CN111620933B - Application of protein GmNAC2 in regulating plant salt tolerance - Google Patents

Abstract

The invention discloses the application of protein GmNAC2 in regulating the salt tolerance of plants. The present invention provides the application of GmNAC2 protein or its related biological materials in regulating plant salt tolerance; GmNAC2 protein is the protein shown in SEQ ID No. 1 or its substitution and/or deletion of one or several amino acid residues and/or Or added, or a protein whose sequence has 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 obtained by linking the N-terminal and/or C-terminal with a tag protein. The present invention proves that the reduced expression of the protein GmNAC2 and its encoding gene GmNAC2 can significantly improve the salt tolerance of plants. The salt-tolerance-related protein and its encoding gene of the present invention are of great significance for cultivating salt-tolerant plant varieties, thereby increasing crop yield.

Description

Application of protein GmNAC2 in regulating plant salt tolerance

technical field

The invention relates to the field of biotechnology, in particular to the application of a protein GmNAC2 in regulating the salt tolerance of plants.

Background technique

Changes in physical and chemical factors in the environment, such as drought, salinity, low temperature and other stress factors, have an important impact on the growth and development of plants. In severe cases, large-scale crop yield reductions will occur. Cultivating stress-tolerant crops is one of the main goals of the planting industry. At present, genetic engineering breeding has become one of the important methods to enhance the stress tolerance of crops. Higher plant cells have multiple pathways to respond to various environmental stresses, among which transcription factors play a role in regulating the expression of stress tolerance-related effector genes. Many types of transcription factors have been found to be related to plant stress tolerance in plants, such as DREB in EREBP/AP2, bZIP, MYB, WRKY and so on.

The NAC ( N AM/ A TAF1/2/ C UC2 ) family is a unique transcription factor family in plants, and the NAC genes that have been studied play a very important role in different life processes. Some NAC family members positively regulate agronomic traits, while some negatively regulate, such as defense against pathogens, plant decline, morphogenesis, and responses to abiotic stresses.

In soybean, Gai Junyi, Yu Deyue, etc. cloned the GmNAC2 gene based on bioinformatics (Meng, Q.C., Yu, D.Y. and Gai, J.Y., Journal of Plant Physiology 164, 2007, 1002-1012,) and identified GmNAC2 in soybean. Expression characteristics in organs and during development, but the function of the GmNAC2 gene and its encoded protein GmNAC2 has not been studied.

Soybean is an important oil crop and the main source of plant protein. It is of great theoretical and practical significance to clarify its stress tolerance mechanism and improve its stress tolerance.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the application of the protein GmNAC2 in regulating the salt tolerance of plants.

In the first aspect, the present invention claims the application of GmNAC2 protein or related biomaterials in regulating the salt tolerance of plants.

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

The GmNAC2 protein is any one of the following proteins:

(A1) the protein whose amino acid sequence is SEQ ID No.1;

(A2) A protein that has the amino acid sequence shown in SEQ ID No. 1 through the substitution and/or deletion and/or addition of one or several amino acid residues and has the same function;

(A3) A protein that has 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology with the amino acid sequence defined in any of (A1)-(A2) and has the same function;

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

In (A2), the substitution and/or deletion and/or addition of one or several amino acid residues refers to the substitution and/or deletion and/or addition of no more than ten amino acid residues.

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

In the application, the activity and/or expression level of the GmNAC2 protein or the gene encoding it in the plant is reduced, and the salt tolerance of the plant is improved; the activity and/or expression of the GmNAC2 protein or the gene encoding the GmNAC2 protein in the plant / Or the expression level is increased, and the salt tolerance of the plant is decreased.

In the second aspect, the present invention claims to protect a method for cultivating plant varieties, which is method A or method B:

Method A: A method of breeding plant varieties with increased salt tolerance, which may include the step of reducing the expression level and/or activity of GmNAC2 protein in recipient plants. The GmNAC2 protein is any of the proteins shown in (A1)-(A4) above.

Method B: A method of breeding a plant variety with reduced salt tolerance, which may include the step of increasing the expression level and/or activity of the GmNAC2 protein in the recipient plant. The GmNAC2 protein is any of the proteins shown in (A1)-(A4) above.

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

Method C: a method for cultivating transgenic plants with improved salt tolerance, which may include the following steps: inhibiting expression of the gene encoding the GmNAC2 protein in the recipient plant to obtain a transgenic plant; the transgenic plant is similar to the recipient plant; Higher than salt tolerance. The GmNAC2 protein is any of the proteins shown in (A1)-(A4) above.

Method D: a method for cultivating transgenic plants with reduced salt tolerance, which may include the following steps: introducing a nucleic acid molecule capable of expressing GmNAC2 protein into a recipient plant to obtain a transgenic plant; the transgenic plant is similar to the recipient plant; Lower than salt tolerance. The GmNAC2 protein is any of the proteins shown in (A1)-(A4) above.

In the method C, the "inhibiting the expression of the gene encoding the GmNAC2 protein in the recipient plant" can be achieved by introducing an interference vector containing the DNA fragment shown in formula (I) into the recipient plant;

SEQ _forward - X - SEQ _reverse (I)

The _forward sequence of the SEQ is positions 426-873 of SEQ ID No.2;

The _reverse sequence of the SEQ is reverse complementary to the _forward sequence of the SEQ;

Described X is the spacer sequence between described SEQ _forward and described SEQ _reverse , in sequence, described X and described

Neither the SEQ _{forward nor} the SEQ _reverse is complementary.

In an embodiment of the present invention, in the method C, the interference vector is specifically inserted into the DNA fragment shown in positions 426-873 of SEQ ID No. 2 between SadI and KpnI of the pZH01 vector, and at the same time in the SadI The recombinant vector obtained by inserting the DNA fragment shown in the reverse complementary sequence of positions 426-873 of SEQ ID No. 2 between XbaI and XbaI.

Given that GmNAC2 negatively regulates the salt tolerance of plants, the methods used to reduce the expression of GmNAC2 and plant interference expression vectors are all protected, for example, RNA interference (RNAi), clustered regularly spaced short palindromic repeats (Clustered regularly spaced short palindromic repeats) Interspaced short palindromic repeats, CRISPR) technology and other technology systems for gene editing.

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

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

The recombinant expression vector can be constructed using existing plant expression vectors. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, and the like. The plant expression vector may also contain the 3' untranslated region of the exogenous gene, i.e., containing the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The poly(A) signal can guide the addition of poly(A) to the 3' end of the mRNA precursor, such as Agrobacterium crown gall inducing (Ti) plasmid genes (such as Nos synthase Nos gene), plant genes (such as soybean storage) The untranslated regions transcribed at the 3' end of protein genes) have similar functions.

When using GmNAC2 to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter (such as cauliflower mosaic virus (CAMV) 35S promoter, maize ubiquitin promoter (Ubiquitin)), or tissue-specific expression promoters (eg, seed-specific expression promoters), which can be used alone or in combination with other plant promoters. In addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG initiation codons or adjacent region initiation codons, etc., but must be Identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The translation control signals and initiation codons can be derived from a wide variety of sources, either natural or synthetic. The translation initiation region can 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 used can be processed, such as adding a gene (GUS gene, luciferase gene, luciferase gene) that can be expressed in plants encoding an enzyme that can produce a color change or a luminescent compound. Gene, etc.), antibiotic markers with resistance (gentamycin marker, kanamycin marker, etc.) or anti-chemical reagent marker gene (such as herbicide resistance gene) and so on.

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

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

In the above method, the interference vector or the recombinant expression vector is introduced into the recipient plant, specifically: by using Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrical conductivity, agricultural Bacillus-mediated and other conventional biological methods transform plant cells or tissues, and the transformed plant tissues are grown into plants.

A transformed cell, tissue or plant is understood to include not only the end product of the transformation process, but also its transgenic progeny.

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

Further, the encoding gene of the GmNAC2 protein can be any of the following DNA molecules:

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

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

(B3) A DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more or 80% or more 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°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO ₄ and 1 mM EDTA, 50°C, 2×SSC, 0.1% Rinse in SDS; also: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M NaPO ₄ and 1mM EDTA, rinse at 50°C, 1×SSC, 0.1% SDS; also: 50°C, Hybridize in a mixture of 7% SDS, 0.5M NaPO and ₁ mM EDTA, rinse at 50°C in 0.5×SSC, 0.1% SDS; also: 50°C in 7% SDS, 0.5M NaPO and ₁ mM Hybridize in a mixed solution of EDTA, rinse in 0.1×SSC, 0.1% SDS at 50°C; also: hybridize in a mixed solution of 7% SDS, 0.5M NaPO ₄ and 1mM EDTA at 50°C, at 65°C, Wash in 0.1×SSC, 0.1% SDS; also: in 6×SSC, 0.5% SDS solution, hybridize at 65°C, then use 2×SSC, 0.1%SDS and 1×SSC, 0.1%SDS each Wash the membrane once.

SEQ ID No. 2 contains 900 nucleotides.

In a third aspect, the present invention claims DNA fragments or interfering vectors, recombinant bacteria or transgenic cell lines containing said DNA fragments.

The DNA fragment provided by the present invention is the DNA fragment shown in the foregoing formula (I). Correspondingly, the interference carrier is the aforementioned interference carrier.

In the fourth aspect, the present invention claims the use of the DNA fragment or the interference vector or the recombinant bacteria or the transgenic cell line described in the third aspect above in improving the salt tolerance of plants.

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

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

Further, the dicotyledonous plant may be a legume.

Still further, the legume can be soybean.

In a specific embodiment of the present invention, the plant is soybean (G.max), specifically soybean variety Kefeng No. 1.

Experiments show that the reduced expression of the salt tolerance-related protein GmNAC2 and its encoding gene GmNAC2 of the present invention can significantly improve the salt tolerance of plants. The salt-tolerance-related protein and its encoding gene of the present invention are of great significance for cultivating salt-tolerant plant varieties, thereby increasing crop yield.

Description of drawings

Figure 1 is a schematic diagram of the plant expression vector pBin438-GmNAC2.

Figure 2. Molecular identification of GmNAC2 transgenic hairy roots.

Figure 3 shows the phenotypic identification of GmNAC2 transgenic hairy roots. A is the growth comparison of control plants and GmnAC2 overexpression and GmnAC2-RNAi hairy roots under 100 mM NaCl stress; B is the chimeric leaf phenotype of transgenic hairy roots and controls under salt stress.

Figure 4 shows the root length statistics of control and transgenic hairy roots after salt stress. In the figure, * indicates a significant difference compared with the control at the P<0.05 level. 1, control; 2, GmnAC2-RNAi; 3, GmnAC2-OE.

Detailed ways

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

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The % in the following examples, unless otherwise specified, are all mass percentages. Quantitative experiments in the following examples are all set up three repeated experiments, and the data are the average or the mean ± standard deviation of the three repeated experiments.

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

Plant binary expression vector pBin438: recorded in Li Taiyuan, Tian Yingchuan, Qin Xiaofeng, et al. Research on high-efficiency insect-resistant transgenic tobacco [J]. Chinese Science (Series B), 1994, 24(3): 276-282, by the Chinese Academy of Sciences Microbiology Provided by Academician Fang Rongxiang of the Institute. The public can obtain it from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences with the consent of Academician Fang Rongxiang.

Agrobacterium rhizogenes K599: described in Attila Kereszt, et al., Agrobacterium rhizogenes-mediaded transformation of soybean to study of root biology, Nature Protocols, 2007, 2(4), 549-552), publicly available from Peter M Gressnon Professor, The University of Queensland, St Lucia, Queensland 4072, Australia, obtained, or obtained from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences with the consent of Professor Peter M Gressnon (written consent).

Wild soybean Y20 and Y55 were collected and evaluated by researcher Lai Yongcai from the Institute of Tillage and Cultivation, Heilongjiang Academy of Agricultural Sciences, and the public can obtain them from the Institute of Tillage and Cultivation, Heilongjiang Academy of Agricultural Sciences.

Glycine max L.Merr.Kefeng 1: recorded 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 agronomictraits on the soybean (Glycine max L. Merr.) Genetic map and their associationwith EST markers, Theor.Appl.Genet,2004,108:1131-1139, the public can obtain from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

Vector pZH01: recorded in Han Xiao, et al. Functional analysis of the rice AP3homologue OsMADS16 by RNA interference, Plant Molecular Biology, 2003, 52, 957-966, public available from Institute of Genetics and Developmental Biology, Chinese Academy of Sciences and Heilongjiang Academy of Agricultural Sciences Obtained from the Institute of Cultivation.

Example 1. Cloning of GmNAC2 encoding gene GmNAC2

The NAC transcription factor family NAC2 was induced by salt stress in the transcriptome analysis of wild soybean Y20 and Y55 after 200 mM NaCl stress.

According to the information of the full-length cDNA sequence of GmNAC2 in the soybean genome sequence of PlantGDB, primers were designed, and the primer sequences are as follows:

Upstream primer with BamH I restriction site:

GmNAC2-up: 5'-cg GGATCC ATGGCATCAGAGCTTGAATTGC-3';

Downstream primer with Kpn I restriction site:

GmNAC2-dp: 5'-gg GGTACC TCAAAAGGACTTGTTGGGCCAG-3'.

Using Y20 cDNA as template and GmNAC2-up and GmNAC2-dp as primers, PCR amplification was performed to obtain a PCR product of about 900 bp. After sequencing, the PCR product is 900bp and has the nucleotides shown in SEQ ID No.2. After checking the database, the above sequence is the same as the protein and nucleotide sequence listed in AAY46122. But there is no functional description of this protein/gene in the database. The gene shown by the above nucleotide is GmNAC2, the protein encoded by the gene is named GmNAC2, and the amino acid sequence of the protein is SEQ ID No.1.

Example 2, the acquisition of transgenic hairy roots

1. Construction of plant transgenic vector

1. Construction of GmNAC2 overexpression transgenic vector

The fragment obtained by PCR amplification in Example 1 (the fragment can also be artificially synthesized) was inserted into the vector obtained between the BamH I and KpnI restriction sites of pBin438, and the vector was named pBin438-GmNAc2, and the recombinant expression vector pBin438 - A schematic diagram of the structure of GmNAC2 is shown in Figure 1.

2. Construction of GmNAC2-RNAi vector

The 448bp-long gene fragment at the 3' end of the GmNAC2 gene was inserted into the bidirectional expression vector pZH01 in both forward and reverse directions to construct an RNAi vector. Specifically, using the above recombinant plasmid vector as a template, the primer RNAi-F/RNAi-R was used to amplify, and the nucleotides from 426 to 873 from the 5' end of SEQ ID No.2 were inserted into the SacI and KpnI of the pZH01 vector The enzyme cleavage site was inserted, and the reverse complementary sequence of nucleotides 426 to 873 from the 5' end of SEQ ID No. 2 was inserted between the SalI and XbaI sites of the pZH01 vector to obtain the vector pZH01-GmNAC2-RNAi.

RNAi-F: 5'- TCTAGAGAGCTC CACCCTAAGGTTGGATGATTGGGTG-3';

RNAi-R: 5'- GTCGACGGTACC GAACATGTCC TGCAGCGGC-3'.

2. Overexpression and acquisition of RNAi-GmNAC2 hairy roots

1. The two recombinant expression vectors pBin438-GmNAC2 and pZH01-GmNAC2-RNAi obtained in the above step 1 were respectively introduced into Agrobacterium rhizogenes K599 by electroporation to obtain recombinant Agrobacterium.

The plasmid overexpressing the recombinant Agrobacterium was extracted, and sequencing showed that the plasmid was pBin438-GmNAC2, and the recombinant Agrobacterium containing the plasmid was named K599/pBin438-GmNAC2. The recombinant Agrobacterium containing pZH01-GmNAC2b-RNAi identified by sequencing was named K599/GmNAC2-RNAi.

2. The recombinant Agrobacterium K599/pBin438-GmNAC2 and K599/GmNAC2-RNAi were inoculated with two true leaves for 6 days respectively with a syringe, and the seedlings of soybean Kefeng No. 1 with two true leaves were grown for 6 days. %. After 2 weeks, the hairy roots that grow out are called transformed hairy roots. 60 pBin438-GmNAC2 and GmNAC2-RNAi hairy root systems were obtained respectively, which were marked as OE and RNAi, respectively, which could be used for further identification of transgene and detection of stress tolerance.

In the same way, the empty vector pBin438 was transformed into soybean Kefeng No. 1 seedlings, and 60 hairy root systems of the empty vector were obtained, which were used as the experimental control and marked as K599.

3. Molecular identification of the above-mentioned transgenic hairy roots. The total RNA of hairy roots transfected with pBin438-GmNAC2, GmNAC2-RNAi and empty vector hairy roots were extracted and reverse transcribed into cDNA. Using cDNA as a template, primers Primer-F and Primer-R were used to analyze the expression of GmNAC2 gene. Real-Time PCR reactions were performed using the RealTimePCR Master Mix kit from TOYOBO, and the instructions were followed. The primers used for the detection of GmNAC2 gene expression were the same as above; the soybean GmTubulin gene was used as the internal standard, and the primers used were Primer-TF and Primer-TR. The experiment was repeated three times, and the results were taken as the mean ± standard deviation.

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

Primer-R: 5'-TCAAAAGGACTTGTTGGGCCAG-3'.

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

Primer-TR: 5'-TTGAGTGGATTCCCAACAACG-3'.

Figure 2 shows the RT-PCR detection results of GmNAC2 expression in GmNAC2-OE and GmNAC2-RNAi hairy roots and in hairy roots K599 with empty vector. Using soybean GmTubulin gene as the internal standard, it showed that the expression of endogenous GmNAC2 was about 0.98 in K599, while the expression of GmNAC2 in GmNAC2-RNAi hairy roots was about 0.76, which was lower than the control. The relative expression level of GmNAC2 in pBin438-GmNAC2 hairy roots was about 3.96; in pBin438-GmNAC2 hairy roots, the expression level of GmNAC2 was much higher than that in roots transfected with empty vector. The expression of GmNAC2 in GsHSF2b-RNAi hairy roots was lower than that in the control.

Example 3. Salt tolerance identification of transgenic GsHSF2b hairy roots

The experimental samples were the root system of the empty vector (K599), the hairy root of overexpressing GmNAC2 (GmNAC2-OE) and the hairy root of the transfected GmNAC2-RNAi (GmNAC2-RNAi).

Twelve each of the transgenic GmNAC2-OE, GmNAC2-RNAi hairy roots and the empty vector K599 hairy roots were taken, 6 were immersed in 100 mM NaCl solution, and 6 were placed in water as a hydroponic control, and treated at 25°C for 3 days. The length of hairy roots was calculated, and the average length and standard deviation of each hairy root were calculated. Experimental biology was repeated three times.

After 3 days of treatment, photographed and observed, the results are shown in Figure 3: the phenotypes of the empty vector hairy root K599 and the transgenic hairy roots GmNAC2-OE and GmNAC2-RNAi under the condition of hydroponics, the growth of the three hairy roots There were no differences, nor were the leaves of the transgenic hairy root chimeras significantly different. 100mM NaCl treatment for 3 days inhibited the growth of the three types of hairy roots. The growth of GmNAC2-OE hairy roots was slower than that of the control, while the growth of GmNAC2-RNAi hairy roots was better than that of the control. Chimeric leaves showed different effects under salt stress. The degree of wilting was lower in GmNAC2-RNAi, followed by the control, while the GmNAC2-OE chimera had the highest degree of wilting.

The measurement of hairy root phase growth is as follows: Measure each group of roots, first calculate the length of each hairy root, then calculate the average value, and then take the average ± standard deviation;

Under hydroponics, the hairy root lengths of empty vector K599, GmNAC2-RNAi and GmNAC2-OE were 2.3±0.49, 2.38±0.56 and 2.28±0.57, respectively, with no significant difference. After treatment with 100 mM NaCl, the hairy root lengths of control, GmNAC2-RNAi and GmNAC2-OE were 0.98±0.48, 1.21±0.39 and 0.75±0.41, respectively, and there were significant differences among them. GmNAC2-RNAi grew significantly better than control, while GmNAC2 -OE hairy roots grew significantly slower than controls (Figure 4). The above statistical data showed that the decreased expression of GmNAC2 significantly increased the tolerance of hairy roots to salt stress, while the overexpression of GmNAC2 significantly decreased the salt tolerance of hairy roots.

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

<110> Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

Application of <120> protein GmNAC2 in regulating plant salt tolerance

<130> GNCLN190297

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 299

<212> PRT

<213> Glycine max (L.) Merrill

<400> 1

Met Ala Ser Glu Leu Glu Leu Pro Pro Gly Phe Arg Phe His Pro Thr

1 5 10 15

Asp Glu Glu Leu Val Leu His Tyr Leu Cys Arg Lys Cys Ala Ser Gln

20 25 30

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

35 40 45

Pro Trp Asp Leu Pro Gly Leu Ala Thr Tyr Gly Glu Lys Glu Trp Tyr

50 55 60

Phe Phe Ser Pro Arg Asp Arg Lys Tyr Pro Asn Gly Ser Arg Pro Asn

65 70 75 80

Arg Ala Ala Gly Thr Gly Tyr Trp Lys Ala Thr Gly Ala Asp Lys Pro

85 90 95

Ile Gly Gln Pro Lys Pro Val Gly Ile Lys Lys Ala Leu Val Phe Tyr

100 105 110

Ala Gly Lys Ala Pro Lys Gly Asp Lys Ser Asn Trp Ile Met His Glu

115 120 125

Tyr Arg Leu Ala Asp Val Asp Arg Ser Val Arg Lys Lys Asn Thr Leu

130 135 140

Arg Leu Asp Asp Trp Val Leu Cys Arg Ile Tyr Asn Lys Lys Gly Thr

145 150 155 160

Ile Glu Lys Leu Gln Pro Ser Ser Asp Val Ala His Ser Arg Asn Ile

165 170 175

Glu Ser Ser Glu Ile Glu Asp Arg Lys Pro Glu Ile Leu Lys Ser Gly

180 185 190

Gly Gly Cys Leu Pro Pro Pro Val Pro Val Pro Ala Pro Pro Gln Ala

195 200 205

Thr Ala Lys Thr Asp Tyr Met Tyr Phe Asp Pro Ser Asp Ser Ile Pro

210 215 220

Lys Leu His Thr Asp Ser Ser Cys Ser Glu Gln Val Val Ser Pro Glu

225 230 235 240

Phe Ala Ser Glu Val Gln Ser Glu Pro Lys Trp Asn Glu Trp Glu Lys

245 250 255

Ser Leu Glu Phe Pro Phe Asn Tyr Val Asp Ala Thr Leu Asn Asn Ser

260 265 270

Phe Met Ala Gln Phe Gln Gly Asn Asn Gln Met Leu Ser Pro Leu Gln

275 280 285

Asp Met Phe Met Tyr Trp Pro Asn Lys Ser Phe

290 295

<210> 2

<211> 900

<212> DNA

<213> Glycine max (L.) Merrill

<400> 2

atggcatcag agcttgaatt gcccccaggc ttcagattcc atccaacgga cgaggagctg 60

gtgttgcact atctctgccg caaatgcgcg tcgcagccaa tcgccgttcc catcatcgcc 120

gaaatcgacc tctacaaata cgacccctgg gacttacccg gattggctac ttatggagag 180

aaagagtggt acttcttttc accacgggac cggaaatacc caaacggttc gaggccgaac 240

cgggcggctg gcaccggtta ctggaaggca accggggcgg ataagcccat tggtcagccc 300

aaaccggttg ggattaaaaa agctttggtg ttttacgcag ggaaagctcc taaaggggac 360

aaaagcaatt ggatcatgca cgagtatcgt ctcgcagacg tagatcgctc cgttcgcaaa 420

aagaacaccc taaggttgga tgattgggtg ctttgccgta tttacaacaa gaagggcacg 480

atcgagaaac tgcaaccaag cagcgatgtt gctcatagcc gaaatatcga atcctcggag 540

atcgaagaca ggaagccgga gattctgaaa agcggaggag gttgtcttcc gccgcctgtg 600

ccggtgcctg cgccgccgca agcgacggcg aagacggatt acatgtactt cgacccgtcg 660

gattcaatcc cgaagctgca cacggactcg agctgttcgg agcaggtggt atcgccggaa 720

ttcgcgagcg aggtgcaaag cgagcccaag tggaacgagt gggagaaaag cctcgaattt 780

ccatttaatt acgtggatgc cactctcaac aacagcttca tggcccaatt ccagggcaat 840

aatcagatgt tgtcgccgct gcaggacatg ttcatgtact ggcccaacaa gtccttttga 900

Claims (6)

1. Application of GmNAC2 protein in regulating plant salt tolerance; The GmNAC2 protein is any one of the following proteins: (A1) The protein whose amino acid sequence is SEQ ID No.1; (A2) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein as defined in (A1); The activity and/or expression level of the GmNAC2 protein or its encoding gene in the plant is reduced, and the salt tolerance of the plant is improved; The plant is soybean; The salt tolerance is reflected in the salt tolerance of the transgenic hairy roots. 2. A method for cultivating a plant variety with improved salt tolerance, comprising the step of reducing the expression of GmNAC2 protein in a recipient plant; The GmNAC2 protein is any one of the following proteins: (A1) The protein whose amino acid sequence is SEQ ID No.1; (A2) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein as defined in (A1); The plant is soybean; The salt tolerance is reflected in the salt tolerance of the transgenic hairy roots. 3. A method for cultivating a transgenic plant with improved salt tolerance, comprising the steps of: suppressing expression of the gene encoding the GmNAC2 protein in the recipient plant to obtain a transgenic plant; the transgenic plant is resistant to the recipient plant compared to the recipient plant. increased salinity; The GmNAC2 protein is any one of the following proteins: (A1) The protein whose amino acid sequence is SEQ ID No.1; (A2) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein as defined in (A1); The plant is soybean; The salt tolerance is reflected in the salt tolerance of the transgenic hairy roots. 4. The method according to claim 3, characterized in that: inhibiting expression of the gene encoding GmNAC2 protein in the recipient plant is performed by introducing into the recipient plant a DNA containing the formula (I) Fragmented by the interference vector; seq _forward - X - seq _reverse (I) The _forward sequence of the SEQ is the 426th-873rd position of SEQ ID No.2; The _reverse sequence of the SEQ is reverse complementary to the _forward sequence of the SEQ; The X is the spacer sequence between the SEQ _forward and the SEQ _reverse , and in sequence, the X is not complementary to the SEQ _forward and the SEQ _reverse . 5. The method according to claim 3, wherein the encoding gene of the GmNAC2 protein is a DNA molecule as shown in any of the following: (B1) DNA molecule shown in SEQ ID No.2; (B2) A DNA molecule having more than 80% homology with the DNA sequence defined in (B1) and encoding the GmNAC2 protein. 6. Application of DNA fragments or interference vectors, recombinant bacteria or transgenic cell lines in improving plant salt tolerance; The DNA fragment is shown in formula (I); seq _forward - X - seq _reverse (I) The _forward sequence of the SEQ is the 426th-873rd position of SEQ ID No.2; The _reverse sequence of the SEQ is reverse complementary to the _forward sequence of the SEQ; The X is the spacer sequence between the SEQ _forward and the SEQ _reverse , and in sequence, the X and the SEQ _forward and described SEQ _reverse are not complementary; The interference carrier is an interference carrier containing the DNA fragment shown in formula (I); The recombinant bacteria are recombinant bacteria containing the DNA fragment shown in formula (I); The transgenic cell line is a transgenic cell line containing the DNA fragment shown in formula (I); The plant is soybean; The salt tolerance is reflected in the salt tolerance of the transgenic hairy roots.

Images