8%, the content of glycine is 1.3%, and the content of isoleucine and proline are both 3.4%. The fat index was 27.80. The average hydrophilic number was-0.376.

1.3 hydrophobicity analysis of GRP Gene protein

Studies have shown that amino acids with higher positive values have stronger hydrophobicity, while amino acids with lower negative values have stronger hydrophilicity. The results of the hydrophobicity analysis of the GRP gene are shown in FIG. 2, with the lowest value of-1.7 and the strongest hydrophilicity; the highest value is 1.25, and the hydrophobicity is strongest. As a whole, the GRP gene is known to be a hydrophilic protein because the amino acid sequence of the GRP gene contains more hydrophilic amino acids than hydrophobic amino acids.

1.4 transmembrane region analysis of GRP Gene

The protein sequence contains a transmembrane region, which is shown in FIGS. 2 and 3, and the results show that: the amino acids of the GRP gene protein are all located on the surface of a cell membrane, and a typical transmembrane helical region is not formed, so that the analysis result is basically consistent with the analysis result of a hydrophobic region of the protein. Indicating that the GRP gene protein is not a membrane receptor protein related to cell signaling.

2. Material method

2.1 materials

The soybean seed mutant M18, Jinong 18, Escherichia coli DH5 strain, Agrobacterium EHA105 strain, cloning recombinant vector PMD18-T-GRP, pCAMBIA3301, cloning vector PMD18-T are all provided and preserved by Jilin agriculture university plant biotechnology center.

2.2 methods

2.2.1 obtaining fragments of interest

Through previous research work, a soybean drought-resistant mutant M18 is screened. M18 research shows that the root characteristics are obviously different from wild type Jinong 18. Screening differential expression genes by using RNA-seq to obtain an expression fragment which is homologous with GRP. Extracting genome of soybean drought-resistant mutant M18 seedling stage leaf, amplifying by using specific primer to obtain target fragment, cloning target gene by homologous pcr amplification technology to obtain gene sequence with target fragment size about 848bp, wherein the pcr amplification system is 25 mul, and comprises: the amplification conditions were: pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 45s, renaturation at 60 ℃ for 45s, extension at 72 ℃ for 45s, and 42 cycles; finally, the extension is carried out for 10min at 72 ℃ and the product is stored at 4 ℃.

And (3) after 1% agarose gel electrophoresis recovery of the pcr product, sequencing the recovered product, connecting the recovered product with correct sequencing result to a cloning vector pMD18T to obtain a recombinant vector pMD18-T-GRP, transforming into an escherichia coli competent cell, selecting a single colony, extracting a plasmid, and sending the plasmid to Jilin province, Kuume corporation for sequencing.

2.2.2 construction of plant overexpression vectors

Carrying out double enzyme digestion of bgliI and Bste II on recombinant cloning vector pMD18-T-GRP and plant expression vector pCAMBIA3301 respectively, recovering small fragment pMD18-T-GRP and large fragment pCAMBIA3301, and using T₄And (3) DNA ligase connection, replacing a GUS fragment in pCAMBIA-3301 by a small fragment of PMD18-T-GRP, and constructing a recombinant plant overexpression vector PCAMBIA 3301-GRP-over-expression. PCR detection and double enzyme digestion identification are carried out on the vector, and the construction diagram of the vector is shown in figure 4.

2.2.3 construction of RNAi interference expression vectors

pcr amplifies positive-sense fragment with intron, the amplified product is electrophoresed through 1% agarose gel, the target fragment is recovered and purified, the sense fragment and basic expression vector pCAMBIA3301 are subjected to double enzyme digestion by Bgl-II and BstEII (restriction enzymes are products of the company), the vector and the sense fragment are recovered and are connected according to the volume ratio of 1:3, the connection system is as follows, 12 muL of target fragment, 4uL of vector, 1uLT 4 ligase, 2 mu L T4Buffer and 1 muL of ddH₂And O. The reaction was carried out at 22 ℃ overnight. The vector obtained at this time was named pCAMBIA-zy. The antisense fragment is connected into a pCAMBIA-zy vector by adopting the same method and carrying out double enzyme digestion by Bgl II and BstE II to obtain an RNAi interference expression vector PCAMBIA3301-GRP-RNAi with herbicide resistance (bar) group as a screening marker. And carrying out PCR detection and double enzyme digestion identification on the vector. The vector construction scheme is shown in FIG. 5.

2.2.4 genetic transformation of Soybean

The recombinant plasmid PCAMBIA-GRP-

RNAi DNA and PCAMBIA-GRP-over expression DNA are respectively transferred into a receptor soybean variety Jinong 18, and a transformation plant is obtained.

2.2.5 progeny detection of transgenic plants

2.2.5.1 PCR detection

Primer5.0 software was used to design the following primers: herbicide resistance (bar),35s promoter, nos terminator and target Gene (GRP). The primer sequences of BarS/BarAS, 35S/35AS, nosSS/nosAS, GRP of the target gene are shown in Table 1.

Young and tender transformed soybean Plant leaves are picked, a Nuclean Plant Genomic DNA Kit provided by Jilin province, Kuumei, Biotech, Inc. is used for extracting a genome, pcr detection is carried out, and untransformed recipient soybean (Jinong 18) leaf genome is used as a negative control. The herbicide resistance (bar), the 35s gene, the nos terminator and the target Gene (GRP) are 25 mul systems, and the PCR amplification conditions are shown in Table 2.

TABLE 1 primer sequences

TABLE 2

PCR amplification conditions

2.2.5.2 southern blotting detection of transgenic plants

Extracting the T which is positive by PCR detection₂Genomic DNA of soybean leaf was digested with restriction enzyme Hind III, and the purified bar was used as a probe, and the probe was prepared by labeling the genomic DNA with DIG DNALabeling and detection Kit I Kit (product of Roche Co.) according to the instructionPreparing a sample, transferring a membrane, pre-hybridizing, washing the membrane, dyeing and developing and the like, and carrying out Southern blotting detection.

2.2.5.3 drought stress test of transgenic plants

The potting method is a mature mode for the physiological and ecological control experiment of plant water. The experiment uses a potting method, controls water to simulate drought stress, and for 3 transgenic materials: jinong 18 (untransformed receptor plant as negative control CK), recombinant expression vector PCAMBIA-GRP-over expression plant, and recombinant expression vector PCAMBIA-GRP-RNAi plant. And (3) carrying out difference significance analysis through the change of 5 indexes, namely plant height, fresh weight of the overground part, fresh weight of the underground part, dry weight of the overground part and dry weight of the underground part, so as to compare the drought resistance of 3 parts of materials.

2.2.5.4 fluorescent quantitative PCR detection of transgenic plants

Extracting the transgenic plant with hybridization signal in southern test and the RNA of receptor soybean (Jinong 18), reverse transcribing into cDNA with reverse transcription kit, and diluting 5 times for use. And using cDNA diluted by 5 times as a template, selecting a beta-action (Genbank accession number: NM001252731.2) gene as an internal reference gene of a qRT-PCR experiment, simultaneously designing quantitative PCR primers QNCAS and QNCS and fluorescent quantitative PCR primers QCGRP and QACGRP primer sequences by using primer5.0, and then carrying out fluorescent quantitative PCR. The primer sequences required for fluorescence quantification are shown in Table 3.

RNA extraction: the steps are carried out according to the operation steps of an RNAiSoPlus kit produced by TaKaRa company.

qPCR reaction: the reaction system is 20 mu L according to the operation in the specification of q RT-PCRmix produced by GeneCopoeia, wherein 2 xAll-in-oneqRT-PCRmix 10 mu L; 1.0. mu.L each of the corresponding primers (20 umol/L); cDNA2.0. mu.L; 6.0 mu.L of nucleic-free water; the mRNA detection of each sample and the control receptor is repeated for 3 times, and the amplification condition is 95 ℃ for 3 min; 40s at 95 ℃; 40s at 55 ℃; 35 cycles to obtain corresponding Ct value, outputting data and storing after finishing the reaction program, and passing through 2 in relative quantification^-△△CtThe method calculates the quantitative result.

The final calculation formula: the experimental group (Ct target gene-Ct reference) is a control group.

TABLE 3 primer sequences

3 results and analysis

3.1 detection and verification of Glycine-Rich Glycine protein Gene (GRP) cloning vector in Soybean

As shown in FIG. 6, the total DNA of the young leaves of the mutant Jinong 18 plant is used as a template for PCR amplification, and the amplified single amplification band with the size of 848bp and strong specificity is consistent with the expected result. And (4) carrying out electrophoretic recovery on the PCR product, and then connecting the PCR product into a cloning vector. The PCR result based on the cloning vector PMD18T-GRP showed that the obtained fragment length was consistent with the length of the target fragment, indicating that the cloning fragment had been successfully ligated into the cloning vector.

3.2 detection and verification of Glycine-Rich Gene (GRP) expression vector in Soybean

A double enzyme digestion test is carried out by using BglII enzyme and BstEII enzyme, and gel electrophoresis is carried out to ensure that the recombinant plasmid vector is correct. As shown in FIG. 7, the size of the plant over-expression vector pCAMBIA3301-GRP-over expression double-enzyme digestion fragment is consistent with the predicted fragment size 900bp (including the sum of homologous arm fragments), which primarily indicates that the expression vector is successfully constructed, and then the constructed expression vector plasmid is used as a template to carry out conventional PCR detection, and the size and the position of the GRP target gene fragment are the same as the expected fragment, which indicates that the plant over-expression vector is successfully constructed.

As shown in FIG. 8, the size of the plant RNAi expression vector pCAMBIA3301-GRP-RNAi double enzyme digestion fragment is consistent with the expected fragment size 1170bp (including the sum of the positive, the antisense fragments and the intron fragments), which primarily indicates that the construction of the expression vector is successful, and then the constructed expression vector plasmid is used as a template to carry out conventional PCR detection, and the positive fragment, the antisense fragment and the intron fragment of the GRP gene are all the same as the expected fragment size, which indicates that the construction of the plant interference expression vector is successful.

3.3 T₂Generation and detection of transgenic plants

Respectively introducing the overexpression vector PCAMBIA3301-GRP-over expression plasmid DNA into a soybean receptor variety Jinong 18 by an agrobacterium infection method. Obtaining 4T strains by pcr detection₀Generation of positive plant, receiving T₀30 seeds with positive generation. Performing generation-adding treatment by indoor and outdoor continuous planting to obtain 11T plants₁Generation of positive plant, receiving T₁Generating 60 positive seeds, continuously carrying out generation-adding treatment by indoor and outdoor continuous planting to obtain T₂And (3) generating a positive plant 14, extracting leaf genome DNA, respectively utilizing herbicide resistance (bar), a 35s promoter, a not terminator and gene specific primers of a target gene GRP to carry out detection one by one, taking a recombinant expression vector PCAMBIA3301-GRP-over expression plasmid DNA as a positive control, taking an untransformed receptor soybean plant 'Jinong 18' as a negative control and taking water as a blank control, and the pcr detection result is shown in figure 9.

The interference vector PCAMBIA3301-GRP-RNAi plasmid DNA is respectively introduced into a soybean receptor variety Jinong 18 through an agrobacterium infection method. Obtaining 3T strains by pcr detection₀Replacing positive plants and harvesting T₀And 22 generation positive seeds. Carrying out generation-adding treatment by indoor and outdoor continuous planting to obtain T₁Generating positive plants 9 to obtain T₁Replacing 53 positive seeds, continuously planting indoors and outdoors to perform generation-adding treatment to obtain T₂And (3) generating 12 positive plants, extracting leaf genome DNA, detecting one by respectively utilizing gene specific primers of screening markers bar and 35s promoters, non terminators and target genes GRP, taking the recombinant expression vector PCAMBIA3301-GRP-RNAi plasmid DNA as a positive control, taking an untransformed receptor soybean plant 'Jinong 18' and water as a negative control, and taking the pcr detection result as shown in figure 10.

As can be seen from FIGS. 9 and 10, T is₂The size of a band amplified by a gene specific primer in a generation transgenic plant is consistent with that of a target gene, and the fact that a plant glycine-rich protein (GRP) gene is successfully integrated into a transformed positive plant is preliminarily proved.

3.4 T₂Southern blotting detection of transgenic plants

And (3) extracting leaf genome DNA of the transgenic plant with positive pcr detection result in sequence, performing enzyme digestion by using restriction enzyme Hind III, using a screening marker bar as a probe, and performing southern blotting detection by using recombinant expression vector PCAMBIA3301-GRP-over expression plasmid DNA and interference vector PCAMBIA3301-GRP-RNAi plasmid DNA as positive control and plant receptor (Jinong 18) in non-transformed soybean as negative control.

As shown in FIG. 11, only the transformed plants showed hybridization signals, and 2 of the detected GRP overexpression vector gene plants showed significant hybridization signals, so that 7 transgenic plants which were detected to be positive by southern blotting were obtained. It can be seen from the figure that the functional elements are integrated in the genome of the recipient soybean in a single copy, and the integration sites are not the same.

As shown in FIG. 12, only the transformed plants showed hybridization signals, and 1 of the tested GRP interference vector gene-transfected plants showed significant hybridization signals, and 6 transgenic plants that were positive by southern blotting were obtained. It can be seen from the figure that the functional elements are integrated in the genome of the recipient soybean in a single copy, and the integration sites are not the same.

3.5 test detection of drought resistance of transgenic plants in seedling stage

Nutrient soil (microbial matrix) is used as a cultivation medium, unified flowerpots (with the diameter of 12cm multiplied by 10cm) are selected as a container, soil is uniformly mixed before sowing, the pots are filled, bottom water is thoroughly poured, and an electronic scale is used for weighing, so that the weight of each flowerpot is ensured to be consistent after soil is mixed, and the weight of each flowerpot is ensured to be consistent after bottom water is thoroughly poured. Planting 6 pots of each material, 5 grains per pot, watering 550ml every 3 days after seedling emergence, and starting to cut off water for the stress group in the three-leaf period. 4 water-break drought stress groups with 5 days, 8 days, 11 days and 15 days of stress treatment are set according to the conditions of soybean plants in the stress groups. Each water-cut drought stress component is: CK treatment, overexpression treatment and RNAi interference treatment. And control group (normally watered 600ml every 3 days): CK untreated, overexpression untreated, RNAi untreated controls were compared. And continuously measuring each index of the plants of the stress group and the plants of the control group after each stress group is treated for corresponding days and rehydrated for 3 days.

The indexes were measured after 15 days of drought stress treatment and three days of rehydration, as can be seen in table 4: the overground fresh weight and the overground dry weight after the GRP over-expression gene treatment and the CK treatment are obviously different and reach an extremely obvious level. Compared with the strain height, the overground fresh weight, the underground fresh weight, the overground dry weight and the underground dry weight after the CK treatment after the GRP interfering gene treatment, the strain height, the overground fresh weight, the overground dry weight and the underground dry weight have significant differences and reach extremely significant levels; the result shows that the transgenic GRP overexpression vector gene plant after drought stress treatment can show stronger drought resistance than CK through gene expression under drought stress.

TABLE 4 significance analysis of stress and control groups

3.6 T₂Fluorescent quantitative pcr detection of transgenic plants

And (3) continuously carrying out fluorescent quantitative pcr detection on the transgenic plants with positive southern blotting detection results. Separately over-expression of GRP and interference of GRP gene at T₂The expression quantity of the generation transformed plants and the untransformed plants (Jinong 18CK) under the drought stress on different days is analyzed. The results are shown in FIGS. 13 and 14: in 15 days of the soybean plants simulated by drought treatment, the expression quantity of the GRP over-expression genes in the soybean leaves is increased and then decreased, and in 8 days of the drought treatment, the expression quantity of the GRP genes in the soybean leaves is the highest and is 5.2 times of that of a control. The expression level of GRP gene in the root and stem of soybean has no obvious change. The expression level of the GRP interference gene in the rhizome leaves is gradually reduced in 15 days after simulated drought treatment, and the expression level in the roots is the minimum in 15 days after the drought treatment.

The GRP gene is present in many monocotyledonous and dicotyledonous plants. The invention clones GRP gene in mutant (M18), uses bioinformatics analysis, successfully constructs plant expression vector (plant over-expression vector and plant interference vector), transfers functional elements into acceptor soybean (Jinong 18) by agrobacterium-mediated method, and southern blotting detection shows that the functional elements are integrated into soybean genome in single copy form. The drought resistance of the GRP overexpression gene is better than that of a receptor plant Jinong 18(CK) and the expression is obvious through a plant seedling stage drought resistance test; the GRP interference gene does not show drought resistance superior to that of receptor plant Jinong 18(CK) under a stress test; the difference between GRP overexpression genes, GRP interference genes and CK has irregularity and instability, so that the drought resistance identification and detection of the GRP overexpression genes and the GRP interference genes and the CK can be comprehensively analyzed by combining more indexes.

The detection of pcr by fluorescence quantitative found that: the expression level of the GRP overexpression gene in the soybean leaves is highest, and the expression level in the leaves tends to increase firstly and then decrease with the increase of drought stress days; the expression level in roots and stems is low, and the change of the expression level is not obvious. The expression quantity of the GRP interference gene in the roots, stems and leaves of the soybean is less, and the expression quantity is gradually reduced along with the increase of drought stress days.

Biological identification of the GRP transgenic plant shows that the GRP transgenic overexpression vector plant has stronger drought resistance compared with CK in the drought stress environment, and the significance of the overground fresh weight and the overground dry weight is more obvious, which is consistent with the result that the expression quantity of the GRP gene in stems and leaves is higher in the fluorescent quantitative PCR detection result.

The invention lays a foundation for further understanding the molecular mechanism of GRP gene participating in adverse reactions such as drought, and provides conditions for researching GRP gene in soybean.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Sequence listing

<110> Jilin university of agriculture

<120> glycine-rich protein gene of soybean plant and application thereof

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gctggcggag gtggtggagc tgggggaggc tatggaggtg gtgttggtgg aggttatgga 180

ggcgataagg gtggaggtgc tggaggtggc atagaaggtg gacatggtgg tattggagga 240

gaaggtggtg gtgttggagg aggagctgga ggaggtggtg ctggtgggat aggaggagga 300

catgctggtg gcattggagg aggagggggt ggaggtggtg gagccggaag tggtgctgca 360

ggcaacgtag gaggtggaca tggtggtggc attggagggg gagctggagg tgctggtgga 420

ggtggtcaag ccggaggtgg acatggtggt ggcattggag ggggagctgg aggtgctggt 480

ggaggtggtc aagccggagg tggacatggt ggtggcattg gaggtggaca tggtagtgga 540

attggaggag gagctggtgg tggtagtggg gctggaggca acataggagg tggacatggt 600

ggtggtgttg gaggaggagg agttggtgtt ggtggcggtt atggaggtgg taaaggagga 660

ggagctggtg gatgtggtgg aattaataat ggtggcatgg gaggtggaca t 711

<210> 2

<211> 18

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<213> Soybean (Glycine max)

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tagaggacct aacagaac 18

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ccgtgttctc tccaaatg 18

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atgagcccag aacgacgc 18

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cccccagtgt caccttcagc 20

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gttcagtgat gggaaaatta cg 22

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atcttgactg agcgtggtta ttcc 24

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gctggtcctg gctgtctcc 19

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<213> Soybean (Glycine max)

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tgttggagga ggaggagtt 19

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<213> Soybean (Glycine max)

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cctcccatgc caccattatt a 21

Claims

1. An application of a glycine protein-rich gene of a soybean plant in improving drought resistance of soybeans is characterized in that a nucleotide sequence of the glycine protein-rich gene of the soybean plant is shown as SEQ ID No. 1.