CN113249393B - Soybean GmPCBER4 Gene, Protein and Application - Google Patents

Abstract

The invention discloses a soybean GmPCBER4 gene, protein and application. The nucleotide sequence of the GmPCBER4 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein is shown in SEQ ID No.2. The gene was introduced into the target plant to obtain transgenic plants. It was found that the overexpression gene GmPCBER4 could significantly increase the yield of the plants. Under the same stress treatment, the transgenic plants were found to have stronger salt resistance and drought resistance than wild plants. This shows that the GmPCBER4 gene can be used to regulate plant stress resistance, and the invention has great application value for plant breeding.

Description

Soybean GmPCBER4 gene, protein and application

Technical Field

The invention belongs to the field of biotechnology, and in particular relates to soybean GmPCBER4 gene, protein and application.

Background Art

Soybean (Glycine max (L.) Merr.) is an important source of high-quality protein and edible oil for humans, and plays an important role in agricultural production. With the deterioration of the global environment, drought and soil salinization are becoming increasingly serious, and soybean production has been seriously affected. Exploring soybean salt- and drought-tolerant genes and cultivating new stress-resistant soybean varieties are effective ways to solve the current soybean production development in my country.

Under stress conditions, NADPH oxidase on the plasma membrane of plant cells is activated and ROS (including O ^2- , OH ^- and H ² O ² ) is overproduced. A large amount of accumulated ROS can cause a decrease in enzyme activity, increased membrane permeability, increased mutations, and even lead to cell necrosis and programmed cell death (PCD). Therefore, improving the efficiency of the active oxygen scavenging system and enhancing the level of antioxidant metabolism are one of the important ways to enhance plant stress resistance. Phenylcoumaranbenzylic ether reductase (PCBER) is a member of the SDR460A family, which can catalyze the 7-0-4 reduction reaction of phenylcoumaran lignans dependent on NADP to generate the corresponding diphenols. PCBER protein is the most abundant protein in the xylem of plants, and plays an important role in the synthesis of lignin, lignans, and flavonoids and in maintaining the balance of active oxygen in plants. At present, there are relatively few studies on this enzyme, and there are no reports on its correlation with plant yield and stress resistance.

Summary of the invention

One of the objectives of the present invention is to provide a soybean GmPCBER4 gene.

The second object of the present invention is to provide a protein encoded by the above gene.

The third object of the present invention is to provide an expression cassette containing the above gene, a transgenic cell line, a recombinant bacterium, a recombinant virus, a recombinant vector, an expression vector and a host cell containing the expression vector.

A major purpose of the present invention is to provide the application of the GmPCBER4 gene or protein.

In order to achieve the above object, the technical solution adopted by the present invention is summarized as follows:

A GmPCBER4 gene, whose nucleotide sequence is shown in SEQ ID NO.1, the nucleotide sequence consists of 933 bases, or a DNA molecule that hybridizes with the DNA sequence defined by SEQ ID NO.1 under stringent conditions.

The amino acid sequence of the protein (1) encoded by the above gene is shown in SEQ ID No. 2. The sequence consists of 310 amino acid residues.

The protein encoded by the above-mentioned GmPCBER4 gene can also include a protein derived from (1) which is formed by replacing, deleting or adding one or more ((such as 1-30; preferably 1-20; more preferably 1-10; such as 5, 3)) amino acid residues of the amino acid sequence of SEQ ID NO: 2, and has the protein function of (1); or a protein derived from (1) which has 80% ((preferably more than 90%, such as 95%, 98%, 99% or higher)) homology with the protein sequence defined in (1) and has the protein function of (1).

That is to say, the functions of the genes protected by the present invention include not only the above-mentioned GmPCBER4 gene, but also homologous genes having higher homology with SEQID NO: 1 (such as the homology is higher than 40%; preferably higher than 50%; preferably higher than 60%; more preferably higher than 70%; more preferably higher than 80%; more preferably higher than 90%; more preferably higher than 95%; more preferably higher than 98%).

The expression cassette, transgenic cell line, recombinant bacteria, recombinant virus, recombinant vector, expression vector and host cell containing the expression vector and the construction method thereof containing the above-mentioned gene also fall within the protection scope of the present invention.

The recombinant expression vector can be a recombinant plasmid obtained by inserting the GmPCBER4 gene into the multiple cloning site of the vector pCAMBIA3301. The recombinant expression vector can be a recombinant plasmid obtained by replacing the small fragment between the two restriction sites of BamH I and Xba I of the vector pCAMBIA3301 with the GmPCBER4 gene.

The application of the GmPCBER4 gene or protein in improving plant stress resistance, including salt resistance and/or drought resistance. It also includes application in improving plant germination rate and/or yield under adverse stress. The gene or protein also has important applications in regulating plant height or cultivating plants with changed plant type.

The present invention also discloses a method for cultivating transgenic plants, wherein the GmPCBER4 gene is introduced into a target plant to obtain a transgenic plant, wherein the transgenic plant satisfies at least one of the following phenotypes (1) to (4):

(1) Stronger stress resistance than the target plant;

(2) the germination rate under adverse stress is higher than that of the target plant;

(3) The growth status under stress is better than that of the target plant, such as plant height, stem thickness, etc.;

(4) The yield is higher than that of the target plant.

Specifically, the GmPCBER4 gene can be introduced into the target plant through the recombinant expression vector. In the method, the recombinant expression vector can be used to transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc., and the transformed plant tissues can be cultivated into plants.

Specifically, in order to improve the excellent traits of plants, the present invention also protects a new plant breeding method, comprising the following steps (1) and/or (2):

(1) By increasing the activity of the GmPCBER4 protein in the target plant, a plant having the following traits is obtained: stronger stress resistance than the target plant/higher yield under stress than the target plant/better growth state under stress than the target plant, such as plant height, stem thickness, etc.;

(2) Promoting the expression of the GmPCBER4 gene in the target plant to obtain a plant having the following traits: stronger stress resistance than the target plant/higher yield under stress than the target plant/better growth state under stress than the target plant, such as plant height;

The implementation method of "promoting the expression of GmPCBER4 gene in target plants" can be as follows (1) or (2) or (3):

(1) Introducing the GmPCBER4 gene into the target plant;

(2) introduction of strong promoters and/or enhancers;

(3) Other common methods in this field.

In the present invention, there is no particular limitation on the plants applicable to the present invention, as long as they are suitable for gene transformation operations, such as various crops, flower plants, or forestry plants, etc. The plants can be, for example (but not limited to): dicotyledons, monocotyledons or gymnosperms.

As an embodiment, the "plant" includes but is not limited to soybeans, and any plant having the gene or a homologous gene thereof is applicable. It is particularly suitable for plants that need to improve salt tolerance and drought resistance. In actual application, for plants that need to improve salt tolerance and drought resistance, strains with the gene can be cultivated by genetically modifying the gene.

The "plant" mentioned in the present invention includes the whole plant, its parent and progeny plants and different parts of the plant, including seeds, fruits, buds, stems, leaves, roots (including tubers), flowers, tissues and organs, and our target gene or nucleic acid is present in these different parts. The "plant" mentioned here also includes plant cells, suspension cultures, callus, embryos, meristem regions, gametophytes, sporophytes, pollen and microspores, and similarly, each of the aforementioned objects contains the target gene/nucleic acid.

The present invention includes any plant cell, or any plant obtained or obtainable by the method therein, and all plant parts and propagules thereof. This patent also includes transfected cells, tissues, organs or whole plants obtained by any of the aforementioned methods. The only requirement is that the offspring exhibit the same genotypic or phenotypic characteristics, and the offspring obtained using the method of this patent have the same characteristics.

The invention also extends to the harvestable parts of the plants as described above, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. It also further relates to other derivatives of the plants after harvest, such as dry granules or powders, oils, fats and fatty acids, starch or proteins. The invention also relates to foods or food additives obtained from the relevant plants.

Advantages of the present invention:

The present invention discovered the GmPCBER4 protein and its gene from soybean, introduced them into target plants, and obtained transgenic plants. When the transgenic plants were treated with the same stress, it was found that the salt resistance and drought resistance of the transgenic plants were stronger than those of wild plants, which shows that the GmPCBER4 gene can be used to regulate plant stress resistance. In addition, the GmPCBER4 protein and its encoding gene also have the potential to increase plant yield.

For saline-alkali or arid areas, it is necessary to cultivate some salt-tolerant or drought-resistant plants to adapt to the growth in the area. Some new varieties can be bred by introducing this gene, which has great application value for plant breeding.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a transformation vector map;

FIG2 is a PCR detection diagram of the BAR gene of transgenic soybean;

FIG3 is a PCR detection diagram of the transgenic soybean GmPCBER4 gene;

FIG4 shows the phenotype of transgenic soybean under salt treatment;

FIG5 is the result of the drought tolerance experiment of transgenic soybean;

Figure 6 shows the results of the transgenic soybean yield experiment.

DETAILED DESCRIPTION

The present invention will be described in detail below through specific embodiments. These embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be obtained through commercial channels.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

Unless otherwise specified, the implementation of the present invention will use conventional botanical techniques, microorganisms, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination and bioinformatics techniques that are obvious to those skilled in the art. These techniques are fully explained in the published literature. In addition, the methods of DNA extraction, construction of phylogenetic trees, gene editing methods, construction of gene editing vectors, and obtaining gene-edited plants used in the present invention can be achieved by using methods already disclosed in existing literature, except for the methods used in the following examples.

The terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" used herein are meant to include isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), natural types, mutant types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, single-stranded or double-stranded structures. These nucleic acids or polynucleotides include gene coding sequences, antisense sequences and regulatory sequences of non-coding regions, but are not limited to this. These terms include a gene. "Gene" or "gene sequence" is widely used to refer to a functional DNA nucleic acid sequence. Therefore, a gene may include introns and exons in a genomic sequence, and/or include a coding sequence in a cDNA, and/or include a cDNA and its regulatory sequences. In specific embodiments, such as with respect to an isolated nucleic acid sequence, it is preferably assumed to be cDNA.

In addition, in order to have a more intuitive understanding of the technical solution of the present invention, some professional terms involved in the present invention are explained as follows:

"Expression vectors" refers to a vector that adds expression elements (such as promoter, RBS, terminator, etc.) to the basic skeleton of a cloning vector to enable the expression of the target gene.

"Agrobacterium-mediated transformation" refers to the technology of inserting the target gene into the modified T-DNA region, using the infection of Agrobacterium to achieve the transfer and integration of foreign genes into plant cells, and then regenerating transgenic plants through cell and tissue culture technology.

Target plant: target plant. The target plant described in the present invention is soybean.

Target gene: target gene, also known as target gene, is a gene used for gene recombination, changing the characteristics of receptor cells and obtaining the expected expression product in genetic engineering design and operation. It can be from the organism itself or from a different organism.

Example

1. Acquisition of GmPCBER4 gene

A soybean GmPCBER4 gene was screened using a variety of technical means such as transcriptomics and metabolomics. The full-length coding frame nucleotide sequence of the gene is 933bp long and consists of 310 amino acids. After sequencing, its nucleotide sequence is shown in SEQ ID NO.1, and its protein sequence is shown in SEQ ID NO.2.

1. Extraction of total RNA:

(1) Grind with liquid nitrogen, take 100 mg of powder into a 2 ml centrifuge tube (not too much, about the bottom of the centrifuge tube), add 500 ul Buffer RCL/β-mercaptoethanol. Mix quickly and make sure there are no lumps (vortex mix immediately).

(2) 55°C water bath for 3 min, centrifugation at maximum speed (>14.000 g) for 5 min at room temperature; 13000 g for 5 min;

(3) Transfer the supernatant (approximately 450 μl) into the gDNA Filter Colum (blue) containing a 2 ml collection tube, centrifuge at 12,000 g for 2 min at room temperature, and retain the filtrate;

(4) Add an equal volume (400ul) of Buffer RCB to the collection tube (new) and mix by inverting 5-10 times. 12000g, 1min.

(5) Pour the entire mixture into a new 2 ml collection tube containing HiBindTM RNA Mini Colum (red). Centrifuge at 12000g for 1 min at room temperature. Remove the mobile phase and place the column back into the collection tube.

(6) Place the remaining mixed system in the column and centrifuge at 10,000 g for 1 min at room temperature. Remove the mobile phase and return the column to the collection tube.

(7) Add 400 μl of RWC Wash Buffer and centrifuge at 12,000 g for 1 min at room temperature to remove the mobile phase.

(8) Place the column on an original 2 ml collection tube, add 500 ul RNA Wash Buffer II (diluted with ethanol), centrifuge at 12000 g for 1 min at room temperature, remove the mobile phase, and place the column back into the collection tube.

(9) Repeat step (8), remove the mobile phase, empty the collection tube, put the column back into the collection tube, and centrifuge at 1000°C for 2 min.

(10) RNA elution: Place the column in a new 1.5 ml centrifuge tube, dry the column for 3 min, add 35 ul of DEPC water for elution, making sure the water is added in the center of the column membrane, stand at room temperature for 2 min, and centrifuge at 12,000 g for 2 min. The liquid obtained in the 1.5 ml centrifuge tube is the RNA.

2. Cloning and sequence analysis of GmPCBER gene

(1) Reverse transcribe RNA into cDNA and use it as a PCR template.

(2) Design of full-length cDNA amplification primers:

5’ primer: P4-F: 5’-GGATCCATGGCAGGGGACAGCAAGAGCAAG-3’

3’ primer: P4-R: 5’-GCTCTAGAGCTTAGACAAACTGATTAAGGTACTCATCCACAGTG-3’

PCR reaction system (20 μl): DNA template 50 ng; 10× buffer, 2.0 μl; 10 mM dNTP, 0.5 μl; 25 mM MgCl ₂ , 2.0 μl; 10 μM primers, 1 μl each; Taq enzyme 1 unit; add sterile ddH2O to 20 μl. PCR reaction program: 95°C, denaturation 5 min; 94°C, denaturation 1 min, 60°C annealing 1 min, 72°C extension 1.5 min, 35 cycles; 72°C extension 10 min.

(3) After optimizing the PCR conditions according to the Tm values of the primers, PCR amplification was performed according to the method described in the first aspect of the invention, and the obtained product was connected to Escherichia coli. After PCR and enzyme digestion identification, the sequence was determined and the product size was 933 bp.

2. Construction of plant expression vector of phenylcoumaran benzyl ether reductase GmPCBER4 gene

(1) Primers were designed based on the nucleotide sequence of the isolated phenylcoumarane benzyl ether reductase GmPCBER4 gene, and polymerase chain reaction was performed using the cDNA reverse transcribed from total RNA as a template. 1 μl of PCR product was connected to the pCAMBIA3301 vector, and the operation steps were carried out according to the product instructions of TakaRa (Figure 1). Then, Escherichia coli DH5α strain was transformed and grown on LB plates overnight. Plaques were picked for colony PCR. White colonies were picked and cultured in LB liquid medium overnight to preserve the bacteria, extract plasmid DNA, and transform EHA105.

3. Functional identification of phenylcoumaran benzyl ether reductase GmPCBER4 gene

(1) Explant disinfection (DN50):

Place a number of seeds (a batch of about 100 seeds, select clean and complete seeds) in a tissue culture bottle (sterilized);

Pour in an appropriate amount of sterile water, shake for 1 minute, and discard the waste liquid;

Pour in an appropriate amount of 75% ethanol, shake for 1 minute and discard the waste liquid;

Pour in an appropriate amount of 0.1% mercuric chloride, shake for 15 minutes and pour out the waste liquid (toxic and recyclable, raw mercury and waste liquid should be poured out separately);

Pour in sterile water and wash for 1 minute, discard the waste liquid, and repeat three times;

Pour in sterile water and wash for 3 minutes, discard the waste liquid, and repeat twice;

Pour sterile water to cover the seeds, label with the date and name, and soak overnight (in a dark incubator).

(2) Bean germination (GM):

a. Place the tissue culture bottles and beans soaked overnight into a sterilized clean bench and pour out the soaking liquid;

b. Use tweezers to take out the beans and place them into the prepared solidified germination medium, and insert them into the GM solid medium with the navel facing downwards;

c. Heat the lid of the culture dish with the outer flame of an alcohol lamp, seal it with a sealing film, and mark the date and name;

d. Place the beans in the tissue culture room.

(3) Shaking bacteria:

Small shake:

a. Take 3 ml of LB (with corresponding resistance) in a centrifuge tube or test tube, use a gun to pick up the GmPCBER4 strain and add it to the LB liquid in the test tube, and mix it by blowing and sucking with the gun;

b. Note the strain name and date, and place in a shaker for overnight culture.

Big shake:

Open the clean bench and pour about 100 ml of LB (with antibiotics) liquid into the sterilized conical flask or shake bottle;

a. Pour 3 ml of the small shaking bacterial solution into a conical flask and indicate the name of the strain and the date;

b. Place the bacterial solution in a shaker and shake until OD 600-0.8 (ratio: 100ml LB for Agrobacterium + 2ml small shaking bacterial solution); measure the OD value after 4-8 hours.

(4) Preparation of infection solution:

a. After the bacterial solution is shaken (OD value 600 = 0.8-1.0), transfer the bacterial solution to two 50 ml centrifuge tubes;

b. Centrifuge at 4000 rpm for 10 min at room temperature;

c. Pour off the supernatant, add a small amount of CCM liquid and mix by pipetting, and make up the volume to 45-50 ml to form the infection solution for use.

(5) Cutting beans

Use tweezers to take the beans out of the tissue culture bottle and place them in a culture dish. Cut the cotyledons, cleanly remove the main buds, and use a knife to make 4-5 cuts at the cotyledon node.

(6) Infection

Use tweezers to clamp the cut soybean cotyledons into a conical flask, pour in the infiltration dye solution, seal the conical flask with sealing film, and shake it in a 28℃ shaker for 30 minutes; after shaking, pour out the liquid, then wash it with CCM liquid, wipe the cotyledons with filter paper and dry them thoroughly, lay them on the filter paper with the cut surface facing down, seal it with a breathable membrane, and place it in the tissue culture room for dark culture for 4-5 days.

(7) Bud induction:

Take out the cotyledons that have been cultured in the dark, insert them obliquely into the bud induction medium at 45° (cut surface facing up), and insert about 15 per 15cm plate. Seal with a breathable film, mark the name and date, and culture in the tissue culture room for 10 days.

(8) Bud screening:

Remove the cotyledons after bud induction, cut off the main buds (especially thick ones), keep the clustered buds, and perform section processing (remove the culture medium on them) to allow the fresh tissue to fully contact the culture medium. Those that have no main buds or cannot be distinguished should also be moved to SIM+ culture medium, and the explants should be inserted into the bud screening culture medium (SIM ⁺ ) at a 45-degree angle. Seal with a breathable film, write the name and date, and place in the tissue culture room for 14 days.

(9) Bud elongation:

a. After bud screening, select explants with well-differentiated cluster buds and remove dead buds and cotyledons;

b. Make a cut surface on the bottom of the explant (scrape out the green tissue and try to minimize the damage to the clustered buds) to ensure that the fresh tissue is in full contact with the culture medium;

c. Move the explants into SEM culture medium, about three per bottle, write the name and date, and culture in the tissue culture room.

(10) Rooting (done simultaneously with subculture):

When the seedlings grow to about the height of the bottle mouth, they can be rooted and subcultured (about 15 days, the first time may take longer). When the regenerated buds reach 2-3cm in length, they can be transferred to rooting medium (RM). Cut the elongated seedlings and insert them vertically into the RM medium. Scrape out the remaining fresh tissue and insert it into new SEM medium. Put them in the tissue culture room, mark the name and date until they grow enough roots, which is the regenerated seedling.

(11) Hardening of seedlings:

a. Kill the vermiculite and soil in advance, mix the soil and vermiculite in a 1:1 ratio, put them in small flower pots, and pour water on them;

b. Use tweezers to remove the regenerated seedlings from the tissue culture bottle, wash off the culture medium, and plant them in a small flower pot with a hole dug (try not to break the roots);

c. Write the name, batch and date with a marker and label, insert a bamboo stick, put on a fresh-keeping tape and tie it with a rubber band (be careful not to block the water outlet below);

d. Place the transferred soybean seedlings in the greenhouse, remove the fresh-keeping bags after seven days, and water them on time;

e. Remove the fresh-keeping bag and move it to a larger basin after about five days.

(12) Screening of positive strains

PPT (160 mg/L) was sprayed on the leaves of transgenic soybean seedlings transferred to pots. The positive strains were harvested if the leaves did not turn yellow. The leaf DNA of T0 plants was extracted using the CTAB method.

PCR detection of marker gene BAR and target gene GmPCBER4:

The primers for BAR gene amplification were: forward primer BAR-F: 5'-GCACCATCGTCAACCACTACATCGAG-3', reverse primer BAR-R: 5'-TGAAGTCCAGCTGCCAGAAACCCAC-3', and the amplified fragment length was 441bp. The reaction system was 25μl: 10mmol/L dNTP, 0.5μl; 10×PCR buffer, 2.5μl; 10μmol/mL forward and reverse primers, 1μl each; template DNA, 1μl; Taq enzyme 0.2μl (5U/u1); ddH ₂ O, 18.8μl. The amplification program was: 94℃ 5min; 94℃, 45s; 55℃ 45s; 72℃ 1min; 35 cycles; 72℃ extension 10min.

PCR detection of target gene GmPCBER4: 5' end primer: P4JC2-F:

5'-CTGCCCCCAGAGACAGAGT-3', 3' end primer: P4JC2-R: 5'-CATGCGATCATAGGCGTCTC-3', product size is 462bp. PCR reaction system (20μl): DNA template 50ng, 10mM dNTP 0.5μl, 25mM MgCl ₂ 2.0μl, 10μM primer 1μl each, Taq enzyme 1 unit, add sterile ddH ₂ O to 20μl. PCR reaction program: 95℃, denaturation 5min; 94℃, denaturation 1min, 60℃ annealing 1min, 72℃ extension 1.5min, 35 cycles; 72℃ extension 10min. 1% agarose gel electrophoresis to detect PCR results.

PCR detection of the BAR gene in different strains of transgenic soybeans showed that the amplified fragment size was 441 bp, indicating that the exogenous gene marker gene BAR can be stably inherited in transgenic soybeans (Figure 2). At the same time, PCR detection of different strains of transgenic soybeans detected the target gene GmPCBER4, and the amplified fragment size was 462 bp, indicating that the exogenous gene can be stably inherited in the offspring of transgenic soybeans (Figure 3). Plant PCR detection showed that the target gene was passed to the offspring in the transgenic soybeans through sexual reproduction, and after homozygosity, a homozygous body in which the target gene can be stably inherited to the offspring can be obtained.

(13) Salt tolerance test

The harvested seeds of the T3 generation and the control strain DN50 were planted in vermiculite for germination, and then moved to nutrient soil (vermiculite: nutrient soil = 1:1) at the V1 stage. When the soybean seedlings grew to the stage of three compound leaves, they were watered with salt. The experimental group was watered with 1L of nutrient solution containing 200mM NaCI, and the control group was watered with 1L of nutrient solution. They were placed under the same experimental conditions and observed after three days. The experimental results showed that after the salt treatment, the transgenic strains were taller, had thicker stems, and had better growth conditions than the DN50 plants; after the salt treatment, the control variety DN50 had short plants and gradually yellowed leaves. The experimental results showed that the transgenic strains were able to maintain a good growth condition under salt stress, that is, the GmPCBER4 gene improved the salt tolerance of soybeans (Figure 4).

(14) Drought tolerance treatment

The harvested seeds of the T3 generation and the control strain DN50 were planted in vermiculite for germination, and then moved to nutrient soil (vermiculite: nutrient soil = 1:1) at the V1 stage. The soybean seedlings were treated for drought tolerance when they grew to the stage of three compound leaves. The experimental group was not watered, and the control group was watered normally. The two groups of materials were placed under the same experimental conditions. The growth status was observed after about 12 days. As shown in the figure, the transgenic strains were able to maintain good growth conditions after water shortage treatment, with bright green leaves, tall plants, and normal flowering and fruiting, while the control variety DN50 gradually shrank due to water shortage, and the leaves gradually lost their green and turned yellow. The experimental results showed that the overexpressed gene GmPCBER4 significantly improved the drought tolerance of the plants (Figure 5).

(15) Production statistics

The transgenic strains and the control strain DN50 were planted in the natural field environment, and the test records were taken after normal maturity. The experimental results showed that the transgenic strains were significantly higher than the control in terms of plant height, number of pods per plant, number of grains per plant and yield per plant (Table 1, Figure 6). The experimental results show that the over-expressed gene GmPCBER4 significantly increased the yield of the plant.

Table 1 Statistics of transgenic soybean yield

Sequence Listing

<110> Ludong University

<120> Soybean GmPCBER4 gene, protein and application

<130> 2021

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Tyr Ile Gly Lys Phe Ile Val Glu Ala Ser Ala Lys Ala Gly His Pro

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Thr Phe Leu Leu Val Arg Glu Ser Thr Leu Ser Asn Pro Ala Lys Ser

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Pro Leu Ile Asp Asn Phe Lys Gly Leu Gly Val Asn Leu Val Leu Gly

50 55 60

Asp Leu Tyr Asp His Gln Ser Leu Val Ser Ala Ile Lys Gln Val Asp

65 70 75 80

Val Val Ile Ser Thr Val Gly His Leu Gln Leu Ala Asp Gln Asp Lys

85 90 95

Ile Ile Ser Ala Ile Lys Glu Ala Gly Asn Val Lys Lys Phe Tyr Pro

100 105 110

Ser Glu Phe Gly Asn Asp Val Asp Arg Thr His Ala Val Glu Pro Ala

115 120 125

Lys Ser Ala Phe Ala Thr Lys Ala Lys Val Arg Arg Ala Ile Glu Ala

130 135 140

Glu Gly Ile Pro Phe Thr Tyr Val Ser Ser Asn Phe Phe Ala Gly Tyr

145 150 155 160

Phe Leu Pro Asn Leu Ser Gln Pro Gly Ala Thr Ala Ala Pro Arg Asp

165 170 175

Arg Val Ile Ile Leu Gly Asp Gly Asn Pro Lys Ala Val Phe Asn Lys

180 185 190

Glu Glu Asp Ile Gly Thr Tyr Thr Ile Asn Ser Val Asp Asp Pro Arg

195 200 205

Thr Leu Asn Lys Ile Leu Tyr Ile Arg Pro Pro Ala Asn Thr Leu Ser

210 215 220

Phe Asn Glu Leu Val Thr Leu Trp Glu Gly Lys Ile Gly Lys Thr Leu

225 230 235 240

Glu Arg Ile Tyr Val Pro Glu Glu Gln Leu Leu Lys Gln Ile Glu Glu

245 250 255

Ser Ala Pro Pro Val Asn Val Ile Leu Ser Ile Asn His Ser Ser Tyr

260 265 270

Val Lys Gly Asp His Thr Asn Phe Glu Ile Glu Ser Ser Phe Gly Val

275 280 285

Glu Ala Ser Ala Leu Tyr Pro Asp Val Lys Tyr Ile Thr Val Asp Glu

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Tyr Leu Asn Gln Phe Val

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Claims (3)

1. SoybeanGmPCBER4The application of the gene in improving the salt resistance and/or drought resistance of soybean is characterized in thatGmPCBER4The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

2. SoybeanGmPCBER4Use of a gene for increasing soybean yield, characterized in thatGmPCBER4The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

3. A method for growing transgenic plants, characterized in that the plant of claim 1GmPCBER4Introducing a gene into a plant of interest to obtain a transgenic plant satisfying at least one phenotype of (1) or (2) as follows:

(1) Stress resistance is stronger than that of the target plant; the stress resistance is salt resistance and/or drought resistance;

(2) The yield is higher than that of the target plant;

the target plant is soybean.

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