CN111620933B

CN111620933B - Application of protein GmNAC2 in regulation and control of salt tolerance of plants

Info

Publication number: CN111620933B
Application number: CN201910140506.5A
Authority: CN
Inventors: 张劲松; 陈受宜; 卞潇华; 韦伟; 张万科; 陶建军; 林晴; 阴翠翠; 何锶洁
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2019-02-26
Filing date: 2019-02-26
Publication date: 2022-05-17
Anticipated expiration: 2039-02-26
Also published as: CN111620933A

Abstract

The invention discloses application of protein GmNAC2 in regulation and control of salt tolerance of plants. The invention provides application of GmNAC2 protein or related biomaterials thereof in regulating and controlling the salt tolerance of plants; the GmNAC2 protein is a protein shown in SEQ ID No.1 or a protein which is substituted and/or deleted and/or added by one or more amino acid residues, or a protein which has a sequence with more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology and has the same function, or a fusion protein obtained by connecting a label at the N end and/or the C end of the protein. The invention proves that the salt tolerance of plants can be obviously improved by reducing the expression of the protein GmNAC2 and the encoding gene GmNAC2 thereof. The salt tolerance related protein and the coding gene thereof have important significance for cultivating salt tolerant plant varieties so as to improve the crop yield.

Description

Application of protein GmNAC2 in regulation and control of salt tolerance of plants

Technical Field

The invention relates to the technical field of biology, in particular to application of protein GmNAC2 in regulation and control of plant salt tolerance.

Background

The change of physical and chemical factors in the environment, such as drought, saline alkali, low temperature and other stress factors, has important influence on the growth and development of plants, can cause large-scale yield reduction of crops in severe cases, and the cultivation of stress-tolerant crops is one of the main targets of the planting industry. At present, genetic engineering breeding has become one of the important methods for enhancing the stress tolerance of crops. Higher plant cells respond to various stresses in the environment in multiple ways, with transcription factors playing a role in regulating the expression of stress-tolerance-related effector genes. Several classes of transcription factors have been found to be associated with plant stress tolerance in plants, for example: DREB class in EREBP/AP2, bZIP, MYB, WRKY, etc.

NAC(NAM/ATAF1/2/CUC2) family is a family of transcription factors that are characteristic in plants, and the NAC gene that has been studied plays a very important role in different life processes. Some members of the NAC family positively regulate agronomic traits, while some play a negative regulatory role, such as defense against pathogenic bacteria, plant senescence, morphogenesis, and response to abiotic stress.

In soybean, gaijun ytterbium, jun and the like cloned the GmNAC2 gene (Meng, q.c., Yu, d.y.and Gai, j.y., Journal of Plant Physiology 164, 2007, 1002-1012) and identified the expression characteristics of GmNAC2 in soybean organs and during development, but the function of the GmNAC2 gene and its encoded protein, GmNAC2, was not studied.

Soybean is an important oil crop, is a main source of plant protein, clarifies the stress tolerance mechanism of the plant protein, further improves the stress tolerance of the plant protein, and has important theoretical and practical significance.

Disclosure of Invention

The invention aims to provide application of protein GmNAC2 in regulating and controlling salt tolerance of plants.

In a first aspect, the present invention claims the use of GmNAC2 protein or a related biomaterial thereof for modulating salt tolerance in plants.

The related biological material can be a nucleic acid molecule capable of expressing the GmNAC2 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

the GmNAC2 protein is any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No. 1;

(A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.1 and has the same function;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

In (a2), the substitution and/or deletion and/or addition of one or several amino acid residues means substitution and/or deletion and/or addition of not more than ten amino acid residues.

SEQ ID No.1 consists of 299 amino acid residues. The GmNAC2 protein is a NAC-type transcription factor in soybean.

In the application, the activity and/or the expression quantity of the GmNAC2 protein or the coding gene thereof in the plant are reduced, and the salt tolerance of the plant is improved; the activity and/or expression level of the GmNAC2 protein or the coding gene thereof in the plant is increased, and the salt tolerance of the plant is reduced.

In a second aspect, the invention claims a method of growing a plant variety, method a or method B:

the method A comprises the following steps: a method for breeding a plant variety with increased salt tolerance may comprise the step of reducing the expression level and/or activity of GmNAC2 protein in a recipient plant. The GmNAC2 protein is a protein shown in any one of the above (A1) - (A4).

The method B comprises the following steps: a method for breeding a plant variety with reduced salt tolerance may comprise the step of increasing the expression level and/or activity of GmNAC2 protein in a recipient plant. The GmNAC2 protein is a protein shown in any one of the above (A1) - (A4).

Further, the present invention provides a method for breeding a transgenic plant, which is method C or method D:

the method C comprises the following steps: a method of breeding transgenic plants with increased salt tolerance comprising the steps of: inhibiting and expressing a coding gene of GmNAC2 protein in a receptor plant to obtain a transgenic plant; the transgenic plant has increased salt tolerance as compared to the recipient plant. The GmNAC2 protein is a protein shown in any one of the above (A1) - (A4).

The method D comprises the following steps: a method of breeding transgenic plants with reduced salt tolerance comprising the steps of: introducing a nucleic acid molecule capable of expressing GmNAC2 protein into a recipient plant to obtain a transgenic plant; the transgenic plant has reduced salt tolerance as compared to the recipient plant. The GmNAC2 protein is a protein shown in any one of the above (A1) - (A4).

In the method C, the 'suppression of the expression of the coding gene of the GmNAC2 protein in the receptor plant' can be realized by introducing an interference vector containing a DNA fragment shown as the formula (I) into the receptor plant;

SEQ_{forward direction}-X-SEQ_{Reverse direction} (I)

Said SEQ_{Forward direction}Has the sequence of the 426-873 position of SEQ ID No. 2;

said SEQ_{Reverse direction}And the sequence of SEQ_{Forward direction}Is complementary in reverse direction;

said X is said SEQ_{Forward direction}And said SEQ_{Reverse direction}In the sequence, the X and the

SEQ_{Forward direction}And said SEQ_{Reverse direction}Are not complementary.

In one embodiment of the present invention, the interference vector in the method C is specifically a recombinant vector obtained by inserting the DNA fragment shown in the 426-873 rd position of SEQ ID No.2 between SacI and KpnI of the pZH01 vector and simultaneously inserting the DNA fragment shown in the reverse complement of the 426-873 rd position of SEQ ID No.2 between SacI and XbaI.

Since GmNAC2 negatively regulates plant salt tolerance, methods for reducing the expression of GmNAC2 and plant interference expression vectors are protected, and for example, RNA interference (RNAi), a technical system for gene editing using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology, and the like are developed.

When GmNAC2 is used to construct a recombinant plant interference expression vector, any promoter such as cauliflower mosaic virus (CAMV)35S promoter, maize Ubiquitin promoter (Ubiquitin) may be added before its transcription initiation nucleotide, and they may be used alone or in combination with other plant promoters.

In said method D, said "introducing into a recipient plant a nucleic acid molecule capable of expressing said GmNAC2 protein" can be effected by introducing into said recipient plant a recombinant expression vector comprising a gene encoding said GmNAC2 protein.

The recombinant expression vector can be constructed using existing plant expression vectors. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment 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 polyadenylation signal can direct polyadenylation to the 3 'end of the mRNA precursor, and untranslated regions transcribed from the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (e.g., nopalin synthase Nos), plant genes (e.g., soybean storage protein genes) all have similar functions.

When GmNAC2 is used to construct a recombinant plant expression vector, any one of an enhanced promoter or a constitutive promoter (e.g., cauliflower mosaic virus (CAMV)35S promoter, Ubiquitin promoter from maize (Ubiquitin)) or a tissue-specific expression promoter (e.g., seed-specific expression promoter) may be added before the transcription initiation nucleotide, and they may be used alone or in combination with other plant promoters. In addition, when the gene of the present invention is used to construct plant expression vectors, 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 proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound which can produce a color change (GUS gene, luciferase gene, etc.), an antibiotic marker having resistance (gentamicin marker, kanamycin marker, etc.), or a chemical-resistant marker gene (e.g., herbicide-resistant gene), etc., which can be expressed in plants.

In the present invention, the promoter for promoting transcription of the encoding gene in the recombinant expression vector is a 35S promoter.

More specifically, the recombinant vector is a recombinant plasmid (named pBin438-GmNAC2) obtained by inserting the coding gene of the GmNAC2 protein into a multiple cloning site (such as BamH I and KpnI) of a pBin438 vector.

In the above method, the introduction of the interference vector or the recombinant expression vector into the recipient plant may specifically be: plant cells or tissues are transformed by conventional biological methods using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, conductance, agrobacterium mediation, etc., and the transformed plant tissues are grown into plants.

Transformed cells, tissues or plants are understood to comprise not only the end product of the transformation process, but also transgenic progeny thereof.

In each of the above aspects, the "nucleic acid molecule capable of expressing the GmNAC2 protein" is a gene encoding the GmNAC2 protein.

Further, the gene encoding the GmNAC2 protein can be a DNA molecule described in any one of the following items:

(B1) DNA molecule shown in SEQ ID No. 2;

(B2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) and encodes the GmNAC2 protein;

(B3) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of homology with the DNA sequence defined in (B1) or (B2) and encodes the GmNAC2 protein.

In the above genes, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with a mixed solution of 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄And 1mM EDTAWashing at 50 deg.C in 0.1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and then washed once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

SEQ ID No.2 comprises 900 nucleotides.

In a third aspect, the invention claims a DNA fragment or an interfering vector, a recombinant bacterium or a transgenic cell line containing said DNA fragment.

The DNA fragment provided by the invention is the DNA fragment shown in the formula (I). Accordingly, the interfering vector is an interfering vector as described herein before.

In a fourth aspect, the invention claims the use of a DNA fragment or an interference vector or a recombinant bacterium or a transgenic cell line as described in the third aspect hereinbefore for increasing salt tolerance in plants.

In each of the above aspects, the salt tolerance may be embodied as the salt tolerance of the transgenic hairy root or the chimera thereof. In one embodiment of the invention, the salt stress of the hairy roots or chimeras thereof is specifically 100mM NaCl for 3 days.

In each of the above aspects, the plant may be a dicotyledonous plant or a monocotyledonous plant.

Further, the dicot may be a leguminous plant.

Further, the leguminous plant may be soybean.

In a particular embodiment of the invention, the plant is soybean (g.max), in particular soybean variety kefeng No. 1.

Experiments prove that the salt tolerance of plants can be obviously improved by reducing the expression of the salt tolerance related protein GmNAC2 and the encoding gene GmNAC2 thereof. The salt tolerance related protein and the coding gene thereof have important significance for cultivating salt tolerant plant varieties so as to improve the crop yield.

Drawings

FIG. 1 is a schematic representation of the plant expression vector pBin438-GmNAC 2.

Fig. 2 is a molecular characterization of GmNAC2 transgenic hairy roots.

Fig. 3 is a phenotypic characterization of GmNAC2 transgenic hairy roots. A is the growth comparison of control plants and GmNAC2 overexpression and GmNAC2-RNAi hairy roots under 100mM NaCl stress; b is the leaf phenotype of the chimera after salt stress of transgenic hairy roots and a contrast.

FIG. 4 is a control and transgenic hairy root length statistics after salt stress. In the figure, indicates significant differences at P <0.05 levels compared to control. 1, comparison; 2, GmNAC 2-RNAi; 3, GmNAC 2-OE.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the following examples,% is by mass unless otherwise specified. In the quantitative tests in the following examples, three replicates were set, and the data are the mean or the mean ± standard deviation of the three replicates.

All plant material was grown at 25 ℃ with 16h/8h light per day (light/dark).

Plant binary expression vector pBin 438: the research on highly effective insect-resistant transgenic tobacco is described in Litaiyuan, Tian Ying Chuan, Qin Xiao Feng, et al, China science (edition B), 1994,24(3):276-282, provided by honor academy of sciences of the institute of microbiology, China. The public was informed by the consent of the Fang Rong Xiang academy and could be obtained from the institute of genetics and developmental biology, the Chinese academy of sciences.

Agrobacterium rhizogenes K599: described in Attila Kereszt, et al, Agrobacterium rhizogenes-mediated transformation of root biology, Nature Protocols,2007,2(4), 549-Straus, publicly available from The professor Peter M Gresson, The University of Queenland, St. Lucia, Queenland 4072, Australia, or obtained from The institute of genetics and developmental biology, Inc., after The Provisions of Peter M Gresson (written consent).

The wild soybeans Y20 and Y55 are collected and evaluated by researchers from the research institute of farming and cultivation of the academy of agricultural sciences of Heilongjiang province, and the public can obtain the wild soybeans from the research institute of farming and cultivation of the academy of agricultural sciences of Heilongjiang province.

Leguminous feng No.1 (Glycine max l.merr.kefeng 1): described in W.K.Zhang, Y.J.Wang, G.Z.Luo, J.S.Zhang, C.Y.He, X.L.Wu, J.Y.gai, S.Y.Chen, QTL mapping of ten analytic trajectories on the sobean (Glycine max L.Merr.) genetic map and the upper association with EST markers, which is publicly available from the institute of genetics and developmental biology, China academy of sciences, 2004,108: 1131-1139.

Vector pZH 01: described in Han Xiao, et al, functional analysis of the rice AP3 homologue OsMADS16 by RNA interference, Plant Molecular Biology,2003,52, 957-.

Example 1 cloning of GmNAC2 encoding Gene GmNAC2

NAC transcription factor family NAC2 was obtained in a transcriptome analysis of wild soybean Y20 and Y55 after 200mM NaCl stress for induction by salt stress.

Based on the information on the full-length cDNA sequence of GmNAC2 in the soybean genomic sequence of plantagdb, primers were designed with the following sequences:

upstream primer with BamH I cleavage site:

GmNAC2-up：5’-cgGGATCCATGGCATCAGAGCTTGAATTGC-3’；

downstream primer with Kpn I cleavage site:

GmNAC2-dp：5’-ggGGTACCTCAAAAGGACTTGTTGGGCCAG-3’。

and carrying out PCR amplification by using Y20 cDNA as a template and GmNAC2-up and GmNAC2-dp as primers to obtain a PCR product of about 900 bp. After sequencing, the PCR product is 900bp and has the nucleotide shown in SEQ ID No. 2. The above sequence was found to be identical to the protein and nucleotide sequence as listed in AAY46122 by database inspection. But there is no description of the function of the protein/gene in the database. The gene shown by the nucleotide is GmNAC2, the protein coded by the gene is named as GmNAC2, and the amino acid sequence of the protein is SEQ ID No. 1.

Example 2 obtaining of transgenic hairy roots

Firstly, plant transgenic vector construction

1. Construction of GmNAC2 overexpression transgene vector

The vector obtained by inserting the fragment obtained by PCR amplification in example 1 (which can also be artificially synthesized) between BamH I and KpnI cleavage sites of pBin438 was named pBin438-GmNAC2, and the schematic structural diagram of the recombinant expression vector pBin438-GmNAC2 is shown in FIG. 1.

2. GmNAC2-RNAi vector construction

A gene fragment 448bp long at the 3' end of the GmNAC2 gene is inserted into a bidirectional expression vector pZH01 in the positive and negative directions, so that an RNAi vector is constructed. Specifically, the recombinant plasmid vector is used as a template, a primer RNAi-F/RNAi-R is used for amplification, the nucleotide 426 to 873 from the 5 'end of the SEQ ID No.2 is inserted into the SacI and KpnI enzyme cutting sites of the pZH01 vector, and the reverse complementary sequence of the nucleotide 426 to 873 from the 5' end of the SEQ ID No.2 is inserted between the SalI and XbaI sites of the pZH01 vector, so that the vector pZH01-GmNAC2-RNAi is obtained.

RNAi-F：5’-TCTAGAGAGCTCCACCCTAAGGTTGGATGATTGGGTG-3’；

RNAi-R：5’-GTCGACGGTACCGAACATGTCC TGCAGCGGC-3’。

Second, overexpression and obtaining of RNAi-GmNAC2 hairy roots

1. And (3) respectively introducing the 2 recombinant expression vectors pBin438-GmNAC2 and pZH01-GmNAC2-RNAi obtained in the step one into the transformed Agrobacterium rhizogenes K599 by an electric shock method to obtain the recombinant Agrobacterium tumefaciens.

Extracting a plasmid for over-expressing the recombinant agrobacterium, and sequencing to show that the plasmid is pBin438-GmNAC2, and the recombinant agrobacterium containing the plasmid is named as K599/pBin438-GmNAC 2. And the recombinant agrobacterium containing pZH01-GmNAC2b-RNAi identified by sequencing is named as K599/GmNAC 2-RNAi.

2. Respectively inoculating recombinant agrobacterium tumefaciens K599/pBin438-GmNAC2 and K599/GmNAC2-RNAi to a seedling of the Megalobacillus buehi No.1 which grows for 6 days and contains two true leaves by using a syringe, and carrying out moisture preservation and growth: the light is irradiated for 16 hours, the temperature is 25 ℃, and the humidity is 50%. After 2 weeks, hairy roots were grown, i.e., transformed hairy roots. 60 trans-pBin 438-GmNAC2 and GmNAC2-RNAi hairy root systems are obtained respectively and are marked as OE and RNAi respectively, and the trans-pBin-GmNAC 2 and GmNAC2-RNAi hairy root systems can be further used for transgene identification and stress tolerance detection.

The empty vector pBin438 was transferred to the seedling of Leguminosae Feng No.1 in the same way to obtain 60 root systems of empty vector hairy roots, which were used as experimental control and labeled as K599.

3. The transgenic hairy roots are subjected to molecular identification. Total RNAs of the trans-pBin 438-GmNAC2, GmNAC2-RNAi hairy roots and the trans-empty vector hairy roots are extracted respectively and are reversely transcribed into cDNA. The expression level of GmNAC2 gene was analyzed using cDNA as a template and primers Primer-F and Primer-R. Real-Time PCR reaction Using the RealTime PCR Master Mix kit from TOYOBO, the procedure was as described. The primer used for detecting the expression level of the GmNAC2 gene is the same as above; the soybean GmTubulin gene is used as an internal standard, and the primers are Primer-TF and Primer-TR. The experiment was repeated three times and the results were averaged ± standard deviation.

Primer-F：5’-ATGGCATCAGAGCTTGAATTGC-3’；

Primer-R：5’-TCAAAAGGACTTGTTGGGCCAG-3’。

Primer-TF：5’-AACTCCATTTCGTCCATTCCTTC-3’；

Primer-TR：5’-TTGAGTGGATTCCCAACAACG-3’。

FIG. 2 shows the results of RT-PCR assays for GmNAC2-OE and GmNAC2-RNAi hairy roots and GmNAC2 expression in the transgenic empty vector hairy root K599. With the soybean GmTubulin gene as an internal standard, the expression of endogenous GmNAC2 detected in K599 is about 0.98, while the expression of GmNAC2 in GmNAC2-RNAi hairy roots is about 0.76, which is reduced compared with the control. The relative expression amount of GmNAC2 in the transferred pBin438-GmNAC2 hairy roots is about 3.96; in the trans-pBin 438-GmNAC2 hairy root, the expression level of GmNAC2 is much higher than that of GmNAC2 in the trans-empty vector root system. Whereas GsHSF2b-RNAi hairy roots were transformed to have lower GmNAC2 expression than the control.

Example 3 salt tolerance identification of GsHSF2b transgenic hairy roots

Experimental samples are transferred empty vector root (K599), overexpressed GmNAC2 hairy roots (GmNAC2-OE) and transferred GmNAC2-RNAi hairy roots (GmNAC 2-RNAi).

12 transgenic GmNAC2-OE, GmNAC2-RNAi hairy roots and the transgenic empty vector K599 hairy roots are respectively taken, 6 plants are immersed in 100mM NaCl solution, 6 plants are placed in water as a water culture control, and the water culture control is treated for 3 days at 25 ℃. The length of the hairy roots is calculated, and the average length and standard deviation of each hairy root are calculated. The experimental biology was repeated three times.

After 3 days of treatment, the results are shown in FIG. 3: the phenotype of the empty transgenic hairy root K599, the transgenic hairy roots GmNAC2-OE and GmNAC2-RNAi has little difference in growth of the hairy roots of the three under the condition of water culture, and the leaves of the transgenic hairy root chimera have no obvious difference. After being treated by 100mM NaCl for 3 days, the growth of three types of hairy roots is inhibited, the GmNAC2-OE hairy roots grow more slowly than a control, the GmNAC2-RNAi hairy roots grow better than the control, chimeric leaves show wilting to different degrees under salt stress, the GmNAC2-RNAi wilting degree is lower than the control, and the GmNAC2-OE chimeric leaves have the highest wilting degree.

The amount of hairy root phase growth was measured specifically as follows: specifically measuring each group of root systems, firstly calculating the length of each hairy root, then calculating the average value, and then taking the average value plus or minus standard deviation;

in water culture, the trans-empty vectors K599, GmNAC2-RNAi and GmNAC2-OE hairy root lengths are respectively 2.3 +/-0.49, 2.38 +/-0.56 and 2.28 +/-0.57, and no obvious difference exists. After 100mM NaCl treatment, the hair root lengths of the control, GmNAC2-RNAi and GmNAC2-OE are respectively 0.98 +/-0.48, 1.21 +/-0.39 and 0.75 +/-0.41, the three are obviously different, the growth of GmNAC2-RNAi is obviously superior to that of the control, and the growth of the GmNAC2-OE hair root is obviously slower than that of the control (figure 4). The above statistics show that the reduced expression of GmNAC2 significantly increases the tolerance of hairy roots to salt stress, while the excessive expression of GmNAC2 gene significantly reduces the salt tolerance of hairy roots.

The above examples demonstrate that reducing the expression of the soybean transcription factor NAC family member GmNAC2 can improve tolerance of plants to salt stress.

<110> institute of genetics and developmental biology of Chinese academy of sciences

Application of <120> protein GmNAC2 in regulation and control of salt tolerance of plants

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Claims

The application of GmNAC2 protein in regulating and controlling the salt tolerance of plants;

the GmNAC2 protein is any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No. 1;

(A2) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in (A1);

the activity and/or the expression level of the GmNAC2 protein or the coding gene thereof in the plant are reduced, and the salt tolerance of the plant is improved;

the plant is soybean;

the salt tolerance is embodied as the salt tolerance of the transgenic hairy roots.
2. A method for breeding a plant variety with improved salt tolerance, comprising the step of reducing the expression level of GmNAC2 protein in a recipient plant;

the GmNAC2 protein is any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No. 1;

(A2) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in (A1);

the plant is soybean;

the salt tolerance is embodied as the salt tolerance of the transgenic hairy roots.
3. A method of breeding transgenic plants with improved salt tolerance comprising the steps of: inhibiting and expressing a coding gene of GmNAC2 protein in a receptor plant to obtain a transgenic plant; the transgenic plant has increased salt tolerance as compared to the recipient plant;

the GmNAC2 protein is any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No. 1;

(A2) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in (A1);

the plant is soybean;

the salt tolerance is embodied as the salt tolerance of the transgenic hairy roots.
4. The method of claim 3, wherein: the suppression expression of the coding gene of the GmNAC2 protein in the receptor plant is realized by introducing an interference vector containing a DNA fragment shown as a formula (I) into the receptor plant;

SEQ_{forward direction}- X - SEQ_{Reverse direction} （I）

Said SEQ_{Forward direction}The sequence of (1) is the 426-873 position of SEQ ID No. 2;

said SEQ_{Reverse direction}And the sequence of SEQ_{Forward direction}Is complementary in reverse direction;

said X is said SEQ_{Forward direction}And said SEQ_{Reverse direction}In the sequence, the X and the SEQ_{Forward direction}And said SEQ_{Reverse direction}Are not complementary.
5. The method of claim 3, wherein: the coding gene of the GmNAC2 protein is a DNA molecule shown in any one of the following lists:

(B1) DNA molecule shown in SEQ ID No. 2;

(B2) and (3) a DNA molecule which has more than 80% homology with the DNA sequence limited by (B1) and encodes the GmNAC2 protein.
6, the application of the DNA fragment or the interference vector, the recombinant bacterium or the transgenic cell line in improving the salt tolerance of the plant;

the DNA fragment is shown as a formula (I);

SEQ_{forward direction}- X - SEQ_{Reverse direction} （I）

Said SEQ_{Forward direction}Has the sequence of the 426-873 position of SEQ ID No. 2;

said SEQ_{Reverse direction}And the sequence of SEQ_{Forward direction}Is complementary in reverse direction;

said X is said SEQ_{Forward direction}And said SEQ_{Reverse direction}In the sequence, the X and the

SEQ_{Forward direction}And said SEQ_{Reverse direction}Are not complementary;

the interference vector is an interference vector containing a DNA fragment shown in a formula (I);

the recombinant bacterium is a recombinant bacterium containing a DNA fragment shown as a formula (I);

the transgenic cell line is a transgenic cell line containing a DNA segment shown in a formula (I);

the plant is soybean;

the salt tolerance is embodied as the salt tolerance of the transgenic hairy roots.