CN113493501B - Plant stress tolerance-related GmNTF2B-1 protein, and coding gene and application thereof

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CN113493501B

Publication number: CN113493501B
Application number: CN202010258233.7A
Authority: CN
Inventors: 陈明; 马有志; 陈凯; 周永斌; 徐兆师; 陈隽
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2020-04-03
Filing date: 2020-04-03
Publication date: 2023-01-03
Anticipated expiration: 2040-04-03
Also published as: CN113493501A

The invention discloses a plant stress tolerance related protein GmNTF2B-1, and a coding gene and application thereof. The protein provided by the invention is the protein of a) or b) or c) or d) as follows: a) A protein consisting of an amino acid sequence shown as a sequence 1 in a sequence table; b) A fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 1 in the sequence table; c) The protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1 in the sequence table; d) A protein having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the amino acid sequence defined in any one of a) to c) and having the same function. Experiments prove that: the GmNTF2B-1 can improve the drought tolerance of plants, provides a foundation for artificially controlling the expression of stress resistance and stress tolerance related genes, and plays an important role in breeding of plants with enhanced stress resistance and stress tolerance.

GmNTF2B-1 protein related to plant stress tolerance, and coding gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a GmNTF2B-1 protein related to plant stress tolerance, and a coding gene and application thereof.

Background

Soil drought is an important environmental factor affecting plant growth and development. Under the stress conditions of drought 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. 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. They play an important role in the regulation of gene expression in plant stress responses.

Disclosure of Invention

The invention aims to solve the technical problem of how to regulate and control the stress tolerance of plants.

In order to solve the technical problems, the invention firstly provides a protein related to plant stress tolerance.

The protein related to plant stress tolerance provided by the invention is derived from soybean (Glycine max) in the genus of Glycine, is named GmNTF2B-1, and is a) or B) or c) or d) as follows:

a) A protein consisting of an amino acid sequence shown as a sequence 1 in a sequence table;

b) A fusion protein obtained by connecting labels to the N terminal or/and the C terminal of the protein shown in the sequence 1 in the sequence table;

c) The protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1 in the sequence table;

d) A protein having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the amino acid sequence defined in any one of a) to c) and having the same function.

Wherein, the sequence 1 in the sequence table is composed of 123 amino acid residues.

The tag of protein GmNTF2B-1 of B) above is specifically shown in Table 1.

TABLE 1 sequences of tags

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

The protein GmNTF2B-1 in the above c), wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein GmNTF2B-1 of a) or B) or c) or d) can be artificially synthesized, or can be obtained by synthesizing a coding gene and then carrying out biological expression.

In order to solve the technical problems, the invention also provides a biological material related to the GmNTF2B-1 protein.

The biological material related to the GmNTF2B-1 protein provided by the invention is any one of the following A1) to A12):

a1 Nucleic acid molecules which code for the GmNTF2B-1 protein;

a2 An expression cassette comprising the nucleic acid molecule according to A1);

a3 A recombinant vector containing the nucleic acid molecule according to A1);

a4 A recombinant vector containing the expression cassette of A2);

a5 A recombinant microorganism containing the nucleic acid molecule according to A1);

a6 A recombinant microorganism containing the expression cassette described in A2);

a7 A recombinant microorganism containing the recombinant vector of A3);

a8 A recombinant microorganism containing the recombinant vector of A4);

a9 A transgenic plant cell line containing the nucleic acid molecule according to A1);

a10 A transgenic plant cell line containing the expression cassette of A2);

a11 A transgenic plant cell line containing the recombinant vector of A3);

a12 A transgenic plant cell line containing the recombinant vector of A4).

In the above biological material, the nucleic acid molecule of A1) is a gene represented by the following 1) or 2) or 3):

1) The coding sequence is a cDNA molecule shown in a sequence 2 in a sequence table or a genome DNA molecule shown in a sequence 3 in the sequence table;

2) A cDNA molecule or a genome DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by 1) and codes GmNTF2B-1 protein;

3) A cDNA molecule or a genome DNA molecule which hybridizes with the nucleotide sequence limited by 1) or 2) under strict conditions and codes GmNTF2B-1 protein.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The nucleotide sequence encoding GmNTF2B-1 of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified and have 75% or more identity to the nucleotide sequence of GmNTF2B-1 isolated in the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode GmNTF2B-1 and have the same function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 1 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to assess the identity between related sequences.

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

In the above biological material, the expression cassette containing a nucleic acid molecule encoding GmNTF2B-1 (GmNTF 2B-1 gene expression cassette) described in A2) refers to a DNA capable of expressing GmNTF2B-1 in host cells, and the DNA may include not only a promoter which initiates transcription of GmNTF2B-1 but also a terminator which terminates transcription of GmNTF2B-1. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: a constitutive promoter; tissue, organ and development specific promoters and inducible promoters. Suitable transcription terminators include, but are not limited to: an agrobacterium nopaline synthase terminator (NOS terminator), a cauliflower mosaic virus CaMV 35S terminator, a tml terminator and a pea rbcS E9 terminator.

The existing expression vector can be used for constructing a recombinant vector containing the GmNTF2B-1 gene expression cassette. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Corp.) and the like. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The sources of the translational control signals and initiation codons are wide ranging from natural to synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound capable of producing a color change (GUS gene, luciferase gene, etc.), a marker gene for antibiotics (e.g., nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to phosphinothricin as an herbicide, hph gene conferring resistance to hygromycin as an antibiotic, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or a marker gene for chemical resistance (e.g., herbicide resistance), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose, which can be expressed in plants. From the safety of transgenic plants, the transformed plants can be screened directly in stress without adding any selective marker gene.

In the above biological material, the vector may be a plasmid, a cosmid, a phage, or a viral vector. In the invention, the recombinant expression vector can be specifically a recombinant plasmid 16318hGFP-GmNTF2B-1, pCAMBIA1302-GmNTF2B-1, pCAMBIA3301-GmNTF2B-1 or pCAMBIA1305-GmNTF2B-1promoter.

Furthermore, the 16318hGFP-GmNTF2B-1 is a recombinant plasmid obtained by inserting the GmNTF2B-1 gene into a multiple cloning site of 16318hGFP, preferably a recombinant plasmid obtained by inserting a DNA fragment shown by a sequence 2 in a sequence table into a BamHI enzyme cutting site of 16318 hGFP. The pCAMBIA1302-GmNTF2B-1 is a recombinant plasmid obtained by inserting a GmNTF2B-1 gene into a multiple cloning site of the pCAMBIA1302, and preferably is a recombinant plasmid obtained by inserting a DNA fragment shown in a sequence 2 in a sequence table into an NcoI enzyme cutting site of the pCAMBIA 1302. The pCAMBIA3301-GmNTF2B-1 is a recombinant plasmid obtained by inserting a GmNTF2B-1 gene into a multiple cloning site of pCAMBIA3301, and is preferably a recombinant plasmid obtained by inserting a DNA fragment shown in a sequence 2 in a sequence table between Nco I and BstE II enzyme cutting sites of pCAMBIA 3301. The pCAMBIA1305-GmNTF2B-1promoter is a recombinant plasmid obtained by replacing a 35s promoter between Nco I enzyme cutting sites of a pCAMBIA1305 vector with a DNA fragment shown by 1 st to 2000 th nucleotides from the 5' end of a sequence 4 in a sequence table.

In the above biological material, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium.

In the above biological material, none of the transgenic plant cell lines comprises propagation material.

In order to solve the technical problems, the invention also provides a new application of the GmNTF2B-1 protein or the biological material.

The invention provides application of the GmNTF2B-1 protein or the biological material in regulation and control of plant stress tolerance.

The invention also provides application of the GmNTF2B-1 protein or the biological material in cultivating transgenic plants with improved stress tolerance.

The invention also provides application of the GmNTF2B-1 protein or the biological material in plant breeding.

The invention also provides application of the GmNTF2B-1 protein or the biological material in regulating and controlling plant growth.

In the above application, the regulation is enhancement or promotion.

The application of the GmNTF2B-1 protein in serving as a nuclear transport protein also belongs to the protection scope of the invention.

In the above application, the stress tolerance is drought tolerance. The improvement of the drought tolerance of the plant is embodied in any one of the following (1) to (7): (1) improving the survival rate of plants under drought treatment; (2) under drought treatment, increasing the dry weight of plant roots; (3) under drought treatment, increasing the surface area of the plant roots; (4) Reducing the hydrogen peroxide content in the roots and/or leaves of the plants under drought treatment; (5) Under drought treatment, the damaged area proportion of plant leaves is reduced; (6) Reducing the content of superoxide anion free radicals in plant leaves under drought treatment; (7) Under drought treatment, the relative conductivity of the plant root system is reduced. The drought treatment may be PEG treatment or water control treatment.

In the above application, the plant is a dicotyledonous plant. The dicotyledonous plant can be soybean; the soybean is Willimas82.

In order to solve the technical problems, the invention also provides a method for cultivating the transgenic plant with improved stress tolerance.

The method for cultivating the transgenic plant with improved stress tolerance comprises the steps of improving the content and/or activity of GmNTF2B-1 protein in a receptor plant to obtain the transgenic plant; the transgenic plant has higher stress tolerance than the recipient plant.

In the method, the method for improving the expression level and/or activity of the GmNTF2B-1 protein in the recipient plant is to overexpress the GmNTF2B-1 protein in the recipient plant.

In the method, the overexpression method is to introduce a gene coding for GmNTF2B-1 protein into a recipient plant;

the nucleotide sequence of the coding gene of the GmNTF2B-1 protein is a DNA molecule shown as a sequence 2 in a sequence table.

In one embodiment of the invention, the gene coding for the GmNTF2B-1 protein is introduced into the recipient plant by means of a recombinant plasmid pCAMBIA3301-GmNTF2B-1. The recombinant plasmid pCAMBIA3301-GmNTF2B-1 is a vector obtained by inserting a DNA fragment shown in a sequence 2 in a sequence table between Nco I and BstE II enzyme cutting sites of a pCAMBIA3301 vector.

In the above method, the stress tolerance is drought tolerance. The transgenic plant has higher stress tolerance than the acceptor plant in any one of the following X1) -X7):

x1) the hydrogen peroxide content in the transgenic plant root and/or leaf is lower than that of the recipient plant;

x2) the root surface area of the transgenic plant is greater than that of the recipient plant;

x3) the survival rate of the transgenic plant is higher than that of the receptor plant;

x4) the transgenic plant has a higher dry root weight than the recipient plant;

x5) the damaged area ratio of the leaves of the transgenic plant is lower than that of the receptor plant;

x6) the superoxide anion radical content of the transgenic plant leaves is lower than that of the recipient plant;

x7) the relative conductivity of the transgenic plant root system is lower than that of the recipient plant.

In order to solve the technical problems, the invention finally provides a method for promoting the growth and development of plants.

The method for promoting the growth and development of the plant comprises the steps of improving the content and/or activity of GmNTF2B-1 protein in a receptor plant to obtain a transgenic plant; the transgenic plant grows at a higher level than the recipient plant.

In one embodiment of the invention, the gene coding for the GmNTF2B-1 protein is introduced into the recipient plant by means of a recombinant plasmid pCAMBIA1302-GmNTF2B-1. The recombinant plasmid pCAMBIA1302-GmNTF2B-1 is a vector obtained by inserting a DNA fragment shown in a sequence 2 in a sequence table between Nco I enzyme cutting sites of a pCAMBIA1302 vector.

In the above method, the transgenic plant has higher growth development level than the recipient plant in any one of the following Y1) or Y2):

y1) the transgenic plant has a longer root than the recipient plant;

y2) the root surface area of the transgenic plant is greater than that of the recipient plant.

In the above method, the expression vector carrying the encoding gene can be transformed into plant cells or tissues by using conventional biological methods such as Ti plasmid, ri plasmid, plant viral vector, direct DNA transformation, microinjection, conductance, agrobacterium-mediated transformation, etc., and the transformed plant tissues can be cultured into plants.

In the above method, the transgenic plant is understood to include not only the first generation transgenic plant obtained by transforming the GmNTF2B-1 gene into a recipient plant, but also the progeny thereof. For transgenic plants, the gene can be propagated in the species, and can also be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

In the above method, the plant is a dicotyledonous plant. The dicotyledonous plant can be soybean; the soybean can be specifically cultivar Willimas82.

Experiments prove that the GmNTF2B-1 is expressed under drought induction, and the encoded protein is positioned on cell nucleus. The GmNTF2B-1 can improve the drought tolerance of plants, promote the growth and development of the plants, provide a foundation for artificially controlling the expression of stress tolerance related genes, and play an important role in breeding the plants with enhanced stress tolerance.

Drawings

FIG. 1 shows the fluorescent Real-time quantitative (Real-time) PCR result of the drought stress induced expression of GmNTF2B-1.

FIG. 2 shows the localization of GmNTF2B-1 in Arabidopsis protoplasts. A:16318hGFP empty vector control; b: gmNTF2B-1 localizes in the nucleus.

FIG. 3 is a graph showing the effect of GmNTF2B-1 on the growth of soybean hairy roots under normal conditions. A: phenotype of empty vector soybean hairy roots; b: transforming the phenotype of soybean hairy roots of GmNTF 2B-1; c: under normal conditions, carrying out statistical analysis on the total root length of the empty carrier soybean hairy roots and the GmNTF2B-1 soybean hairy root system; d: under normal conditions, the surface areas of the empty carrier soybean hairy roots and the GmNTF2B-1 soybean hairy root systems are statistically analyzed; e: the vertical section of the root tip meristematic region of the empty transgenic carrier soybean hairy root; f: the root tip meristematic region of the soybean hairy root is transferred into GmNTF2B-1 in a longitudinal section. Wherein, the 35S: GFP is transferred empty vector soybean hairy root; gmNTF2B-1 pro: gmNTF2B-1-GFP was transferred into GmNTF2B-1 soybean hairy roots.

FIG. 4 is a graph showing the effect of GmNTF2B-1 on drought tolerance of soybean. A: phenotype of soybean with transferred GmNTF2B-1 and soybean with transferred empty carrier on the 5 th day of drought treatment (the drought treatment mode is water control); b: phenotype of soybean with GmNTF2B-1 and soybean with empty carrier at day 6 after drought treatment; c: hairy root phenotype of GmNTF2B-1 transferred soybean and empty carrier transferred soybean; d: survival rates of the soybeans with the GmNTF2B-1 and the soybeans with the empty carrier after drought treatment; e: after drought treatment, the root surface area of the GmNTF2B-1 soybean and the empty carrier soybean is transferred; f: after drought treatment, transferring the root dry weights of the GmNTF2B-1 soybean and the empty carrier soybean; g: relative expression quantity of GmNTF2B-1 in the soybean hairy root transferred with the GmNTF2B-1 and the soybean hairy root transferred with the empty carrier; h: DAB dyeing results of leaves of soybeans transferred with GmNTF2B-1 and soybeans transferred with empty carriers after drought treatment; i: after drought treatment, NBT dyeing results of leaves of the soybeans transferred with GmNTF2B-1 and the soybeans transferred with empty carriers; j and K are partial enlargements of H and I, respectively; l: after drought treatment, the hydrogen peroxide content in the leaves of the soybeans transferred with GmNTF2B-1 and the soybeans transferred with empty carriers; m: after drought treatment, the damage ratio of the leaves of the GmNTF 2B-1-transferred soybean and the empty carrier transferred soybean is changed. Wherein WT is empty carrier soybean; gmNTF2B-1 pro: gmNTF2B-1 is transferred to GmNTF2B-1 soybean.

FIG. 5 is a qualitative and quantitative experiment of root active oxygen content under drought conditions. A: under normal conditions, performing DAB dyeing on roots of the soybeans transferred with the GmNTF2B-1 and the soybeans transferred with empty carriers; b: after drought treatment, performing DAB dyeing on roots of the soybeans converted to GmNTF2B-1 and the soybeans converted to empty carriers; c: after drought treatment, the root hydrogen peroxide content of the soybeans transferred with GmNTF2B-1 and the soybeans transferred with empty carriers; d: after drought treatment, the relative conductivity of the root systems of the soybeans with the GmNTF2B-1 and the soybeans with the empty carrier is increased.

FIG. 6 shows the positive detection and the determination of soil water content and water potential of empty carrier soybean hairy roots and GmNTF2B-1 soybean hairy roots. A: positive detection of empty carrier soybean hairy roots and GmNTF2B-1 soybean hairy roots; b: during the drought treatment, the water content of the soil changes; c: change of soil water potential during drought treatment.

FIG. 7 shows soybean hairy roots to verify whether the expression of GmNTF2B-1 is induced by drought. A: under normal conditions, the expression condition of a reporter gene GUS in soybean hairy roots of pCAMBIA1305 is transferred; b: under normal conditions, the expression condition of a reporter gene GUS in soybean hairy roots of pCAMBIA1305-GmNTF2B-1promoter is transferred; c:20% PEG simulation of the expression of the reporter gene GUS in soybean hairy roots transformed with pCAMBIA1305 under drought conditions; d:20% PEG to mimic the expression of the reporter gene GUS in the hairy root of soybean transformed with pCAMBIA1305-GmNTF2B-1promoter under drought conditions; e: A. relative expression level of GUS gene in four cases, B, C and D.

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. In the quantitative tests in the following examples, three replicates were set up and the results averaged.

The reagent formulations used in the following examples are as follows:

TABLE 1 cellulase hydrolysate formulation

TABLE 2 PEG4000 solution formulation

TABLE 3W 5 solution formulation

TABLE 4 MMG solution formulation

TABLE 5 WI solution formulation

TABLE 6 GM (Germination Medium) Germination Medium

TABLE 7 IM (Infection Medium) Infection Medium formulation

TABLE 8 Co-cultured Medium formula for CCM

TABLE 9 RIM (Root Induction Medium) Root Induction Medium

The sources of the kits used in the following examples are as follows:

DAB dye liquor kit: the product name is as follows: a DAB color development kit; the commodity product number: DA010; and (4) commodity brands: solarbio; specification: 2X 10mL.

NBT dye liquor kit: the product name is as follows: NBT color kit; the commodity goods number: i023-1-1; and (4) commercial manufacturers: nanjing was built into bioengineering institute Co., ltd; specification: 2X 10mL.

Hydrogen peroxide content determination kit: the product name is as follows: hydrogen peroxide assay kit (colorimetric method); the commodity product number: a064-1-1; and (4) commercial manufacturers: nanjing was established into bioengineering institute Co., ltd; specification: 50 tubes/48 samples.

GUS staining kit: the product name is as follows: a GUS staining kit; the commodity goods number: RTU4032; and (4) commercial manufacturers: zhongke ruitai (Beijing) Biotech limited.

The 16318hGFP vector, pCAMBIA3301 vector and Agrobacterium K599 strain in The following examples are described in The literature "Gao, Y., ma, J., ZHEN, J.C., chen, J.J., chen, M.M., ZHOU, Y.B., et al (2019) The Electron Factor GmEF4 Is Involved in The Response to Drought and Salt Tolerance in Soybean. Int J Mol Sci.20 (12). Doi: 10.3390/ij20123001", publicly available, for The sole purpose of repeating The experiments related to The present invention, and not for other uses.

The pCAMBIA1302 and pCAMBIA1305 vectors described in the following examples are described in "Li, B., liu, Y., cui, X.Y., fu, J.D., zhou, Y.B., zheng, W.J., et al (2019) Genome-Wide characteristics and Expression Analysis of Soybean TGA transformation Factors Identified a Novel TGA Gene analyzed in driven and Salt Tolernce.front Plant Sci.doi: 10.3389/fpls.2019.00549", and are publicly available from the applicant, and the biomaterials are used only for repeating the experiments related to the present invention and are not used for other purposes.

Example 1, obtaining of GmNTF2B-1 protein and its coding gene and analysis of GmNTF2B-1 expression characteristics

1. GmNTF2B-1 protein and obtaining of coding gene thereof

The Williams82 soybean seedlings growing in sand for about 14 days are quickly frozen by liquid nitrogen and stored at-80 ℃ for later use. Total RNA from soybean leaves was extracted by Trizol method (Tianggen) 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 full-length cDNA sequence of the GmNTF2B-1 gene is obtained by the method of 5'RACE and 3' RACE, the nucleotide sequence of the cDNA sequence is shown as a sequence 2 in a sequence table, the protein consisting of 123 amino acid residues is coded and named as GmNTF2B-1, and the amino acid sequence of the protein is shown as a sequence 1 in the sequence table. The genome sequence of the GmNTF2B-1 gene is shown as a sequence 3 in a sequence table.

2. Real-time fluorescent quantitative PCR analysis of expression characteristics of GmNTF2B-1

1. Stress handling

Taking soybean seedlings (only two true leaves) under normal growth conditions, and carrying out the following treatment:

(1) Drought treatment: taking out the soil-cultured soybean seedlings with consistent growth vigor, sucking water on roots, placing on dry filter paper, taking out materials after 1 hour, 3 hours, 6 hours, 12 hours and 24 hours of drought treatment, quickly freezing by using liquid nitrogen, and storing at-80 ℃ for later use.

(2) And (4) comparison treatment: soybean seedlings without any treatment were directly frozen at-80 ℃ as a control (0 hour).

2. Extraction of RNA

Total RNA from soybean leaves was extracted by Trizol method (Tianggen).

3. Obtaining of cDNA

The purified RNA was reverse transcribed into cDNA.

4. Real-time fluorescent quantitative PCR

The cDNA was diluted 50-fold and used as a template for Q-RT-PCR. Q-RT-PCR amplification is carried out on the sample by using a specific primer pair of the non-coding region at the 3' end of the gene, the response condition of the gene to various treatments is analyzed, and actin is used as an internal reference gene. The primer sequences are as follows:

the target gene: f5' GTGGAGCACTACAGCAC-;

R:5’-GGTGGAGATTGAGTGGTGGC-3’；

internal reference gene: f5 'CGGTGGTTCTATCTTGGCATC-3';

R:5’-GTCTTTCGCTTCAATAACCCTA-3’。

Q-RT-PCR in ABI

7000 real-time fluorescence quantitative PCR instrument, one parallel experiment set 3 times of repetition. The method reported by Livak KJ and Schmittgen TD (2001), 2 ^-ΔΔCT And calculating the relative expression amount. Delta C _T ＝(C _T.Target －C _T.Actin ) _Time _x －(C _T.Target －C _T.Actin ) _Time ₀ . Time x denotes renMean Time point, time ₀ Represents the expression of a 1-fold amount of the target gene after actin correction.

The results are shown in FIG. 1. Under the condition of drought treatment, the expression quantity of the GmNTF2B-1 is obviously increased 1h after the treatment, the peak value is reached 8h after the treatment, the numerical value is about 6 times of that before the treatment, and the expression quantity begins to slowly fall back 12h after the treatment. Therefore, gmNTF2B-1 is obviously expressed by drought induction.

Example 2 GmNTF2B-1 subcellular localization analysis

1. Construction of expression vectors

1. Cloning of GmNTF2B-1 Gene

Primers GmNTF2B-1-16318hGFP-F and GmNTF2B-1-16318hGFP-R are designed according to the GmNTF2B-1 gene sequence, bamH I enzyme digestion recognition sites are respectively introduced into the tail ends of the primers, and the GmNTF2B-1 is amplified by PCR by taking soybean cDNA as a template. The primer sequences are as follows:

GmNTF2B-1-16318hGFP-F：5’-TATCTCTAGAGGATCCATGGATCCAGACGCG-3’；

GmNTF2B-1-16318hGFP-R：5’-TGCTCACCATGGATCCTGCATAGTTTAA-3’。

the PCR amplification product was subjected to 1.2% Agarose Gel electrophoresis, and a band of about 1.6Kb was recovered and purified using Agarose Gel DNA Purification Kit Ver.2.0.

2. Construction of recombinant expression vectors

(1) And (3) digesting the purified PCR product recovered in the step (1) by using a restriction enzyme BamH I, and recovering the digested product.

(2) The 16318hGFP vector is digested with restriction enzyme BamH I, and the vector backbone is recovered.

(3) And (3) connecting the enzyme digestion product in the step (1) with the vector skeleton in the step (2) to obtain a recombinant plasmid 16318hGFP-GmNTF2B-1.

(4) The ligation product obtained in step (3) was electrically shocked to transform TOP10 strain (purchased from Beijing Tiangen corporation) and cultured overnight at 37 ℃, and positive clones were picked for sequencing.

The sequencing result shows that: the recombinant plasmid 16318hGFP-GmNTF2B-1 is a vector obtained by inserting a DNA fragment shown in a sequence 2 in a sequence table between BamH I enzyme cutting sites of the 16318hGFP vector.

2. Transformation of Arabidopsis protoplasts

1. Arabidopsis thaliana (Columbia ecotype (Col-0)) was grown in a soil culture room.

2. Under the condition of good growth, the protoplast is prepared by taking the leaf before blooming.

3. Cutting the middle well-grown leaves, and cutting into 0.5-1mm wide strips with a blade.

4. The cut leaves are put into the prepared enzymolysis liquid (about 10-20 leaves are needed for every 5-10mL of enzymolysis liquid). The leaves were completely immersed in the enzymatic hydrolysate with tweezers.

5. The vacuum pump vacuums in the dark (wrapped in tinfoil) for 30 minutes. At this time, PEG4000 solution can be prepared, 200 muL and 1000 muL of gun heads are sharpened, so that the suction and the beating are mild during the operation, the PEG4000 solution can be stored for five days after one-time preparation, but the preparation is preferably carried out currently, each sample needs 100 muL of PEG4000 solution, and the total preparation amount of the solution can be adjusted according to the amount of the experimental sample.

6. The enzymatic digestion was continued in the dark without shaking at room temperature (28 ℃ C., 50rpm shaking for at least 3 h). When the enzymolysis liquid turns green, the culture dish is slightly shaken to promote the release of the protoplast. At this point a certain amount of W5 solution was pre-cooled.

7. The protoplasts were examined under a microscope in solution, and the Arabidopsis mesophyll protoplasts were approximately 30-50 μm in size.

8. The enzyme solution containing protoplasts was diluted with an equal amount of W5 solution before removing undissolved leaves by filtration.

9. A35-75 μm nylon membrane or a 60-100 mesh sieve is wetted with a W5 solution, and then the enzymatic hydrolysate containing protoplasts is filtered.

10. Centrifuging with 30mL round bottom centrifuge tube at 4 deg.C and 100g for 1-2min to precipitate protoplast, and removing supernatant as much as possible. The protoplasts were then gently resuspended with 10mL of W5 solution pre-cooled on ice.

11. The protoplasts were allowed to stand on ice for 30 minutes.

The following operations were carried out at room temperature 23 ℃:

12. centrifuging for 8-10min at 100g to precipitate protoplast. The W5 solution was removed as much as possible without touching the protoplast pellet. Then using a proper amount of MMG solution (1 mL) resuspended protoplasts to a final concentration of 2X 10 ⁵ one/mL.

13. mu.L or 20. Mu.L of DNA (10-20. Mu.g of about 5-10kb of the recombinant plasmid 16318hGFP-GmNTF2B-1 prepared in step one) was added to a 2mL EP tube. The 16318hGFP empty vector was also used as a control.

14. Add 100. Mu.L protoplasts (2X 10) ⁴ One), gently mix.

15. Add 110. Mu.L of PEG solution and gently tap the tube to mix thoroughly (approximately 6-10 samples can be transformed each time).

16. The transformation mixture was induced for 20-30min (depending on the experimental situation, longer transformation time may be required for higher expression).

17. The conversion reaction was stopped by diluting the conversion mixture with 400-440. Mu.L of W5 solution at room temperature and gently shaking the tube upside down to mix well.

18. Centrifuge at 100g for 2min at room temperature and then remove the supernatant. Then, 1mL of W5 solution was added and the mixture was washed once by suspension, centrifuged at 100g for 2min, and the supernatant was removed.

19. The protoplasts were gently resuspended in the multi-well tissue culture dish with 1mL of WI solution.

20. Protoplasts were induced at room temperature (20-25 ℃) for more than 18 hours. Then GFP tag expression was observed under confocal laser microscopy.

3. Microscopic examination of Arabidopsis protoplasts

Protoplasts after dark culture for 18h were tableted, and then GFP (green fluorescent protein) fluorescence was observed in a Laser scanning confocal microscope (Bio-Rad MicroRadiance) (Laser scanning confocal, LSMC) and scanned. The operating parameters of the LSCM are as follows: ex =488nm, em =525 ± 15nm, power =10%, zoom7, medium speed scan, frame512 × 512. The software is TIME-COARSE and PHOTOSHOP5.0.

The results are shown in FIG. 2. The results show that: compared to the fluorescent signal of the empty vector control, which is dispersed throughout the cell, gmNTF2B-1 is mainly localized in the nucleus.

Example 3 Effect of GmNTF2B-1 protein on growth of Soybean hairy root

1. Construction of recombinant expression vectors

1. Cloning of GmNTF2B-1 Gene

Primer pairs (GmNTF 2B-1-1302-F and GmNTF 2B-1-1302-R) are designed according to the sequence of the GmNTF2B-1 gene, nco I enzyme digestion recognition sites are respectively introduced into the tail ends of the primers, and GmNTF2B-1 is amplified by PCR by taking soybean cDNA as a template. The primer sequences are as follows:

GmNTF2B-1-1302-F：5’-GGGACTCTTGACCATGATGGATCCAGACGCG-3’；

GmNTF2B-1-1302-R：5’-TCAGATCTACCCATGGTGCATAGTTTAA-3’。

2. Construction of recombinant expression vectors

(1) The purified PCR product recovered in step 1 was digested with restriction enzyme Nco I, and the digested product was recovered.

(2) The pCAMBIA1302 vector was digested with restriction enzyme Nco I, and the vector backbone was recovered.

(3) And (3) connecting the enzyme digestion product in the step (1) with the vector skeleton in the step (2) to obtain a recombinant plasmid pCAMBIA1302-GmNTF2B-1.

The sequencing result shows that: the recombinant plasmid pCAMBIA1302-GmNTF2B-1 is a vector obtained by inserting a DNA fragment shown in a sequence 2 in a sequence table between NcoI enzyme cutting sites of a pCAMBIA1302 vector.

2. Obtaining and identifying transgenic soybean hairy root

1. Preparing recombinant agrobacterium: the recombinant plasmid pCAMBIA1302-GmNTF2B-1 is transformed into agrobacterium GV3101 (Beijing Bomaide biotechnology, inc.) to obtain recombinant agrobacterium.

2. Bean sterilization and culture: the whole process is carried out in a fume hood, and 200 beans (variety Williams82, germplasm bank of Chinese academy of agricultural sciences) are placed in a culture dish with the diameter of 15cm and placed in a closed container. Then 100mL of bleech (Huawang bleaching water purchased from underground competitive products supermarket of Bian Shang market in Beijing) is taken and added with 4mL of concentrated hydrochloric acid (purchased from Xilongan chemical industry), the mixed solution is immediately placed in a closed container, and the sealed container is kept stand for 16-18h. Taking out the beans in the closed container the next day, placing on a super clean bench, opening the cover of the culture dish, and blowing for 0.5-1h. Beans are placed in culture dishes containing GM medium, 10 beans/dish, 24 ℃,18h (light)/6 h (dark), and cultured for 3-4 days, and the condition of seeds is seen, and the radicle grows to about 2 cm.

3. Bacterial liquid culture: the recombinant Agrobacterium obtained in step 1 was inoculated into 1mL of LB liquid medium (containing 50mg/mL rifampicin, 100mg/mL kanamycin) and cultured at 28 ℃ and 300rpm for about 30 hours for use. After 2-3 days, 30. Mu.L of the bacterial solution was added to 30mL of LB liquid medium (containing 50mg/mL rifampicin, 100mg/mL kanamycin) and cultured to OD ₆₅₀ And (5) =0.5-0.7. Centrifuging (4000rpm, 10min), adding IM liquid medium (without Agar), and making the OD of the culture solution ₆₅₀ ＝0.6。

4. Obtaining an explant: cutting bean which germinates for 3-5 days from 0.3-0.5cm of hypocotyl, cutting cotyledon into two parts, and removing terminal bud.

5. Transformation and co-culture: the inoculum obtained in step 3 and the explant obtained in step 4 were mixed for 30min and shaken 2-3 times, during which filter paper was placed on CCM (1 sheet/dish). The bacterial solution was decanted, the cotyledonary node was placed on several layers of filter paper, excess bacterial solution was aspirated off, and then placed on CCM with filter paper (25/dish) for 3 days at 28 ℃ in the dark.

6. And (3) sporophyll node degerming: washed 4-5 times with liquid RIM (without Agar) and blotted dry on filter paper. The solid RIM medium is inserted upside down, the hypocotyl is upward, and the hairy root grows after about 10 to 14 days of culture, thus obtaining the hairy root of the soybean transformed with GmNTF2B-1.

The recombinant plasmid pCAMBIA1302-GmNTF2B-1 is replaced by pCAMBIA1302 vector according to the method, and other steps are kept unchanged, so that the empty vector soybean hairy roots are obtained.

7. And performing molecular identification on the transgenic GmNTF2B-1 soybean hairy roots and the transgenic empty vector soybean hairy roots at the DNA level.

The primer sequences identified were as follows:

primer pair for identifying DNA level of empty vector soybean hairy roots:

F：5'-AGTAAAGGAGAAGAACTTTTCACTG-3'；

R：5'-TTTGTATAGTTCATCCATGCCATGAT-3'；

the expected band is 711bp.

Transferring GmNTF2B-1 soybean hairy root DNA level identification primer pair:

F：5’-GACTCTTGACCATGGATGGATCCAGACGCG-3；

R：5’-TGACGAGGGTGTCTCCCTCAAACTT-3’；

the expected band is 751bp.

The results are shown in FIG. 3G. The empty vector soybean hairy roots are amplified to obtain a target band with the size of 711bp, and the GmNTF2B-1 soybean hairy roots are amplified to obtain a target band with the size of 751bp.

8. And detecting the relative expression quantity of the target gene GmNTF2B-1 in the transgenic soybean hairy roots and the transgenic empty vector soybean hairy roots on the cDNA level. CYP2 gene is used as an internal reference gene. The primer sequences are as follows:

the primer sequence of the internal reference CYP2 gene:

F：5'-ACCAGAATTGCATTACCAGTTGA-3'；

R：5'-GGTGCCGAGACTCATGAAGATAT-3'；

the target gene primer sequence:

F：5’-GTGGAGCACTACTACAGCAC-3’；

R：5’-GGTGGAGATTGAGTGGTGGC-3’。

the results are shown in FIG. 3H. The results show that: compared with the expression level of GmNTF2B-1 in the empty vector soybean hairy roots, the expression level of GmNTF2B-1 in the empty vector soybean hairy roots is increased by 14 times.

3. Positive identification of transgenic soybean hairy roots

And (3) cutting the roots of the soybean hairy roots of the transferred GmNTF2B-1 and the transferred empty carrier soybean hairy roots obtained in the step two into slices by a blade by adopting a bare-handed slicing method, and observing the fluorescent signal of the GFP fluorescent protein under a laser confocal microscope (LSM 880with Airyscan, a major engineering building of the institute of crop science of Chinese academy of agricultural sciences).

The results are shown in FIGS. 3E and 3F. The results show that: fluorescent signals are observed in the soybean hairy root with the transferred GmNTF2B-1 and the soybean hairy root with the transferred empty carrier, and the soybean hairy root with the transferred GmNTF2B-1 and the soybean hairy root with the transferred empty carrier are successfully obtained.

4. Phenotypic identification of transgenic soybean hairy roots

And (3) observing the root growth and development conditions of the transgenic GmNTF2B-1 soybean hairy roots (n = 12) obtained in the step two and the transgenic empty vector soybean hairy roots (n = 12), and comparing the root length and the root surface area. The experiment was repeated three times and the results were averaged ± standard deviation.

The root growth and development results are shown in FIGS. 3A and 3B. The result shows that under the normal growth condition, the GmNTF2B-1 can promote the growth of soybean root systems, and is mainly shown in the aspect of promoting the development of the root systems.

The root length and root surface area statistics are shown in fig. 3C and fig. 3D. The results show that: the average root length of the soybean hairy root transferred with the GmNTF2B-1 is 16.5cm; the average root length of the empty transfer carrier soybean hairy roots is 11.1cm; the root surface area of the soybean hairy root transformed with GmNTF2B-1 was 8.7cm on average ² (ii) a The average root surface area of the empty carrier soybean hairy roots is 4.1cm ² . The root length and the root surface area of the soybean hairy root with the transferred GmNTF2B-1 are obviously larger than those of the soybean hairy root with the transferred empty carrier.

Example 4 Effect of GmNTF2B-1 protein on stress tolerance of Soybean

1. Construction of recombinant expression vectors

1. Cloning of GmNTF2B-1 Gene

Designing a primer pair (GmNTF 2B-1-3301-F and GmNTF 2B-1-3301-R) according to the sequence of the GmNTF2B-1 gene, respectively introducing Nco I and BstE II enzyme digestion recognition sites at the tail end of the primer, and carrying out PCR amplification on the GmNTF2B-1 by taking soybean cDNA as a template.

The primer sequences are as follows:

GmNTF2B-1-3301-F：5’-GGACTCTTGACCATGATGGATCCAGACGCG-3’；

GmNTF2B-1-3301-R：5’-ATTCGAGCTGGTCACCTCATGCATAGTTTAA-3’。

2. Construction of recombinant expression vectors

(1) The purified PCR product recovered in step 1 was digested with restriction enzymes Nco I and BstE II, and the digested product was recovered.

(2) The pCAMBIA3301 vector was digested with restriction enzymes Nco I and BstE II, and the vector backbone was recovered.

(3) And (3) connecting the enzyme digestion product in the step (1) with the vector skeleton in the step (2) to obtain a recombinant plasmid pCAMBIA3301-GmNTF2B-1.

(4) The ligation product of step (3) was shocked to transform TOP10 strain (purchased from Beijing Tiangen), cultured overnight at 37 ℃, and positive clones were picked for sequencing.

The sequencing result shows that: the recombinant plasmid pCAMBIA3301-GmNTF2B-1 is a vector obtained by inserting a DNA fragment shown in a sequence 2 in a sequence table between Nco I and BstE II enzyme cutting sites of a pCAMBIA3301 vector.

2. Obtaining transgenic soybean hairy root

1. Agrobacterium transformation

(1) Reactivating Agrobacterium K599 strain stored at-80 deg.C, culturing bacterial liquid OD at 28 deg.C and 200rpm ₆₀₀ To 0.6-1.0.

(2) 1mL of the activated cell suspension was aspirated into 40mL of LB medium containing streptomycin (50 mg/L), and cell suspension OD was cultured at 28 ℃ and 200rpm ₆₀₀ To about 0.6.

(3) Transferring the bacterial liquid into a sterilized 50mL centrifuge tube, inserting the centrifuge tube into ice, carrying out ice bath for 30min, centrifuging the centrifuge tube at 5000rpm for 5min, and removing the supernatant.

(4) With 2mL of sterilized 20mM CaCl ₂ Resuspend the cells, mix well and dispense 200 μ L per tube. Quick freezing with liquid nitrogen, and storing at-80 deg.C.

(5) Separately, 4. Mu.L (about 2. Mu.g) of the recombinant plasmid pCAMBIA3301-GmNTF2B-1 of step one was added to newly prepared 200. Mu.L of the Agrobacterium infected state, and after ice-bath for 5min, it was frozen with liquid nitrogen for 8min, and then rapidly placed in a water bath at 37 ℃ for 5min.

(6) 600. Mu.L of LB medium without antibiotics was added to each tube and cultured on a shaker at 200rpm at 28 ℃ for 4-6h.

(7) Centrifuging at 5000rpm for 2min, removing part of supernatant to leave 100-200 μ L of liquid in each tube, suspending the liquid with a pipette gun, and culturing on LB solid medium uniformly coated with Kan (50 mg/L) and Str (50 mg/L) in an inverted manner at 28 ℃ until a single colony grows out.

(8) And (4) picking a single colony, performing amplification culture, and screening to obtain a positive colony.

(9) The method comprises uniformly sowing Willimas82 soybean seeds in vermiculite sterilized at high temperature and high pressure, adding appropriate amount of nutrient solution, and culturing under high humidity condition until soybean cotyledon forms (about 5 days). Agrobacterium K599 transformed with a vector for expressing a target gene (recombinant plasmid pCAMBIA3301-GmNTF 2B-1) was activated while sowing soybean, and cultured at 28 ℃ on a solid medium resistant to Kan (50 mg/L) and Str (50 mg/L) until colonies grew out.

A colony of Agrobacterium was picked at the R with a 1mL syringe needle and injected at the soybean cotyledon node. Culturing under high temperature and high humidity condition until soybean hairy root grows out, and obtaining the soybean hairy root transformed with GmNTF2B-1.

The recombinant plasmid pCAMBIA3301-GmNTF2B-1 is replaced by pCAMBIA3301 vector according to the above method, and other steps are kept unchanged, so as to obtain the empty vector soybean hairy root.

2. Molecular characterization of transgenic Soybean hairy roots

The trans-GmNTF 2B-1 soybean hairy roots and the trans-empty vector soybean hairy roots obtained in the step 1 were identified on the DNA level. The primer sequences identified were as follows:

primer pair for identifying DNA level of empty vector soybean hairy roots:

F：5'-ATGGTGAGCAAGGGCGAGGAG-3'；

R：5'-TCAAAGATCTACCATGTACAGCTCGT-3'；

the expected band is 700bp.

Transferring GmNTF2B-1 soybean hairy root DNA level identification primer pair:

F：5’-CAAACGAAATGAAAGTTGGTAGCAAAAGGA-3；

R：5’-CTCTCTTCTCTATTCTCTCCCACTTCTCTC-3’；

the expected band is 751bp.

The results of the partial sample identification are shown in FIG. 6A. The results show that: no target bands of 700bp and 751bp are detected in a negative control (Williams 82), 700bp target bands are detected in 5 empty vector soybean hairy roots, and 751bp target bands are detected in 7 transformed GmNTF2B-1 soybean hairy roots; therefore, 3 empty vector-transferred hairy root strains (# 1, #2, # 3) and 3 GmNTF2B-1 soybean hairy root strains (# 1, #2, # 3) were selected for the following experimental analysis.

3. Detection of expression quantity of target gene GmNTF2B-1 in transgenic soybean hairy roots

And (3) detecting the relative expression quantity of the target gene GmNTF2B-1 in the transgenic soybean hairy roots and the transgenic empty vector soybean hairy roots obtained in the step 1 on the cDNA level. CYP2 gene is used as an internal reference gene. The primer sequences are as follows:

an internal reference CYP2 gene primer pair:

F：5'-ACCAGAATTGCATTACCAGTTGA-3'；

R：5'-GGTGCCGAGACTCATGAAGATAT-3'；

the target gene primer pair:

F：5’-GTGGAGCACTACTACAGCAC-3’；

R：5’-GGTGGAGATTGAGTGGTGGC-3’。

the results are shown in FIG. 4G. The results show that: compared with the expression level of GmNTF2B-1 in the empty vector soybean hairy roots, the expression level of GmNTF2B-1 in the empty vector soybean hairy roots is increased by 14 times.

3. Drought tolerance identification of GmNTF2B-1 transgenic soybeans and empty vector transgenic soybeans

To evaluate the drought tolerance effect of the GmNTF 2B-1-transferred soybeans and the empty-carrier-transferred soybeans. And (3) carrying out drought tolerance identification on the transformed GmNTF2B-1 soybean hairy roots and the transformed empty carrier soybean hairy roots (100 in each) obtained in the step two. Three replicates were set and the results averaged. The method comprises the following specific steps: transplanting the plants only containing the hairy root systems of the transgenic soybeans into nutrient soil, carrying out drought water control treatment after the plants normally grow for 10 days, respectively obtaining transgenic GmNTF2B-1 soybeans and transgenic empty carrier soybeans after the plants are treated for 5-6 days, respectively observing phenotypes of the transgenic GmNTF2B-1 soybeans and the transgenic empty carrier soybeans, photographing and carrying out statistical analysis on partial root system parameters (survival rate, root surface area and root dry weight parameters).

1. Determination of soil water content and water potential in drought treatment process

In the drought treatment process, 3 identical positions in the soil are respectively selected at the same time every day and are respectively measured for 3 times. The determination of the soil water content and the water potential is respectively determined by a professional instrument, and the name and the goods number of the professional instrument and a manufacturer are as follows:

the product name is as follows: a soil moisture detector JC-TS; order number: TR-0011; the model is as follows: JC-TS; the manufacturer: qingdao environmental group Limited corporation of Jumbo corporation;

the product name is as follows: a soil water potential determinator JC-TSS-2; order number: TR-0014; the model is as follows: JC-TSS-2; the manufacturer: qingdao Juan environmental protection group Co.

The results are shown in FIG. 6B. The results show that: the water content of the soil is reduced from 46 percent before the beginning of water control to 4 percent at the end, and the water potential of the soil is reduced from-0.02 MPa before the beginning of water control to-6.54 MPa at the end. The drought treatment condition is proved to meet the requirements of practical experiments.

2. Phenotype after drought treatment

Phenotype observations after drought treatment are shown in fig. 4A, fig. 4B, and fig. 4C. The results show that: the empty-carrier transferred soybeans are more sensitive to drought treatment, leaf wilting and withering appear earliest, hairy roots grow slowly, and the growth vigor is obviously weaker than that of the transferred GmNTF2B-1 soybeans; the GmNTF 2B-1-transferred soybeans exhibited a certain tolerance to drought treatment.

3. Survival after drought treatment, root surface area and root Dry weight parameters

The results of the survival rate, root surface area and root dry weight parameters are shown in fig. 4D, 4E and 4F. The results show that: under drought conditions, the average survival rates of the empty carrier soybean and the GmNTF2B-1 soybean are respectively 20% and 80%, and the corresponding average root surface areas are respectively 31.21cm ² And 69.56cm ² The average dry root weights were 0.27g and 0.34g, respectively. Therefore, the drought tolerance of the soybean can be improved by over-expressing GmNTF2B-1.

Example 5 GmNTF2B-1 protein improves drought tolerance in soybeans by reducing the hydrogen peroxide content in leaves

Test materials: example 4 soybeans with transferred GmNTF2B-1 and soybeans with transferred empty carrier after drought treatment.

1. DAB dyeing of leaves

Method for treating H with Diaminobenzidine (DAB) by referring to commercial kit (DAB dye solution kit) ₂ O ₂ Is generated for localization. Six days after drought treatment, leaves of the same part of the same plant of GmNTF 2B-1-transgenic soybean and empty carrier-transgenic soybean were cut, respectively, the cut was immersed in an aqueous solution (pH = 3.8) containing 1mg/mL DAB, and reacted for 8H to allow DAB to be absorbed and reacted with H in the leaves ₂ O ₂ And a peroxidase. Then, the leaves are cut into leaf sections of about 3cm and boiled in 95% ethanol solution for 10min for fixing and decoloring, the leaves are placed in saturated chloral hydrate for transparency after the chlorophyll is completely faded, and the transparent leaves can be placed in 50% glycerol for microscopic observation.

The results are shown in FIGS. 4H and 4J. The results show that: after drought treatment, the leaves of the empty vector-transferred soybeans are obviously dyed more deeply than the leaves of the GmNTF 2B-1-transferred soybeans. The deeper the dyeing, the higher the hydrogen peroxide content in the leaves, and the less drought resistant the plant, indicating that GmNTF2B-1 improves the drought resistance of soybean by reducing the hydrogen peroxide content in the plant.

2. Leaf NBT staining

Detecting superoxide anion free radical O by Nitrogen Blue Tetrazole (NBT) tissue staining method according to method provided by commercial kit (NBT staining solution kit) ₂ ^- Is generated. Six days after the drought treatment, the leaves of the same parts of the same plants of the GmNTF 2B-1-transgenic soybeans and the empty vector-transgenic soybeans were cut, the leaves were placed in 10mM phosphate buffer (pH = 7.8) containing 0.1% NBT (w/v) for vacuum infiltration for 20min, the leaves were then placed in 95% ethanol solution for boiling for 10min for fixation and decolorization, the leaves were placed in saturated chloral hydrate for transparence after complete removal of chlorophyll, and the transparent leaves were placed in 50% glycerol for microscopic observation.

The results are shown in FIG. 4I and FIG. 4K. The results show that: after drought treatment, the leaves of the empty vector-transferred soybeans are obviously dyed more deeply than the leaves of the transferred GmNTF2B-1 soybeans. The deeper the staining, the O in the leaves ₂ ^- Content (wt.)The higher the plant is, the less drought resistant the plant is, which indicates that the GmNTF2B-1 reduces the O in the plant ₂ ^- The content of the soybean protein is increased to improve the drought resistance of the soybean.

3. Leaf hydrogen peroxide content determination

The method provided by a commercial kit (hydrogen peroxide content determination kit) is implemented according to the following steps:

1. preparation of tissue samples

According to the tissue mass (g): homogenizing in ice bath with the volume (mL) of the first reagent being (1) to 10, taking 0.1g of soybean leaves subjected to drought treatment in the experiment, and adding 1mL of the first reagent; transferring to an EP tube, centrifuging for 10min at 4 ℃ with a reagent of which the volume is up to 1mL and 8000g, taking the supernatant, and placing on ice for testing.

2. The following reagents were added in order to the EP tube:

TABLE 10, H ₂ O ₂ Operation table for content measurement

Adding reagent IV to dissolve the precipitate, standing at room temperature for 5min, pouring into a cuvette, and measuring the light absorption value A at 415 nm. The comparison tube can be made only once. Calculate Δ a = a assay-a control.

Note: reagent one is volatile, reagent one must be pre-cooled and then ground on ice.

The results are shown in FIG. 4L. The results show that: the hydrogen peroxide content in the empty transfer carrier soybean leaves is 1.18 mu mol/g & FW, the hydrogen peroxide content in the transferred GmNTF2B-1 soybean is 0.68 mu mol/g & FW, and the hydrogen peroxide content in the empty transfer carrier soybean leaves is obviously higher than that of the transferred GmNTF2B-1 soybean. The GmNTF2B-1 is proved to improve the drought resistance of the soybean by reducing the content of hydrogen peroxide in the plant.

4. Determination of damaged area proportion of blade

The conversion of the damaged area proportion of the blade needs to be completed by using a gray level analysis function of graphic analysis software ImageJ (software official network https:// image j. Nih. Gov/ij /). The method comprises the following specific steps:

1. after the Image J software is opened, a File above the software is clicked, an Open is clicked in a pull-down mode, and the scanned picture is pulled into the software. Then click on the Image above the software, drop down the first Type, click it to change it to 8-bit.

2. Click on analyze, pull down click set measure, click on the four options outlined in the graph. Click ok then determine. And then clicking the process, clicking the subset background downwards, only drawing one option of the light background in the graph, and clicking ok.

3. And (3) after the step 2 is completed, clicking analyze, pulling down to form set scale, clicking the analyze, popping up a small box, changing English in the blank box of unit of length into pixels, and clicking OK.

4. And 3, clicking a plurality of irregular circles below the file, selecting heart-shaped circles, and clicking free selections. And clicking Edit, and then pulling down to generate the invert, and clicking.

5. And 4, carrying out gray level statistics after the step 4 is completed. And selecting the bright and colored areas in the image by using a mouse, and enclosing the bright and colored areas as much as possible. Then clicking the measurement appearing in the anlyze pull-down, and popping up the gray scale statistic value of the selected area, and marking the gray scale statistic value as A1; the gray scale value of the whole area is calculated in the same operation and is marked as A2. Calculating the damaged area proportion of the blade according to the following formula: the ratio of damaged area of the blade = A1/A2 × 100%.

The results are shown in FIG. 4M. The results show that: the damaged area proportion of the leaves of the empty carrier soybean leaves is 32.1 percent, the damaged area proportion of the leaves of the GmNTF2B-1 soybean is 10.8 percent, the damaged area proportion of the leaves of the empty vector-transferred soybean is obviously higher than that of the transferred GmNTF2B-1 soybean.

Example 6 GmNTF2B-1 protein improves drought tolerance in soybeans by reducing hydrogen peroxide content in root systems and relative conductivity

1. DAB dyeing of root system

The experimental procedure was the same as for the DAB staining procedure for leaves in example 5.

The results are shown in FIGS. 5A and 5B. The results show that: there was no significant difference between the two before drought treatment. After drought treatment, the dyeing degree of the empty carrier soybean hairy root system is obviously deeper than that of the GmNTF2B-1 soybean hairy root system. The deeper the dyeing, the higher the hydrogen peroxide content in the leaves, and the less drought resistant the plant, further confirming that GmNTF2B-1 improves the drought resistance of soybean by reducing the hydrogen peroxide content in the plant.

2. Root system hydrogen peroxide content determination

The experimental method is the same as that of the hydrogen peroxide content measurement of the leaf blade in the example 5.

The results are shown in FIG. 5C. The results show that: before drought treatment, the hydrogen peroxide content of the roots of the soybeans transferred with GmNTF2B-1 and the soybeans transferred with empty carriers is not very different, and is about 0.45 mu mol/g.FW, and the hydrogen peroxide content of the roots of the soybeans transferred with GmNTF2B-1 is slightly lower. After drought treatment, the hydrogen peroxide content of the soybean and the soybean obviously rises, but the hydrogen peroxide content (1.48 mu mol/g & FW) of the roots of the soybeans transferred with GmNTF2B-1 is obviously lower than that of the soybeans transferred with empty carriers (2.02 mu mol/g & FW). Further proves that the drought resistance of the soybean is improved by reducing the content of hydrogen peroxide in the plant by the GmNTF2B-1.

3. Determination of relative conductivity of root system

Taking plant root tips with the same size, ensuring the integrity of roots as much as possible, washing the plant root tips with tap water, washing the plant root tips with a steam room water for three times, sucking surface water with filter paper, shearing root tissues into almost the same size, weighing 0.3g, repeating the treatment for 3 times, putting the plant root tips into a clean graduated test tube, adding 10mL of deionized water, and immersing a sample. Placing the test tube in a shaking conductometer (DDS-11A) to measure the conductivity S of deionized water ₀ Then, the test tubes are shaken up fully, and the conductivity S of the solution is measured ₁ Sealing the test tubes, placing in boiling water bath to kill plant tissue, taking out the test tubes, cooling to room temperature with tap water, balancing at room temperature for min, shaking, and measuring final conductivity S ₂ And calculating the relative conductivity according to the following formula: relative conductivity = (S) ₁ -S ₀ )/(S ₂ -S ₀ )。

The results are shown in FIG. 5D. The results show that: before drought treatment, the relative conductivity of the roots of the soybeans transferred with the GmNTF2B-1 and the soybeans transferred with empty carriers is not very different, and is about 0.18, and the relative conductivity of the roots of the soybeans transferred with the GmNTF2B-1 is slightly lower. After drought treatment, the relative conductivity of the soybean and the soybean is obviously improved, but the relative conductivity of the roots of the soybeans transferred with GmNTF2B-1 (0.58) is obviously lower than that of the soybeans transferred with an empty carrier (0.81).

Example 7 verification of whether Gene GmNTF2B-1 is really expressed by drought induction

1. Construction of recombinant expression vectors

1. Cloning of GmNTF2B-1-promoter

A primer pair (GmNTF 2B-1-promoter-1305-F and GmNTF 2B-1-promoter-1305-R) is designed according to the sequence of the GmNTF2B-1 gene, ncoI and BstEII enzyme digestion recognition sites are respectively introduced into the tail end of the primer, and the GmNTF2B-1promoter (GmNTF 2B-1-promoter) is amplified by PCR by taking soybean genome DNA as a template. The primer sequences are as follows:

GmNTF2B-1-promoter-1305-F：

5’-ATGACCATGATTACGAATTCCAAACGAAATGAAAGTTGGT-3’；

GmNTF2B-1-promoter-1305-R：5’-TACCCTCAGATCTACCATGGCTCTCTTCTCTATTCTCTCC-3’。

the PCR amplification product was subjected to 1.2% Agarose Gel electrophoresis, and a band of about 2.0Kb was recovered and purified using Agarose Gel DNA Purification Kit Ver.2.0.

2. Construction of recombinant expression vectors

(2) The pCAMBIA1305 vector was digested with restriction enzyme Nco I, and the vector backbone was recovered.

(3) And (3) connecting the enzyme digestion product in the step (1) with the vector skeleton in the step (2) to obtain a recombinant expression vector pCAMBIA1305-GmNTF2B-1.

The sequencing result shows that: the recombinant expression vector pCAMBIA1305-GmNTF2B-1promoter is a vector obtained by replacing a 35s promoter between Nco I enzyme cutting sites of the pCAMBIA1305 vector with a DNA fragment shown by 1 st to 2000 th nucleotides from 5' end of a sequence 4 in a sequence table.

2. Obtaining of transgenic soybean hairy root with pCAMBIA1305-GmNTF2B-1promoter

The procedure is as in step two of example 4.

The recombinant expression vector pCAMBIA1305-GmNTF2B-1promoter was replaced with pCAMBIA1305 vector to obtain a transgenic soybean hairy root.

3. Drought treatment

The hairy root of the transgenic soybean with good growth condition and similar growth condition and the hairy root of the transgenic soybean with good growth condition and similar growth condition are taken and placed in MS ₀ Liquid medium (purchased from Beijing Mengyeme) and MS plus 20% PEG ₀ In the liquid culture medium, the normal growth condition and the drought growth condition are simulated respectively. After 6 hours of treatment, GUS staining was performed.

4. GUS staining

1. Taking materials

The roots were cut and placed in a10 mL centrifuge tube.

2. Dyeing process

The GUS staining solution in the kit (GUS staining kit: product name: GUS staining kit; commercial product number: RTU4032; commercial product manufacturer: zhongkuitai (Beijing) Biotech Co., ltd.) was added to the complete covering material, wrapped with tinfoil paper and shaken for 10 minutes or more until blue color appeared, and stained for more than 30 minutes for insurance.

3. Decolorization of

Replacing the staining solution with absolute ethyl alcohol, shaking for more than 30 minutes, and repeating for 2-3 times.

4. Observation of

And (4) observing under a body type microscope (brand: karl. Zeiss; model: stemi 508; major engineering building of crop science research institute of Chinese academy of agricultural sciences).

The results are shown in FIGS. 7A-7D. The results show that: under normal growth conditions, there was no significant difference between the two. After drought treatment, the dyeing degree of the soybean hairy root system transformed with pCAMBIA1305 is obviously deeper than that of the soybean hairy root system transformed with pCAMBIA1305-GmNTF2B-1promoter. This staining difference is further exacerbated under drought conditions.

5. Real-time fluorescent quantitative PCR analysis of GUS expression characteristics

The procedure is as in example 1. The primer sequences are as follows:

GUS gene: f:5 'GCTATACGCCTTTGAAGCC-3';

R：5’-TTGACTGGCCTCTTCGCTGTA-3’；

internal reference gene: f:5 'TTACCCGATGGCAAGTC-3';

R：5’-GCTCATACGGTCAGCGATAC-3’。

the results are shown in FIG. 7E. The results show that: under normal growth conditions, the relative expression amount of GUS in the hairy roots of the soybeans transformed with pCAMBIA1305 is slightly lower than that of the hairy roots of the soybeans transformed with pCAMBIA1305-GmNTF2B-1promoter. Under the drought condition, the expression difference is obviously enlarged, and the relative expression quantity of GUS in the hairy root of the transgenic pCAMBIA1305 soybean is obviously lower than that of the transgenic pCAMBIA1305-GmNTF2B-1promoter soybean hairy root. The gene GmNTF2B-1 is expressed by drought induction.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, it is possible to make various improvements and modifications without departing from the technical principle of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention.

Sequence listing

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

<120> plant stress tolerance-related GmNTF2B-1 protein, and coding gene and application thereof

<160>4

<170>PatentIn version 3.5

<210>1

<211>123

<212>PRT

<213>Artificial Sequence

<400>1

Met Asp Pro Asp Ala Leu Ala Lys Ala Phe Val Glu His Tyr Tyr Ser

1 5 10 15

Thr Phe Asp Thr Asn Arg Asn Gly Leu Ala Asn Leu Tyr Gln Glu Gly

20 25 30

Ser Met Leu Thr Phe Glu Gly Gln Lys Ile Gln Gly Ala Ser Asn Ile

35 40 45

Val Ala Lys Leu Thr Ser Leu Pro Phe Gln Gln Cys His His Ser Ile

50 55 60

Ser Thr Val Asp Cys Gln Pro Ser Gly Val Asn Ala Gly Met Leu Val

65 70 75 80

Phe Val Ser Gly Asn Leu Gln Leu Ala Gly Glu Gln His Thr Leu Lys

85 90 95

Phe Ser Gln Met Phe His Leu Ile Pro Thr Pro Gln Gly Ser Tyr Tyr

100 105 110

Val Leu Asn Asp Ile Phe Arg Leu Asn Tyr Ala

115 120

<210>2

<211>372

<212>DNA

<213>Artificial Sequence

<400>2

atggatccag acgcgttggc aaaggcattc gtggagcact actacagcac cttcgacacc 60

aacaggaacg gcttagcgaa tctctaccag gagggttcca tgctcacttt cgaagggcag 120

aagatccaag gcgcttccaa catcgttgcc aagctcacct ccctcccttt tcagcagtgc 180

caccactcaa tctccaccgt cgactgccag ccctccggcg tcaacgccgg catgctcgtc 240

ttcgtcagcg gaaaccttca gctcgccggc gaacagcaca ctctcaagtt cagccagatg 300

ttccatttga taccaacacc tcagggaagc tattatgtgt tgaatgacat attccgttta 360

aactatgcat ga 372

<210>3

<211>1997

<212>DNA

<213>Artificial Sequence

<400>3

agagagagag agagaagtgg gagagaatag agaagagaga tggatccaga cgcgttggca 60

aaggcattcg tggagcacta ctacagcacc ttcgacacca acaggaacgg cttagcgaat 120

ctctaccagg agggttccat gctcactttc gaagggcaga agatccaagg cgcttccaac 180

atcgttgcca agctcacctc cctccctttt cagcagtgcc accactcaat ctccaccgtc 240

gactgccagc cctccggcgt caacgccggc atgctcgtct tcgtcagcgg aaaccttcag 300

ctcgccggcg aacagcacac tctcaagttc agccaggtac tctccctctg ttagggtttt 360

attcgctctc cgatccatcg ctttctgcta taaatactcg attcgcgttt cgatctgcct 420

aatcctgtgt ttcgctgttt agttgtgtga cggcgttctt ttgacgttga cgcgcatgac 480

tggtttcaat acgattgaca tttctttgca taataatata gcgaatattg catgttgctt 540

ttgaatttgg ttgctgattt ggctcgtgtg tttgattgat tatcgtgtcg atttgttcat 600

ctggaaaacc taaattgttt agtgttagga ggttttagat tgttattaat gctattggcg 660

tgggaatcta tcacggagag aaggatgaat tgggaagatt ttcaaatgaa ttggcaattg 720

attgccaatt tgtgatcgag aatctagggt aactgcatag tcaaaggttt gctcacagta 780

gagagtgagt ggcaaaacaa ttatccatcc ttttatcatg ataattttaa tttaatggac 840

tttattaatt ggatcaaagt tgtttttgga tggttatgga ttgtactact gaatatctgt 900

tggatatcca aaggcaactt gatttcttta ctctcttcat tgtgccctca accacactat 960

taaccaggga aagtccttta agctctcaag ttttggatgc tttcgacatg cttcttgatc 1020

tcaacgttcc tcatgtgctc ttacactggc tgttcttggt ttattcattg atatcaattt 1080

gctagcaaaa gttattgcac ttttctttat ctgtgcttgg gtaggttatt cataattccc 1140

gccttccaag ttgccacctg gcctgaatct gagttcaatt tggtgacctg tggcctgtgg 1200

ggtccttcaa aacagagttt tattcaaatt tggctcaaat cttcctgttc ttctcagaag 1260

gactcaaggg aatttcacaa gaattacagt agaatattta atgaaatttc aatctctata 1320

agtagagttt atgatgtact cattttacct tgaagttaga agtgattgcc tcatgttaga 1380

ggatgggttg tccaaatcct gtcattttac tgaaatgatg gtgcctacat ggaagtggca 1440

ttttatttac agttttactt aaacatattt ttgttcaccc ttactataca tgtttacatg 1500

tgctgctctt acactggtaa ttgtacatta tatttttcct gtatatgcag aaatattgtt 1560

tttggtgggc acttcatctt attttcttgc tctattgttt tctcatgtac tgtttctcta 1620

atttctgtcc actctctttt cagatgttcc atttgatacc aacacctcag ggaagctatt 1680

atgtgttgaa tgacatattc cgtttaaact atgcatgaag atgtcatatg accggaaagg 1740

ccatagaaga agtttgccta tagcaagctg tttcagattg cacatttgaa attgaaattg 1800

ggttcttttt ggttggttgt gttgaacgct taattgtgcc tgttgtgttt agaattttct 1860

gttgtgactg gttaaaaacc ctccttcaag gataaattgt tgaaagatta ctggatattg 1920

gagattgaaa acttgaattt aatatatttg tgatccgcga tattgaatcc aagcatcttg 1980

atccagttta tctctac 1997

<210>4

<211>2039

<212>DNA

<213>Artificial Sequence

<400>4

caaacgaaat gaaagttggt agcaaaagga cgtttgcaaa agcattccca tcatttcttt 60

tgcattcagt ttgcgttgga ccattagatt tgtctttaga attcatgcga caacaagctg 120

gaggaaagat cagacatgca ctaagttatt tacctccatt gtctcttaac tagtaataag 180

agctactata gttggtacta gtgtgcttgg tgtacaaggg ccacactgtt tttgaatttg 240

ggaattcgat ctttctcagg ttcattaatt tttctccatt attttgtatt tacaaggttt 300

tgcaagagca atatgacaga agaatatagc ttttgtgaga agtagaaata aataaaacta 360

accctctacc aaatctaaaa ctaaagtctc cataaattga actgatcgat taaactatat 420

tttgtcaata tatgtattaa ggttagattc aatttggtta ttaaaaatat gattttcaaa 480

gcaaatccaa ctgataagat tattcattgg ttacgtgttt tcaaaattac agtagaaact 540

tttgcttggg aggagatgga gttagcatat gtacacaaat agtctcttta aatacttttt 600

ttttttcaca atccactaac caaaatattg aatttcatct taaattaaaa caaaatcttt 660

tactttttag tttcaacctc acactatttt tttttaattt cactaacttt tttttttagt 720

tttgaacctt ttagattatt ttcaatctta aatctaccct aaatctctaa ttatttgaat 780

aggaagtaaa gatttaaact tattatagag aaagtaattg aattacttaa ctataataat 840

caagagaata aactaaaatt aaaactacta agcaataaaa atcagatatt gatcatacta 900

aagggataaa atataacatt tttaaatata atatgctttg caaagttatt gtggttatgt 960

accactgtta aaccattatt aatatgtgag tcgatatgtc cgagtggtta aggagacaga 1020

cttgaaatct gttgggctac gcccgcgcag gttcgaaccc tgctgtcgac gatattttta 1080

ttaataatat atatttttac tttgaatgat ttaatttagg caagatttag taaatattta 1140

atttaagatg aacaaaataa tacttgattt acattattta aaaaatattt attagtacat 1200

aaaatttgtg cgagatctaa taaatcacgc gagtatcaaa tatatatctc actcaataat 1260

aatgatcata atataatttt gtatgctcta ggtgtaaatt tttctacacc gtcaataaat 1320

taaaaattat tgagcgtagc tcagttttta tttatattta tctcagttta taacaactca 1380

cttattcaat gcgataaaat gtatatcgat tttcacaaaa taactgtgat taagttttaa 1440

atttaactta gttgtataat cgatccaaaa aataaaattt ataaaaatat cttgggcctc 1500

attaatttta gaaaaataat tataaaaata aataaactta tcatatactt acttttaata 1560

gataataaga ttatatttaa caaaattaat tgaaaaatta actagaagat gaaaaattaa 1620

aaaaattaaa ggatagttga aaattgaaaa ctaatgtatt aaattaaaag tattaaataa 1680

aattagttat tgaaataatt aaaaagtata aaataacata aaaataataa aattatgttt 1740

tattaaaata gataaaaaaa taaacataat aatagaaaaa atataaaaag ttagaaattc 1800

atattttaaa aaatattaaa atctatcaaa aaaattattt atccgataat caaataaatt 1860

tttaagttaa taaaaataat tagaaattga ctcaaaggtc ttccagaaaa tagcctaata 1920

ataatataat ttggaaaatt gatggttggt acgtaaaaga tacgcgagag ggtgtagacg 1980

tgtagtatat aaaggagggg agagagagag agagaagtgg gagagaatag agaagagag 2039

0.8M Mannitol

1. Use of a protein or a biological material related thereto, wherein the use is any one of the following 1) to 4):

1) Improving the stress tolerance of the soybean;

2) Cultivating transgenic soybean with improved stress tolerance;

3) Soybean breeding;

4) Promoting the growth and development of the soybeans;

the protein is a protein shown in a) or b) as follows:

a) A protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;

b) A fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 1 in the sequence table;

the related biological material is any one of the following A1) to A12):

a1 Nucleic acid molecules encoding the protein;

a3 A recombinant vector containing the nucleic acid molecule according to A1);

a4 A recombinant vector containing the expression cassette of A2);

a6 A recombinant microorganism containing the expression cassette of A2);

a7 A recombinant microorganism containing the recombinant vector of A3);

a8 A recombinant microorganism containing the recombinant vector of A4);

a10 A transgenic plant cell line containing the expression cassette of A2);

a11 A transgenic plant cell line containing the recombinant vector of A3);

a12 A transgenic plant cell line containing the recombinant vector of A4);

in the application, the activity and/or expression level of the protein or the coding gene thereof in soybean is improved.

2. Use according to claim 1, characterized in that: the stress tolerance is drought tolerance.

3. Use of the protein of claim 1 as a nuclear transport protein.

4. The following method one or method two:

the method comprises the following steps: a method for producing a transgenic soybean having improved stress tolerance, comprising the step of increasing the content and/or activity of the protein of claim 1 in a recipient soybean to obtain a transgenic soybean; the stress tolerance of the transgenic soybean is higher than that of the acceptor soybean;

the second method comprises the following steps: a method for promoting soybean growth and development, comprising the steps of increasing the content and/or activity of the protein of claim 1 in a recipient soybean to obtain a transgenic soybean; the transgenic soybean has higher growth and development level than the acceptor soybean.

5. The method of claim 4, wherein: the stress tolerance is drought tolerance.

6. The method of claim 4, wherein: the promotion of soybean growth is the promotion of soybean root growth.

7. The method of claim 4, wherein: the stress tolerance of the transgenic soybean is higher than that of the acceptor soybean, and is embodied in any one of the following X1) -X7):

x1) the hydrogen peroxide content in the transgenic soybean root system and/or leaf is lower than that of the receptor soybean;

x2) the root surface area of the transgenic soybean is larger than that of the receptor soybean;

x3) the survival rate of the transgenic soybean is higher than that of a receptor soybean;

x4) the transgenic soybean has a higher root dry weight than the recipient soybean;

x5) the damaged area proportion of the leaves of the transgenic soybeans is lower than that of the receptor soybeans;

x6) the superoxide anion free radical content of the transgenic soybean leaf is lower than that of the receptor soybean;

x7) the relative conductivity of the transgenic soybean root system is lower than that of the receptor soybean.

8. The method of claim 4, wherein: the transgenic soybean has higher growth development level than the acceptor soybean, and is embodied in any one of the following Y1) or Y2):

y1) the transgenic soybean has a longer root length than the recipient soybean;

y2) the root surface area of the transgenic soybean is greater than that of the recipient soybean.

9. The method of claim 4, wherein: the method for increasing the content and/or activity of the protein of claim 1 in a recipient soybean is to overexpress the protein of claim 1 in the recipient soybean.

10. The method of claim 9, wherein: the method of overexpression is to introduce a gene encoding the protein of claim 1 into a recipient soybean.

11. The method of claim 10, wherein: the nucleotide sequence of the coding gene of the protein is a DNA molecule shown in a sequence 2 in a sequence table.