CN113493501A - Plant stress tolerance-related GmNTF2B-1 protein, and coding gene and application thereof

Info

Abstract

Description

Claims

CN113493501A

Publication number: CN113493501A
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: 2021-10-12
Anticipated expiration: 2040-04-03
Also published as: CN113493501B

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 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; 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 the plants with enhanced stress resistance and stress tolerance.

Plant stress tolerance-related GmNTF2B-1 protein, 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 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;

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 according to b) is specifically shown in Table 1.

TABLE 1 sequence 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

The protein GmNTF2B-1 of 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) a nucleic acid molecule encoding GmNTF2B-1 protein;

A2) an expression cassette comprising the nucleic acid molecule of a 1);

A3) a recombinant vector comprising the nucleic acid molecule of a 1);

A4) a recombinant vector comprising the expression cassette of a 2);

A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);

A6) a recombinant microorganism comprising the expression cassette of a 2);

A7) a recombinant microorganism comprising a3) said recombinant vector;

A8) a recombinant microorganism comprising a4) said recombinant vector;

A9) a transgenic plant cell line comprising the nucleic acid molecule of a 1);

A10) a transgenic plant cell line comprising the expression cassette of a 2);

A11) a transgenic plant cell line comprising the recombinant vector of a 3);

A12) a transgenic plant cell line comprising the recombinant vector of a 4).

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 is hybridized 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 of the present invention encoding GmNTF2B-1 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 with 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 in percent (%), 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 materials, the expression cassette containing a nucleic acid molecule encoding GmNTF2B-1 (GmNTF2B-1 gene expression cassette) described in A2) means a DNA capable of expressing GmNTF2B-1 in a host cell, and the DNA may include not only a promoter which initiates transcription of GmNTF2B-1 but also a terminator which terminates transcription of GmNTF 2B-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: the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator and the pea rbcS E9 terminator.

The recombinant vector containing the GmNTF2B-1 gene expression cassette can be constructed by using the existing expression vector. 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 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 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 transgenic plants can be directly screened and transformed in a stress environment 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-1 promoter.

Furthermore, the 16318hGFP-GmNTF2B-1 is a recombinant plasmid obtained by inserting the GmNTF2B-1 gene into 16318hGFP multiple cloning sites, and is preferably a recombinant plasmid obtained by inserting a DNA fragment shown as a sequence 2 in a sequence table into the 16318hGFP BamHI enzyme cutting sites. The pCAMBIA1302-GmNTF2B-1 is a recombinant plasmid obtained by inserting a GmNTF2B-1 gene into a multiple cloning site of the pCAMBIA1302, and is preferably a recombinant plasmid obtained by inserting a DNA fragment shown by a sequence 2 in a sequence table into a position between NcoI enzyme cutting sites of the pCAMBIA 1302. The pCAMBIA3301-GmNTF2B-1 is a recombinant plasmid obtained by inserting GmNTF2B-1 gene into the multiple cloning site of pCAMBIA3301, and is preferably a recombinant plasmid obtained by inserting a DNA fragment shown by a sequence 2 in a sequence table between the 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, the dry weight of plant roots is increased; (3) under drought treatment, the surface area of the plant roots is increased; (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 can 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 can be Willimas 82.

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 quantity and/or activity of the GmNTF2B-1 protein in the receptor plant is to overexpress the GmNTF2B-1 protein in the receptor 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 in a sequence 2 in a sequence table.

In one embodiment of the invention, the gene encoding GmNTF2B-1 protein is introduced into the recipient plant by means of a recombinant plasmid pCAMBIA3301-GmNTF 2B-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 and is embodied in any one of the following X1) -X7):

x1) hydrogen peroxide content in transgenic plant roots and/or leaves is lower than in recipient plants;

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

x3) the transgenic plant survives more than the recipient plant;

x4) root dry weight of transgenic plants higher than that of recipient plants;

x5) the leaf damage area ratio of the transgenic plant is lower than that of the recipient plant;

x6) leaves of transgenic plants have a lower content of superoxide anion radicals than the recipient plant;

x7) the relative conductivity of the transgenic plant root system was 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 has a higher level of growth development than the recipient plant.

In one embodiment of the invention, the gene encoding GmNTF2B-1 protein is introduced into the recipient plant via the recombinant plasmid pCAMBIA1302-GmNTF 2B-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 grows and develops at a higher level than the recipient plant at any one of the following Y1) or Y2):

y1) root length of the transgenic plant is longer than that of the recipient plant;

y2) 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 Willimas 82.

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 GmNTF 2B-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 soybean hairy root growth under normal conditions. A: phenotype of empty vector soybean hairy roots; b: the phenotype of soybean hairy roots transformed with GmNTF 2B-1; c: under normal conditions, carrying out statistical analysis on the total root length of the empty vector soybean hairy roots and the GmNTF2B-1 soybean hairy root system; d: under normal conditions, the statistical analysis of the root surface areas of empty carrier soybean hairy roots and GmNTF2B-1 soybean hairy roots is carried out; 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 soybean hairy root is transferred into GmNTF2B-1 in longitudinal section. Wherein, the 35S: GFP is transferred empty vector soybean hairy root; GmNTF2B-1 pro: GmNTF2B-1-GFP was transferred to GmNTF2B-1 soybean hairy roots.

FIG. 4 is a graph showing the effect of GmNTF2B-1 on soybean drought tolerance. A: phenotype of soybean transferred with GmNTF2B-1 and soybean transferred with 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; c: hairy root phenotype of GmNTF2B-1 transferred soybean and empty vector 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 soybeans and the empty carrier soybeans; g: relative expression quantity of GmNTF2B-1 in the soybean hairy roots transformed with GmNTF2B-1 and the soybean hairy roots transformed with the empty vector; h: DAB staining results of leaves of soybeans transferred with GmNTF2B-1 and soybeans transferred with empty carriers after drought treatment; i: after drought treatment, NBT staining results of leaves of soybeans transferred with GmNTF2B-1 and soybeans transferred with empty carriers are obtained; 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 leaves of the GmNTF 2B-1-transferred soybeans and the empty carrier transferred soybeans were damaged at a ratio. Wherein WT is empty carrier soybean; GmNTF2B-1 pro: GmNTF2B-1 was soybean transformed with GmNTF 2B-1.

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 soybeans transferred with GmNTF2B-1 and soybeans transferred with empty carriers; b: after drought treatment, performing DAB dyeing on roots of the soybeans transferred with the GmNTF2B-1 and the soybeans transferred with the empty carrier; 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 GmNTF 2B-1-transferred soybeans and the empty carrier transferred soybeans.

FIG. 6 shows the positive detection and soil water content and water potential measurement of empty carrier soybean hairy root and GmNTF2B-1 soybean hairy root. 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 is a graph showing that soybean hairy roots verify whether the expression of GmNTF2B-1 is drought-induced. 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: under the condition of simulating drought by 20 percent PEG, the expression condition of a reporter gene GUS in a hairy root of a soybean of pCAMBIA1305 is transferred; d: under the condition of simulating drought by 20 percent PEG, the expression condition of a reporter gene GUS in soybean hairy roots of pCAMBIA1305-GmNTF2B-1promoter is transferred; e: A. b, C and D, relative expression level of GUS gene.

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.

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 recipe

TABLE 8 CCM (Co-cultured Medium) Co-culture Medium formulation

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: DA 010; and (4) commodity brands: solarbio; specification: 2X 10 mL.

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

Hydrogen peroxide content determination kit: the product name is as follows: hydrogen peroxide assay kit (colorimetric); 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 product number: RTU 4032; 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 only for repeating experiments related to The present invention, and not for other uses.

The pCAMBIA1302 and pCAMBIA1305 vectors described in the following examples are described in the literature "Li, B., Liu, Y., Cui, X.Y., Fu, J.D., Zhou, Y.B., Zheng, W.J., et al (2019) Genome-Wide Characterisation and Expression Analysis of Soybean TGA transformation Factors Identified a Novel TGA Gene analyzed in Dry 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 applicable for other purposes.

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

First, 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 a5 'RACE and 3' RACE method, the nucleotide sequence of the full-length cDNA sequence is shown as a sequence 2 in a sequence table, a 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.

Secondly, 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, performing drought treatment for 1 hour, 3 hours, 6 hours, 12 hours and 24 hours, taking out the material, quickly freezing with 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 to 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:

a target gene: 5'-GTGGAGCACTACTACAGCAC-3' is used as a reference material;

R:5’-GGTGGAGATTGAGTGGTGGC-3’；

internal reference gene: 5'-CGGTGGTTCTATCTTGGCATC-3' is used as a reference material;

R:5’-GTCTTTCGCTTCAATAACCCTA-3’。

Q-RT-PCR in ABI

7000 real-time fluorescence quantitative PCR instrument, a parallel experiment set 3 times. The method reported by Livak KJ and Schmittgen TD (2001), 2^-ΔΔCTAnd 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 an arbitrary Time point, Time₀Represents 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 GmNTF2B-1 is obviously increased 1h after the treatment, the peak value is reached 8h after the treatment, the 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

First, construction of expression vector

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

And (4) 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 cut by restriction enzyme BamH I, and the vector skeleton is recovered.

Thirdly, connecting the enzyme digestion product of the first step with the vector framework of the second step to obtain the recombinant plasmid 16318hGFP-GmNTF 2B-1.

Fourthly, the ligation product obtained in the third step is electrically shocked to transform TOP10 strain (purchased from Beijing Tiangen company), cultured overnight at 37 ℃, and positive clone is 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.

Second, 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 was evacuated for 30 minutes in the dark (foil wrapped). 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 first wetted with a W5 solution, and then the enzymatic hydrolysate containing protoplasts is filtered using it.

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 in 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. The protoplasts were then resuspended in the appropriate amount of MMG solution (1mL) to a final concentration of 2X 10⁵one/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)⁴One), gently mix.

15. Add 110. mu.L of PEG solution and gently tap the tube to mix thoroughly (approximately 6-10 samples can be converted 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 terminated by diluting the conversion mixture with 400-440 μ L 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 to the suspension and washed once, and the suspension was centrifuged at 100g for 2min to remove the supernatant.

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

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

Third, Arabidopsis protoplast microscopic examination

Protoplasts after dark culture for 18h were tabletted, and then GFP (Green fluorescent protein) fluorescence was observed in a Laser scanning confocal microscope (Bio-Rad Microradiance) (Laser scanning confocal, LSMC) and subjected to scanning photography. The operating parameters of the LSCM are as follows: ex 488nm, Em 525 ± 15nm, Power 10%, Zoom7, Frame512 × 512 by medium speed scan. The software is TIME-COARSE and PHOTOSHOP 5.0.

The results are shown in FIG. 2. The results show that: GmNTF2B-1 localizes predominantly in the nucleus, compared to the fluorescent signal of the empty vehicle control, which is diffuse throughout the cell.

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

Construction of recombinant expression vector

1. Cloning of GmNTF2B-1 Gene

Primer pairs (GmNTF2B-1-1302-F and GmNTF2B-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

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

② the pCAMBIA1302 carrier is cut by restriction enzyme Nco I, and the carrier skeleton is recovered.

Thirdly, connecting the enzyme digestion product of the first step with the vector skeleton of the second step to obtain the recombinant plasmid pCAMBIA1302-GmNTF 2B-1.

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

Second, obtaining and identifying transgenic soybean hairy roots

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 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 16-18 h. 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 0.5-1 h. 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 liquid was added to 30mL of LB liquid medium (containing 50mg/mL rifampicin, 100mg/mL kanamycin) and cultured to OD₆₅₀0.5-0.7. Centrifuging (4000rpm, 10min), adding IM liquid culture medium (without Agar), and making OD of bacterial liquid₆₅₀＝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-cultivation: the bacterial suspension obtained in step 3 and the explant obtained in step 4 were mixed for 30min, 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) and cultured in the dark at 28 ℃ for 3 days.

6. And (3) sporophyll node degerming: washed 4-5 times with liquid RIM (without Agar) and blotted dry on filter paper. Inverted and inserted into solid RIM culture medium, hypocotyl is upward, and cultured for about 10-14 days to obtain hairy root of soybean transformed with GmNTF 2B-1.

The recombinant plasmid pCAMBIA1302-GmNTF2B-1 was replaced with pCAMBIA1302 vector according to the above method, and the other steps were kept unchanged to obtain empty vector soybean hairy roots.

7. Molecular identification was performed on transgenic GmNTF2B-1 soybean hairy roots and 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 711 bp.

Primer pair for identifying DNA level of soybean hairy roots transferred with GmNTF 2B-1:

F：5’-GACTCTTGACCATGGATGGATCCAGACGCG-3；

R：5’-TGACGAGGGTGTCTCCCTCAAACTT-3’；

the expected band is 751 bp.

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 751 bp.

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

internal reference CYP2 gene primer sequence:

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.

Third, transgenic soybean hairy root positive identification

And (3) cutting the soybean hairy roots of the transferred GmNTF2B-1 and the transferred empty carrier soybean hairy roots obtained in the step two into thin slices by adopting a free-hand 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 science).

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

Fourth, phenotype identification of transgenic soybean hairy roots

And observing the root growth and development conditions of the transgenic GmNTF2B-1 soybean hairy roots (n is 12) obtained in the step two and the transgenic empty vector soybean hairy roots (n is 12), and comparing the root length with 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, GmNTF2B-1 can promote the growth of soybean root systems, which 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 GmNTF2B-1 is 16.5 cm; the average root length of the empty transfer carrier soybean hairy roots is 11.1 cm; the root surface area of the soybean hairy root transformed with GmNTF2B-1 was 8.7cm on average²(ii) a The average root surface area of the empty carrier soybean hairy roots was 4.1cm². The root length and the root surface area of the soybean hairy root transferred with the GmNTF2B-1 are obviously larger than those of the soybean hairy root transferred with an empty carrier.

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

Construction of recombinant expression vector

1. Cloning of GmNTF2B-1 Gene

Primer pairs (GmNTF2B-1-3301-F and GmNTF2B-1-3301-R) are designed according to the sequence of GmNTF2B-1 gene, Nco I and BstE II 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-1-3301-F：5’-GGACTCTTGACCATGATGGATCCAGACGCG-3’；

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

2. Construction of recombinant expression vectors

Cutting the purified PCR product recovered in the step 1 by restriction enzymes Nco I and BstE II, and recovering the cut enzyme product.

② the pCAMBIA3301 vector is digested with restriction enzymes Nco I and BstE II, and the vector backbone is recovered.

Thirdly, connecting the enzyme digestion product of the first step with the vector skeleton of the second step to obtain the recombinant plasmid pCAMBIA3301-GmNTF 2B-1.

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

Second, obtaining transgenic soybean hairy root

1. Agrobacterium transformation

Activating Agrobacterium K599 strain stored at-80 deg.C, culturing at 28 deg.C and 200rpm to obtain bacterial liquid OD₆₀₀To 0.6-1.0.

② sucking 1mL of activated bacterial liquid into 40mL of LB culture medium containing streptomycin (50mg/L), culturing bacterial liquid OD at 28 ℃ and 200rpm₆₀₀To about 0.6.

Thirdly, 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 discarding the supernatant.

Fourthly, 2mL of sterilized 20mM CaCl₂Resuspend the cells, mix well and dispense 200 μ L per tube. Quick freezing with liquid nitrogen, and storing at-80 deg.C.

Fifthly, respectively adding 4 muL (about 2 mug) of the recombinant plasmid pCAMBIA3301-GmNTF2B-1 in the step one into newly prepared 200 muL of agrobacterium-mediated conditions, freezing for 8min by liquid nitrogen after ice bath for 5min, and rapidly placing in a water bath at 37 ℃ for 5 min.

Sixthly, 600 mu L of LB culture medium without antibiotic is added into each tube, and the mixture is cultured for 4 to 6 hours on a shaking table at the temperature of 28 ℃ and the rpm of 200.

Seventhly, centrifuging at 5000rpm for 2min, removing partial supernatant, leaving 200 mu L of liquid in each tube, suspending the liquid by using a liquid transfer gun, and then performing inverted culture on an LB solid medium uniformly coated with Kan (50mg/L) and Str (50mg/L) at 28 ℃ until single colonies grow out.

Eighthly, selecting a single colony, performing enlarged culture, and screening to obtain a positive colony.

Ninthly, uniformly sowing Willimas82 soybean seeds in the vermiculite subjected to high-temperature and high-pressure sterilization, adding a proper amount of nutrient solution, and culturing under a high-humidity condition until soybean cotyledons are formed (about 5 d). Agrobacterium K599 transformed with a vector for expressing a target gene (recombinant plasmid pCAMBIA3301-GmNTF2B-1) was activated while sowing soybean, and cultured on a solid medium resistant to Kan (50mg/L) and Str (50mg/L) at 28 ℃ 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 conditions until soybean hairy root grows out, and obtaining the soybean hairy root transformed with GmNTF 2B-1.

The recombinant plasmid pCAMBIA3301-GmNTF2B-1 was replaced with pCAMBIA3301 vector according to the above method, and the other steps were kept unchanged to obtain a transgenic empty vector soybean hairy root.

2. Molecular characterization of transgenic soybean hairy roots

The transgenic soybean hairy roots obtained in the step 1 and the transgenic soybean hairy roots with empty vector obtained in the step 1 are 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 700 bp.

F：5’-CAAACGAAATGAAAGTTGGTAGCAAAAGGA-3；

R：5’-CTCTCTTCTCTATTCTCTCCCACTTCTCTC-3’；

the expected band is 751 bp.

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 (Williams82), 700bp in 5 empty vector soybean hairy roots and 751bp in 7 GmNTF2B-1 soybean hairy roots are detected; 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 level 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 GmNTF2B-1 soybean hairy roots and the transgenic empty vector soybean hairy roots obtained in the step 1 on the cDNA level. The CYP2 gene is used as an internal reference gene. The primer sequences are as follows:

internal reference CYP2 gene primer pair:

F：5'-ACCAGAATTGCATTACCAGTTGA-3'；

R：5'-GGTGCCGAGACTCATGAAGATAT-3'；

the target gene primer pair comprises:

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.

Third, drought tolerance identification of GmNTF 2B-1-transferred soybean and empty carrier transferred soybean

To evaluate the drought-enduring effects of the GmNTF 2B-1-transferred soybean and the empty-carrier transferred soybean. And (3) carrying out drought tolerance identification on the transgenic GmNTF2B-1 soybean hairy roots and the transgenic 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 plant only containing the hairy root system of the transgenic soybean into nutrient soil, carrying out drought water control treatment after the plant normally grows for 10 days, respectively obtaining transgenic soybean GmNTF2B-1 and transgenic empty carrier soybean after 5-6 days of treatment, observing the phenotype of the transgenic soybean GmNTF2B-1 and the transgenic empty carrier soybean, 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 actual experiment requirement.

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 vector transferred soybean is more sensitive to drought treatment, leaves are wilted and dried at the earliest, hairy roots grow slowly, and the growth vigor is obviously weaker than that of the transferred GmNTF2B-1 soybean; the GmNTF 2B-1-transferred soybean showed some 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 were 20% and 80%, respectively, and the corresponding average root surface areas were 31.21cm²And 69.56cm²The average dry root weights were 0.27g and 0.34g, respectively. Indicating that the drought tolerance of the soybean can be improved by over-expressing GmNTF 2B-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 dryland treatment.

DAB dyeing of leaves

The method provided by reference to commercial kit (DAB staining solution kit) is to react H with Diaminobenzidine (DAB)₂O₂Is generated for localization. Six days after the drought treatment, the leaves of the same plant of the GmNTF 2B-1-transferred soybeans and the empty carrier-transferred soybeans were cut, the cut was immersed in an aqueous solution (pH 3.8) containing 1mg/mL DAB, and the reaction was carried out for 8 hours to allow DAB to be absorbed and react with H in the leaves₂O₂And a peroxidase. Then, cutting the leaves into leaf sections of about 3cm, boiling in 95% ethanol solution for 10min for fixing and decoloring, placing the leaves in saturated chloral hydrate for transparency after chlorophyll is completely faded, and placing the transparent leaves in 50% glycerin 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 were significantly more stained 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.

Second, NBT staining of leaves

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, GmNTF2B was transferred to-1 cutting off leaves of the same part of the same plant of soybeans and the empty vector soybeans, putting the leaves into 10mM phosphate buffer (pH 7.8) containing 0.1% NBT (w/v) for vacuum infiltration for 20min, then putting the leaves into 95% ethanol solution for boiling for 10min for fixation and decolorization, putting the leaves into saturated chloral hydrate for transparence after chlorophyll is completely faded, and putting the transparent leaves into 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 were significantly more stained than the leaves of the GmNTF 2B-1-transferred soybeans. The deeper the staining, the O in the leaves₂ ^-The higher the content, the less drought resistant the plant, which indicates that GmNTF2B-1 reduces O in the plant₂ ^-The content of the soybean protein is increased to improve the drought resistance of the soybean.

Third, measuring the hydrogen peroxide content in the leaves

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): carrying out ice bath homogenate with the volume (mL) of the first reagent being (1:5) -10, taking 0.1g of the soybean leaves subjected to drought treatment in the experiment, and adding 1mL of the first reagent; transferring to an EP tube, diluting to 1mL with a reagent, centrifuging at 4 ℃ for 10min at 8000g, collecting supernatant, and placing on ice for testing.

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

TABLE 10, H₂O₂Operation 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 carrier transferred 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 carrier transferred 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.

Fourthly, measuring the damaged area proportion of the leaves

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. and after opening the Image J software, clicking a File above the software, pulling down and clicking Open, and pulling the scanned picture into the software. Then click on the Image above the software, pull 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. Then click ok to 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 mode, and popping up a gray scale statistic value of the selected area, wherein the gray scale statistic value is marked 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 damaged area proportion of the leaf is A1/A2 multiplied by 100%.

The results are shown in FIG. 4M. The results show that: the damaged area proportion of the leaves of the empty vector transferred soybean is 32.1 percent, the damaged area proportion of the leaves of the empty vector transferred soybean is 10.8 percent, and the damaged area proportion of the leaves of the empty vector transferred soybean is obviously higher than that of the empty vector transferred soybean of GmNTF 2B-1.

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

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-transferred soybean hairy root system is obviously deeper than that of the empty carrier-transferred soybean hairy root system of GmNTF 2B-1. 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.

Second, measuring the hydrogen peroxide content of root system

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 to the GmNTF2B-1 and the soybeans transferred to the empty carrier 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 to the GmNTF2B-1 is slightly lower. After drought treatment, the hydrogen peroxide content of the soybean and the soybean obviously increases, but the hydrogen peroxide content (1.48 mu mol/g & FW) of the roots of the soybeans transferred to GmNTF2B-1 is obviously lower than that of the soybeans transferred to empty carriers (2.02 mu mol/g & FW). Further proves that GmNTF2B-1 improves the drought resistance of soybeans by reducing the content of hydrogen peroxide in plants.

Third, determination of relative conductivity of root system

Collecting plant root tip with the same size, ensuring root integrity as much as possible, washing with tap water, washing with steam room water for three times, drying surface water with filter paper, cutting root tissue into almost size, weighing 0.3g, and repeating for 3 timesPut into a clean graduated tube, add 10mL of deionized water, and immerse the sample. Placing the test tube in a shaking conductometer (DDS-11A) to measure the conductivity S of deionized water₀Then, the test tubes are shaken up fully, and the conductivity S of the solution is measured₁Sealing 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 S₂And calculating the relative conductivity according to the following formula: relative conductivity ═ S₁-S₀)/(S₂-S₀)。

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 the empty carrier 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 to GmNTF2B-1 (0.58) is obviously lower than that of the soybeans transferred to an empty carrier (0.81).

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

Construction of recombinant expression vector

1. Cloning of GmNTF2B-1-promoter

A primer pair (GmNTF2B-1-promoter-1305-F and GmNTF2B-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 ends of the primers, and the GmNTF2B-1promoter (GmNTF2B-1-promoter) is amplified by PCR by taking soybean genomic 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 is digested with restriction enzyme Nco I, and the vector backbone is recovered.

Thirdly, connecting the enzyme digestion product of the first step with the vector skeleton of the second step to obtain a recombinant expression vector pCAMBIA1305-GmNTF 2B-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 a pCAMBIA1305 vector with a DNA fragment shown by 1 st to 2000 th nucleotides from a 5' end of a sequence 4 in a sequence table.

Second, obtaining of transgenic soybean hairy root of 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.

Third, drought treatment

The hairy root of the transgenic soybean with good growth condition and similar growth condition, namely pCAMBIA1305 soybean hairy root and transgenic soybean hairy root of pCAMBIA1305-GmNTF2B-1promoter are taken and placed in MS₀Liquid medium (purchased from Beijing Mengyei) and MS supplemented with 20% PEG₀In the liquid culture medium, normal growth conditions and drought growth conditions are simulated respectively. After 6 hours of treatment, GUS staining was performed.

Fourth, 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: RTU 4032; 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 of the transgenic pCAMBIA1305 is obviously deeper than that of the soybean hairy root system of the transgenic pCAMBIA1305-GmNTF2B-1 promoter. This difference in staining is further exacerbated under drought conditions.

Fifth, 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', respectively;

R：5’-TTGACTGGCCTCTTCGCTGTA-3’；

internal reference gene: f: 5'-TTACCCGATGGGCAAGTC-3', respectively;

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-1 promoter. Under drought conditions, the expression difference is remarkably enlarged, and the relative expression quantity of GUS in the hairy roots of the soybeans transformed with pCAMBIA1305 is obviously lower than that of the hairy roots of the soybeans transformed with pCAMBIA1305-GmNTF2B-1 promoter. The gene GmNTF2B-1 is expressed by drought induction.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded 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. The protein is the protein of a) or b) or c) or d) as follows:

2. The protein-related biomaterial according to claim 1, which is any one of the following a1) to a 12):

A1) a nucleic acid molecule encoding the protein of claim 1;

A2) an expression cassette comprising the nucleic acid molecule of a 1);

A3) a recombinant vector comprising the nucleic acid molecule of a 1);

A4) a recombinant vector comprising the expression cassette of a 2);

A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);

A6) a recombinant microorganism comprising the expression cassette of a 2);

A7) a recombinant microorganism comprising a3) said recombinant vector;

A8) a recombinant microorganism comprising a4) said recombinant vector;

A9) a transgenic plant cell line comprising the nucleic acid molecule of a 1);

A10) a transgenic plant cell line comprising the expression cassette of a 2);

A11) a transgenic plant cell line comprising the recombinant vector of a 3);

A12) a transgenic plant cell line comprising the recombinant vector of a 4).

3. The related biological material according to claim 2, wherein: A1) the nucleic acid molecule is a gene shown in the following 1) or 2) or 3):

2) a cDNA molecule or genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) and encoding the protein of claim 1;

3) a cDNA molecule or a genomic DNA molecule which hybridizes under stringent conditions with a nucleotide sequence defined in 1) or 2) and encodes a protein according to claim 1.

4. Use of the protein of claim 1 or the related biomaterial of claim 2 or 3 for modulating stress tolerance in a plant;

or, the use of the protein of claim 1 or the related biological material of claim 2 or 3 for the cultivation of transgenic plants with increased stress tolerance;

or, the use of the protein of claim 1 or the related biological material of claim 2 or 3 in plant breeding;

or, the use of the protein of claim 1 or the related biomaterial of claim 2 or 3 for modulating plant growth and development;

or, the use of the protein of claim 1 or the related biomaterial of claim 2 or 3 for promoting plant growth and development.

5. Use according to claim 4, characterized in that: the stress tolerance is drought tolerance;

or, the regulating plant growth development is regulating plant root growth development.

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

7. The following method one or method two:

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

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

8. The method of claim 7, wherein: the stress tolerance is drought tolerance;

or, the plant growth and development is promoted to the plant root growth and development;

or, the transgenic plant has higher stress tolerance than the acceptor plant in any one of the following X1) -X7):

x3) the transgenic plant survives more than the recipient plant;

x4) root dry weight of transgenic plants higher than that of recipient plants;

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

or, the transgenic plant grows and develops at a higher level than the recipient plant and is found in any one of the following Y1) or Y2):

9. The method according to claim 7 or 8, characterized in that: the method for increasing the content and/or activity of the protein of claim 1 in a recipient plant comprises overexpressing the protein of claim 1 in the recipient plant;

or, the method of overexpression is to introduce a gene encoding the protein of claim 1 into a recipient plant;

or the nucleotide sequence of the coding gene of the protein is a DNA molecule shown in a sequence 2 in a sequence table.

10. Use according to any one of claims 4 to 6 or a method according to any one of claims 7 to 9, wherein: the plant is a dicotyledonous plant or a monocotyledonous plant;

alternatively, the dicot is soybean.