CN110922458B - Soybean stress-resistance-related protein GmNF-YB24 and application of coding gene thereof - Google Patents

Abstract

The invention discloses soybean stress resistance-related protein GmNF-YB24 and application of a coding gene thereof. The protein provided by the invention is named as GmNF-YB24 protein and is a protein shown in a sequence 1. The gene coding the GmNF-YB24 protein is named as GmNF-YB24 gene and also belongs to the protection scope of the invention. The invention also protects the application of the GmNF-YB24 protein: regulating and controlling the stress tolerance of the plant; improve the stress tolerance of the plant. The invention also provides a method for preparing a transgenic plant, which comprises the following steps: the GmNF-YB24 gene is introduced into the starting plant to obtain a transgenic plant with higher stress tolerance compared with the starting plant. The protein and the gene provided by the invention provide a basis for human control of expression of stress resistance and stress tolerance related genes, and play an important role in cultivating plants with enhanced stress resistance and stress tolerance.

Description

Soybean stress-resistance-related protein GmNF-YB24 and application of coding gene thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to soybean stress resistance-related protein GmNF-YB24 and application of a coding gene thereof.

Background

Adversity stresses such as drought, high salinity and low temperature are barrier factors affecting the growth and development of soybeans. Therefore, understanding the response and signal transduction mechanism of soybean to stress conditions and improving the stress resistance of soybean varieties become one of the important tasks of soybean genetic research and soybean variety improvement.

Under the stress of adversity, a series of response reactions are generated in plants, and a plurality of physiological, biochemical and developmental changes are accompanied. The reaction mechanism of the plant to the stress is determined, and scientific data is provided for the research and application of the stress-resistant gene engineering. At present, the research on plant stress resistance has been advanced to the cellular and molecular level, and combined with the research on genetics and genetic engineering, the research on improving the growth characteristics of plants by biotechnology is aimed at improving the adaptability of plants to stress.

Under the adverse conditions of environmental stresses such as drought, high salinity, low temperature and the like, the plant can be correspondingly adjusted on the molecular, cellular and overall levels so as to reduce the damage caused by the environment to the maximum extent and survive. Many genes are induced to express by stress, and the products of the genes not only can be directly involved in the stress response of plants, but also can regulate the expression of other related genes or be involved in signal transduction pathways, so that the plants can avoid or reduce damage, and the resistance to the stress environment is enhanced. At present, the salt-tolerant drought-resistant stress signal network of plants is mainly found to comprise a plant hormone signal pathway, a liposome signal pathway, an SnRK2 (cross non-inducing 1-related protein kinase 2) and MAPK (mitogen-activated protein kinase) signal pathway, an ROS (reactive oxygen species) signal pathway and an stomata signal pathway. These signal network systems intimately link the hormonal regulation, metabolism, energy supply and growth and development of plants. The method shows that the plant adapts to the stress such as high salt and drought, and not only depends on the expression of the stress-tolerant related genes, but also depends on the comprehensive regulation and control action of various signal paths induced by the stress such as drought and high salt. Stress-related gene products can be divided into two broad categories: the products coded by the first gene comprise gene products directly participating in plant stress response, such as ion channel protein, aquaporin, osmotic regulatory factor (sucrose, proline, betaine and the like) synthetase and the like; the second class of genes encodes products including protein factors involved in stress-related signaling and regulation of gene expression, such as protein kinases, transcription factors, and the like. Among them, transcription factors play an important role in regulating expression of plant stress-related genes. When abiotic stress comes, the activity of the transcription factor in plants changes, so that the activity of the target gene regulated by the transcription factor is correspondingly changed, namely the expression level of the transcription factor and the protein level is changed. That is, when abiotic stress stimulates a plant body, a secondary signal molecule is produced outside the cell membrane, and then the signal molecule stimulates the intracellular membrane to produce a phosphorylated protein molecule, which in turn activates a transcription factor, so that its activity is increased to regulate a functional gene.

Disclosure of Invention

The invention aims to provide soybean stress resistance related protein GmNF-YB24 and application of a coding gene thereof.

The protein provided by the invention is obtained from soybean (Glycine max (Linn.) Merr.), is named as GmNF-YB24 protein and is (a) or (b) or (c) or (d) as follows:

(a) protein shown in a sequence 1 in a sequence table;

(b) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the sequence 1 and is related to plant stress tolerance and derived from the protein;

(c) a protein derived from soybean, having 95% or more identity to (a) and associated with stress tolerance in plants;

(d) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of (a), (b) or (C).

The labels are specifically shown in table 1.

TABLE 1 sequences of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL
HA	9	YPYDVPDYA

The protein can be synthesized artificially, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

The gene coding the GmNF-YB24 protein is named as GmNF-YB24 gene and also belongs to the protection scope of the invention.

The GmNF-YB24 gene is a DNA molecule as described in the following (1) or (2) or (3) or (4):

(1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

(2) a DNA molecule having an identity of 75% or more to (1) and encoding a protein associated with stress tolerance in plants;

(3) a DNA molecule derived from soybean, having 90% or more identity to (1) and encoding a protein associated with stress tolerance of a plant;

(4) and (2) a DNA molecule which is hybridized with the DNA molecule (1) under strict conditions and encodes a plant stress tolerance related protein.

The stringent conditions are hybridization and washing of the membrane 2 times 5min at 68 ℃ in a solution of 2 XSSC, 0.1% SDS and 2 times 15min at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS.

The identity of 75% or more may be 80%, 85%, 90% or 95% or more.

An expression cassette, a recombinant vector or a recombinant bacterium containing the GmNF-YB24 gene all belong to the protection scope of the invention.

The recombinant vector may specifically be a recombinant expression vector. The recombinant expression vector containing the gene can be constructed by using the existing expression vector. When the gene is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters can be added in front of the transcription initiation nucleotide, and can be used alone or combined with other plant promoters; in addition, when the gene is used to construct a recombinant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codons or adjacent regions initiation codons, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate identification and screening of the transgenic plant or the transgenic microorganism, an expression vector to be used may be processed, for example, a gene for expressing an enzyme or a luminescent compound which produces a color change in the plant or the microorganism, a gene for an antibiotic marker having resistance or a chemical-resistant agent marker, etc. From the perspective of transgenic safety, the transformed plants or microorganisms can be directly screened by adversity stress without adding any selectable marker gene.

The recombinant expression vector can be specifically a recombinant plasmid obtained by inserting a GmNF-YB24 gene into a multiple cloning site (such as a SmaI enzyme cutting site) of the vector pBI 121.

The recombinant expression vector can be specifically a recombinant plasmid obtained by inserting GmNF-YB24 gene into multiple cloning sites (such as PmlI enzyme cutting sites) of the vector pCAMBIA 3301.

The invention also protects the application of the GmNF-YB24 protein, which is (I) or (II) as follows:

regulating and controlling the stress tolerance of plants;

(II) improving stress tolerance of plants.

The invention also provides a plant breeding method, which comprises the following steps: the activity and/or the content of the GmNF-YB24 protein in the target plant are improved, so that the stress tolerance of the plant is improved.

The invention also protects the application of the GmNF-YB24 gene in cultivating transgenic plants with increased stress tolerance.

The invention also provides a method for preparing a transgenic plant, which comprises the following steps: the GmNF-YB24 gene is introduced into the starting plant to obtain a transgenic plant with higher stress tolerance compared with the starting plant. The GmNF-YB24 gene is introduced into the starting plant through a recombinant expression vector.

The invention also protects the application of the GmNF-YB24 protein, the GmNF-YB24 gene or the method in plant breeding. The goal of such breeding is to obtain plants with enhanced stress tolerance.

Any of the stress tolerance described above is drought tolerance and/or salt tolerance.

Any one of the above-mentioned stress tolerance enhancements is embodied as any one or any combination of (e1), (e2), (e3) and (e4) below:

(e1) in an adversity stress environment, the survival rate is high;

(e2) in an adversity stress environment, the conductivity is low;

(e3) in an adversity stress environment, the proline content is high;

(e4) in an adverse stress environment, the malondialdehyde content is low.

The stress is drought stress or salt stress.

Any of the above plants is a monocot or a dicot.

The dicotyledonous plant is specifically arabidopsis thaliana or soybean. The arabidopsis thaliana can be specifically Columbia ecotype arabidopsis thaliana. The soybean can be soybean Williams 82.

The invention discovers that the stress tolerance of a transgenic plant obtained by introducing the GmNF-YB24 gene into the plant is obviously higher than that of a wild plant, the protein and the gene provided by the invention provide a foundation for human control of the expression of stress resistance and stress tolerance related genes, and the protein and the gene play an important role in cultivating the plant with enhanced stress resistance and stress tolerance.

Drawings

FIG. 1 shows the results of drought stress treatment in example 2.

FIG. 2 is the results of high salt stress treatment in example 2.

FIG. 3 shows the results of the relative expression amounts of the GmNF-YB24 genes in example 3.

FIG. 4 is a photograph of the plants after drought/salt treatment in example 3.

FIG. 5 is the relative conductivity of the leaves of the plants after drought/salt treatment in example 3.

FIG. 6 shows the proline content of the leaves of the plants after drought/salt treatment in example 3.

FIG. 7 is the malondialdehyde content of the leaves of the plants after drought/salt treatment in example 3.

FIG. 8 is a DAB photograph of the leaves of the plants after drought/salt treatment in example 3.

FIG. 9 is a photograph of NBT staining of leaves of plants after drought/salt treatment in example 3.

FIG. 10 is a photograph of trypan blue staining of plant leaves after drought/salt treatment in example 3.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples,% is by mass unless otherwise specified.

Soybean "Tiefeng No. 8", abbreviated as Tiefeng No. 8: acquiring a national germplasm resource library (with the number of ZM 242); references to soybeans are: li PS, Yu TF, He GH, Chen M, Zhou YB, Chai SC, Xu ZS and Ma YZ (2014) Genome-wide analysis of the Hsf family in the sobean and functional identification of GmHsf-34 innovative in the hydrogen and heat protocols BMC genomes 15:1009.

The plant eukaryotic expression vector pBI121 is called vector pBI121 for short. Agrobacterium GV 3101: beijing Bylendi Biotech. Columbia ecotype Arabidopsis thaliana, also called wild type Arabidopsis thaliana, is represented by Col-0: arabidopsis thaliana information resources network, SALK corporation, USA.

The plant eukaryotic expression vector pCAMBIA3301 is called vector pCAMBIA3301 for short. Agrobacterium K599: beijing Bylendi Biotech. Soybean Williams 82, abbreviated to Williams 82.

Example 1 obtaining of GmNF-YB24 protein and Gene encoding the same

NaCl treatment is carried out on the four-leaf stage seedling of Tiefeng No. 8 which grows for about 2 weeks under normal conditions for 2 hours, and the seedling is quickly frozen by liquid nitrogen and stored at minus 80 ℃ for later use.

Total RNA from soybean leaves was extracted by Trizol method, and first strand cDNA was synthesized using reverse transcriptase XL (AMV). The ds cDNA was synthesized by SMART method and the PCR product was detected by 1.0% agarose gel electrophoresis.

The sequence 2 in the sequence table was obtained by the methods of 5 'RACE and 3' RACE. The DNA molecule shown in the sequence 2 of the sequence table codes the protein shown in the sequence 1 of the sequence table.

The protein shown in the sequence 1 of the sequence table is named GmNF-YB24 protein. The gene for coding the GmNF-YB24 protein is named as GmNF-YB24 gene, and the open reading frame is shown as a sequence 2 in a sequence table.

Example 2 Effect of GmNF-YB24 protein on stress tolerance of Arabidopsis thaliana

Construction of recombinant expression vector

1. Extracting total RNA of the Tiefeng No. 8 leaf, and carrying out reverse transcription to obtain cDNA.

2. And (3) taking the cDNA obtained in the step (1) as a template, carrying out PCR amplification by adopting a primer pair consisting of B24-F and B24-R, and recovering a PCR amplification product.

B24-F：5'-GTTGTATTTGGTTTGGTGGCATT-3'；

B24-R：5'-CTCAACAACGAAGTTTCAAGCAA-3'。

3. And (3) connecting the PCR amplification product obtained in the step (2) with a Peasky blue vector (all-type gold, Beijing) to obtain a recombinant plasmid Peasky blue-GmNF-YB 24.

4. And (3) carrying out PCR amplification by using a recombinant plasmid Peasyblast-GmNF-YB 24 as a template and a primer pair consisting of GmNF-YB24-121F and GmNF-YB24-121R, and recovering a PCR amplification product.

GmNF-YB24-121F:5'-CTCTAGAGGATCCCCGGGATGGCTGAGTCGGACAA-3'；

GmNF-YB24-121R:5'-ACTAGTGGATCCCCCGGGTTATCTAGTTCTTGCAGAAG-3。

5. And (4) digesting the PCR amplification product obtained in the step (4) by using restriction enzyme SmaI, and recovering a digestion product.

6. The vector pBI121 was digested with restriction enzyme SmaI, and the vector backbone was recovered.

7. And (3) connecting the enzyme digestion product obtained in the step (5) with the vector skeleton obtained in the step (6) to obtain a recombinant plasmid pBI121-GmNF-YB 24. According to the sequencing result, the structure of the recombinant plasmid pBI121-GmNF-YB24 is described as follows: a DNA molecule shown in a sequence 2 of a sequence table is inserted into a SmaI enzyme cutting site of a vector pBI 121.

Second, obtaining transgenic Arabidopsis

1. The recombinant plasmid pBI121-GmNF-YB24 is introduced into agrobacterium GV3101 to obtain recombinant agrobacterium.

2. The recombinant Agrobacterium obtained in step 1 was inoculated in liquid YEP medium and cultured with shaking at 28 ℃ and 3000rpm for about 30 hours.

3. After completion of step 2, the bacterial solution was transferred to a liquid YEP medium containing 50. mu.g/L rifampicin and 50. mu.g/L kanamycin, and cultured at 28 ℃ and 300rpm with shaking to OD_600nm＝1.5-3.0。

4. After completion of step 3, the cells were collected, centrifuged at 4000g for 10min at 4 ℃ and diluted to OD with an aqueous solution containing 10% sucrose and 0.02% silwet_600nmAbout 1.0, the staining solution is obtained.

5. Inversely buckling Columbia ecological arabidopsis thaliana planted in a flowerpot in a container containing a staining solution to immerse the flower for about 50s, then taking out the flowerpot, laterally placing the flowerpot in a tray, covering the flowerpot with black plastic cloth, uncovering the plastic cloth after 24 hours, vertically placing the flowerpot, performing normal illumination culture, and harvesting seeds, namely T₀Seeds of Arabidopsis thaliana were used.

6. Will T₀Seeds of arabidopsis thaliana are sowed on a solid MS culture medium plate containing 50mg/L kanamycin, and the plant obtained by culture is T₁And (4) generation of Arabidopsis thaliana.

7、T₁Selfing arabidopsis thaliana and harvesting seeds to obtain T₁Seeds of Arabidopsis thaliana were used. T is₁The plant obtained by seed culture of arabidopsis thaliana is T₂And (4) generation of Arabidopsis thaliana. T is₂Selfing arabidopsis thaliana and harvesting seeds to obtain T₂Seeds of Arabidopsis thaliana were used. T is₂The plant obtained by seed culture of arabidopsis thaliana is T₃And (4) generation of Arabidopsis thaliana.

8. For T₂Arabidopsis thaliana and sampled T₃PCR identification was performed for Arabidopsis thaliana. If a certain T₂Arabidopsis thaliana and T obtained by selfing₃All the arabidopsis thaliana are positive in PCR identification, and the T is₂The generation of arabidopsis thaliana and the inbred progeny thereof are 1 homozygous GmNF-YB24 transgenic line. The PCR identification method comprises the following steps: extracting genome DNA of the leaves, and carrying out PCR amplification by adopting a primer pair consisting of GmNF-YB24-121F and GmNF-YB24-121R, wherein if an amplification product of about 500bp is obtained, the PCR identification is positive.

Thirdly, obtaining of empty vector Arabidopsis thaliana

Replacing the recombinant plasmid pBI121-GmNF-YB24 with the vector pBI121, and obtaining the empty vector Arabidopsis thaliana by referring to the operation of the second step.

Fourth, stress tolerance identification of Arabidopsis thaliana

1. Drought stress treatment

The seeds to be tested are: GmNF-YB24-1 strain T₃Seed of generation plant, GmNF-YB24-2 strain T₃Seed of generation plant, empty carrier arabidopsis thaliana T₃Seeds of the generation plant and seeds of wild type arabidopsis thaliana. The GmNF-YB24-1 strain and the GmNF-YB24-2 strain are homozygous transgenic GmNF-YB24 strains.

And (3) drought group: sowing seeds to be detected, counting days from the beginning of seed germination, carrying out drought treatment (namely, not watering for one week continuously) after 20 days, then carrying out rehydration treatment (namely, recovering normal watering for one week), then photographing and counting the survival rate;

normal group: sowing seeds to be tested, counting days from the germination of the seeds, continuing normal watering management for two weeks after 20 days, and then photographing.

And setting three repeated tests, and observing and counting 10 plants by each seed to be detected in each repeated test.

The results are shown in FIG. 1. FIG. 1A is a photograph of normal group plants, FIG. 1B is a photograph of drought group plants, and FIG. 1C is a photograph of survival rate of drought group plants.

After drought treatment, plants which are wilted and dead appear, the growth vigor of the plants of GmNF-YB24-1 strain and GmNF-YB24-2 strain is obviously superior to that of wild arabidopsis, and the growth condition of the transgenic empty carrier arabidopsis has no obvious difference with that of the wild arabidopsis. In the drought group, the survival rates of plants of GmNF-YB24-1 strains and GmNF-YB24-2 strains are obviously higher than that of wild arabidopsis, and the survival rate of the transgenic empty carrier arabidopsis is not obviously different from that of the wild arabidopsis.

2. High salt stress management

The seeds to be tested are: GmNF-YB24-1 strain T₃Seed of generation plant, GmNF-YB24-2 strain T₃Seed of generation plant, empty carrier arabidopsis thaliana T₃Seeds of the generation plant and seeds of wild type arabidopsis thaliana. The GmNF-YB24-1 strain and the GmNF-YB24-2 strain are 2 homozygous trans GmNF-YB24 gene strain.

Salt stress group: sowing seeds to be detected, counting days from the beginning of seed germination, carrying out salt stress treatment (namely irrigating with 200mM NaCl aqueous solution for one week continuously) after 20 days, then photographing and counting the survival rate;

normal group: sowing seeds to be tested, counting days from the germination of the seeds, continuing normal watering management for one week after 20 days, and then photographing.

And setting three repeated tests, and observing and counting 10 plants by each seed to be detected in each repeated test.

The results are shown in FIG. 2. Fig. 2A is a photograph of normal group plants, fig. 2B is a photograph of salt stressed group plants, and fig. 2C is a survival rate of salt stressed group plants.

After salt stress treatment, the growth vigor of plants of GmNF-YB24-1 strains and GmNF-YB24-2 strains is obviously superior to that of wild type arabidopsis, and the growth condition of the transgenic empty carrier arabidopsis is not obviously different from that of the wild type arabidopsis. In the salt stress group, the survival rates of plants of GmNF-YB24-1 strains and GmNF-YB24-2 strains are obviously higher than that of wild type Arabidopsis, and the survival rate of the transgenic empty vector plants is not obviously different from that of the wild type Arabidopsis.

Example 3 Effect of GmNF-YB24 protein on stress tolerance of Soybean

Construction of recombinant expression vector

1. And (3) carrying out PCR amplification by using a recombinant plasmid Peasyblast-GmNF-YB 24 as a template and a primer pair consisting of GmNF-YB24-3301F and GmNF-YB24-3301R, and recovering a PCR amplification product.

GmNF-YB24-3301F:5'-CCACCACCACCACCACCACGTGATGGCTGAGTCGGACAA-3'；

GmNF-YB24-3301R:5'-GGTCACCTGTAATTCACACGTGTTATCTAGTTCTTGCAGAAG-3。

2. The vector pCAMBIA3301 was digested with restriction enzyme PmlI, and the vector backbone was recovered.

3. And (3) carrying out In-Fusion seamless cloning on the PCR amplification product obtained In the step (1) and the vector skeleton obtained In the step (2) to obtain a recombinant plasmid pCAMBIA3301-GmNF-YB 24. According to the sequencing result, the structure of the recombinant plasmid pCAMBIA3301-GmNF-YB24 is described as follows: the DNA molecule shown in the sequence 2 of the sequence table is inserted into the PmlI enzyme cutting site of the vector pCAMBIA 3301.

Second, obtaining transgenic soybean

1. And (3) introducing the recombinant plasmid pCAMBIA3301-GmNF-YB24 into agrobacterium K599 to obtain recombinant agrobacterium. The recombinant Agrobacterium was inoculated in liquid YEP medium and cultured with shaking at 28 ℃ and 3000rpm for 18 hours. The bacterial liquid was streaked on a solid YEP medium plate containing 50. mu.g/L streptomycin and 50. mu.g/L kanamycin, and incubated at 28 ℃ for 2 days with standing.

2. The seeds of Williams 82 were sterilized with chlorine for 8 hours, then sown evenly in sterilized vermiculite, and cultured until cotyledons formed.

3. And (3) after the step 1 is finished, picking a bacterial colony by using a needle point of an injector, then pricking the needle point containing the bacterial colony into the cotyledon node of the soybean subjected to the step 2, and culturing until a soybean hairy root grows out to obtain a soybean transgenic GmNF-YB24 gene hairy root complex plant (a transgenic GmNF-YB24 gene plant for short).

Thirdly, obtaining empty carrier soybean

And (3) replacing the recombinant plasmid pCAMBIA3301-GmNF-YB24 with the pCAMBIA3301 vector, and operating according to the second step to obtain the soybean empty-vector hairy root complex plant (the empty-vector-transformed plant for short).

Fourthly, detecting the relative expression quantity of the GmNF-YB24 gene

And after the second step or the third step is finished, taking the hairy roots of the plants, extracting total RNA and carrying out reverse transcription to obtain cDNA, carrying out qRT-PCR by using a cDNA template, and detecting the relative expression quantity of the GmNF-YB24 gene by using an Actin gene as an internal reference gene.

The primers used for detecting the GmNF-YB24 gene were as follows:

GmNF-YB24ygdl-F：5'-GTTGTATTTGGTTTGGTGGCATT-3'；

GmNF-YB24ygdl-R：5'-TTGCGTTCCCCGTGTGACCT-3'。

the primers used to detect the Actin gene were as follows:

Actin-F：5'-CTGTTGGAAGGTGCTGAG-3'；

Actin-R：5'-ACATTGTTCTTAGTGGTGGCT-3'。

the results are shown in FIG. 3. In FIG. 3, control represents a transgenic empty vector plant, 35S is GmNFYB24-1 to 35S, and GmNFYB24-4 represents different transgenic GmNF-YB24 gene plants. The expression quantity of the GmNF-YB24 gene in the hairy root of the transgenic GmNF-YB24 gene plant is 4-6 times of that of the transgenic empty vector plant, and the difference has significance. The result shows that the exogenous gene (GmNF-YB24 gene) is not only successfully integrated on the genome of the soybean, but also can be normally transcribed and expressed in the transgenic soybean.

Stress tolerance identification of soybeans

The plants to be tested are: and step three, obtaining a transgenic GmNF-YB24 gene plant which grows for five weeks, and obtaining a transgenic empty vector plant which grows for five weeks.

Drought stress test group: and carrying out drought treatment on the plant to be detected. The drought treatment method comprises the following steps: a single pour was performed with an aqueous solution containing 25% PEG 6000.

Salt stress test group: and (5) carrying out salt stress treatment on the plant to be detected. Methods of salt stress treatment: a single pour was performed with 200mM NaCl in water.

Control group: the plant to be tested is parallel to the plant to be tested of the test group, and does not carry out salt stress treatment or drought treatment, and the other plants are the same as the test group.

And setting three repeated tests, wherein each plant to be tested in each repeated test is observed and counted on 10 plants.

The photographs of the plants of the test group 7 days after the drought treatment are shown in FIG. 4A, and the photographs of the plants of the control group 7 days after the parallel treatment are shown in FIG. 4B. The photographs of the plants of the test group 7 days after the salt treatment are shown in FIG. 4C, and the photographs of the plants of the control group 7 days after the parallel treatment are shown in FIG. 4D.

The relative conductivities of leaves of the test group plants 3 days after the salt stress treatment, leaves of the test group plants 7 days after the drought treatment, and leaves of the normal group plants 7 days after the parallel treatment are shown in fig. 5. In FIG. 5, A is a control group, B is a salt stress test group, and C is a drought stress test group.

The proline content of leaves of the test group plants 3 days after the salt stress treatment, the proline content of leaves of the test group plants 7 days after the drought treatment, and the proline content of leaves of the normal group plants 7 days after the parallel treatment are shown in FIG. 6. In FIG. 6, A is a control group, B is a salt stress test group, and C is a drought stress test group.

The malondialdehyde content of the leaves of the test group plants after 3 days of salt stress treatment, the malondialdehyde content of the leaves of the test group plants after 7 days of drought treatment, and the malondialdehyde content of the leaves of the normal group plants after 7 days of parallel treatment are shown in FIG. 7. In FIG. 7, A is a control group, B is a salt stress test group, and C is a drought stress test group.

The leaves of the plants of the test group after 3 days of salt stress treatment and 7 days of drought treatment, and DAB staining photographs are shown in FIG. 8.

The NBT staining photographs of leaves of the plants of the test group 3 days after the salt stress treatment and 7 days after the drought treatment are shown in FIG. 9.

The leaves of the plants of the test group after 3 days of salt stress treatment and 7 days of drought treatment are shown in fig. 10.

After the salt stress treatment, the transgenic empty carrier plants have severe wilting, and the growth vigor of the transgenic GmNF-YB24 gene plants is obviously better than that of the transgenic empty carrier plants. After the salt stress treatment, the relative conductivity of the leaves of the plant with the transferred GmNF-YB24 gene is obviously lower than that of the plant with the transferred empty carrier. After the salt stress treatment, the proline content of the plant leaves of the transgenic GmNF-YB24 gene is obviously higher than that of the transgenic empty vector plants. After the salt stress treatment, the malondialdehyde content of the plant leaves of the GmNF-YB24 transgenic plants is obviously lower than that of the plants of the transgenic empty carriers.

After drought treatment, the transgenic empty carrier plants have severe wilting, and the growth vigor of the transgenic GmNF-YB24 gene plants is obviously better than that of the transgenic empty carrier plants. After drought treatment, the relative conductivity of the leaves of the plant transformed with the GmNF-YB24 gene is obviously lower than that of the plant transformed with an empty carrier. After drought treatment, the proline content of the leaves of the plant transformed with the GmNF-YB24 gene is obviously higher than that of the plant transformed with an empty carrier. After drought treatment, the malondialdehyde content of the plant leaves of the transgenic GmNF-YB24 gene is obviously lower than that of the plants of the transgenic empty carriers.

Method for measuring relative conductivity: taking 0.2g of leaves, cutting the leaves into small strips of about 1cm, putting the small strips into a centrifuge tube, adding 5mL of pure deionized water into the centrifuge tube, and standing for 25min at room temperature; secondly, taking a centrifugal tube filled with pure deionized water, measuring potential by using a conductivity meter and recording the potential as Vc; taking the centrifugal tube which is finished with the step I, measuring the potential by using a conductivity meter, and recording the potential as Vo; fourthly, boiling the centrifugal tube which is finished in the third step in boiling water for 30min, then cooling to room temperature, measuring the potential by using a conductivity meter, and recording the potential as Vm; relative conductivity (Vo-Vc) × 100/(Vm-Vc).

The Malondialdehyde (MDA) content detection kit and the proline content detection kit are both products of Beijing Solaibao science and technology Limited company (Beijing), and are operated according to the instruction.

SEQUENCE LISTING

<110> institute of crop science of Chinese academy of agricultural sciences

<120> GNCYX181948

<130> soybean stress resistance-related protein GmNF-YB24 and application of coding gene thereof

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 171

<212> PRT

<213> Glycine max

<400> 1

Met Ala Glu Ser Asp Asn Glu Ser Gly Gly His Thr Gly Asn Ala Ser

1 5 10 15

Gly Ser Asn Glu Phe Ser Gly Cys Arg Glu Gln Asp Arg Phe Leu Pro

20 25 30

Ile Ala Asn Val Ser Arg Ile Met Lys Lys Ala Leu Pro Ala Asn Ala

35 40 45

Lys Ile Ser Lys Glu Ala Lys Glu Thr Val Gln Glu Cys Val Ser Glu

50 55 60

Phe Ile Ser Phe Ile Thr Gly Glu Ala Ser Asp Lys Cys Gln Lys Glu

65 70 75 80

Lys Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr

85 90 95

Leu Gly Phe Glu Glu Tyr Val Glu Pro Leu Lys Val Tyr Leu His Lys

100 105 110

Tyr Arg Glu Leu Glu Gly Glu Lys Thr Ala Met Met Gly Arg Pro His

115 120 125

Glu Arg Asp Glu Gly Tyr Gly His Ala Thr Pro Met Met Ile Met Met

130 135 140

Gly His Gln Gln Gln Gln His Gln Gly His Val Tyr Gly Ser Gly Thr

145 150 155 160

Thr Thr Gly Ser Ala Ser Ser Ala Arg Thr Arg

165 170

<210> 2

<211> 516

<212> DNA

<213> Glycine max

<400> 2

atggctgagt cggacaacga gtccggaggt cacacgggga acgcaagcgg aagcaacgaa 60

ttctccggtt gcagggagca agacaggttc cttccgatag cgaacgtgag caggatcatg 120

aagaaggcgt tgccggcgaa cgcgaagatc tcgaaggagg cgaaggagac ggtgcaggag 180

tgcgtgtcgg agttcatcag cttcataaca ggagaagcgt ccgataagtg ccagaaggag 240

aagaggaaga cgatcaacgg cgatgatctg ctgtgggcca tgaccacgct gggattcgag 300

gagtacgtgg agcctctcaa ggtttatctg cataagtata gggagctgga aggggagaaa 360

actgctatga tgggaaggcc acatgagagg gatgagggtt atggtcatgc aactcctatg 420

atgatcatga tggggcatca acagcagcag catcagggac acgtgtatgg atctggaact 480

actactggat cagcatcttc tgcaagaact agataa 516

Claims (4)

1. The application of the gene of the GmNF-YB24 protein in cultivating transgenic plants with increased stress tolerance;

the GmNF-YB24 protein is (a) or (d) as follows:

(a) protein shown in a sequence 1 in a sequence table;

(d) a fusion protein obtained by connecting a label to the N terminal or/and the C terminal of (a);

the plant is arabidopsis thaliana or soybean;

the stress tolerance is drought tolerance and/or salt tolerance.

2. The use of claim 1, wherein: the gene for coding the GmNF-YB24 protein is a DNA molecule with a coding region as shown in a sequence 2 in a sequence table.

3. A method of making a transgenic plant comprising the steps of: introducing a gene coding a GmNF-YB24 protein into a starting plant to obtain a transgenic plant with higher stress tolerance compared with the starting plant;

the GmNF-YB24 protein is (a) or (d) as follows:

(a) protein shown in a sequence 1 in a sequence table;

(d) a fusion protein obtained by connecting a label to the N terminal or/and the C terminal of (a);

the plant is arabidopsis thaliana or soybean;

the stress tolerance is drought tolerance and/or salt tolerance.

4. The method of claim 3, wherein: the gene for coding the GmNF-YB24 protein is a DNA molecule with a coding region as shown in a sequence 2 in a sequence table.

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