CN112210545B - Salicornia europaea SeSMT2 protein and coding gene and application thereof - Google Patents

Abstract

The invention discloses salicornia europaea SeSMT2 protein and a coding gene and application thereof. The protein provided by the invention is named as SeSMT2 protein and comprises the following components: (1) a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table; (2) the amino acid sequence shown in the sequence 2 or the sequence 4 in the sequence table is substituted and/or deleted and/or added by one or more amino acid residues and is derived from the protein which is derived from the (1) and has the same function as the salicornia europaea. The gene (named SeSMT2) of the coding protein is cloned from salicornia europaea for the first time, and an Arabidopsis strain over-expressing SeSMT2 has better growth, higher lateral root number and main root length compared with an Arabidopsis homologous gene over-expressed strain under the condition of simultaneous treatment of low nitrogen and salt, so that the gene has the function of efficiently utilizing nitrogen in a salinized environment compared with the homologous gene in Arabidopsis.

Description

Salicornia europaea SeSMT2 protein and coding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to salicornia europaea SeSMT2 protein, and a coding gene and application thereof.

Background

Soil salinization is one of the major causes of agricultural ecological environment degradation and is also the major environmental factor limiting crop yield (Sharma et al, 2016). The area of the saline-alkali soil in China is about 9913 ten thousand hectares, and the saline-alkali soil accounts for 26.3% of the area of the saline-alkali soil in the world (Jiajindun and the like, 2014). The important strategy for promoting the sustainable development of agriculture is to research the mechanism of plant response to salt stress, create new species of salt-tolerant crops and fully utilize saline-alkali soil resources.

Nitrogen is a major element essential in the growth and development of plants, and is both a basic substance for protein synthesis and a catalytic substance in the physiological metabolic process (forest Zheng et al, 2011). The salinized soil has high pH value, the effective nitrogen content is generally lower than that of farmland, and a large amount of NaCl is contained. Due to Cl^-And NO₃ ^-NaCl inhibited the uptake of nitrogen by plants, resulting in severe crop losses (Chapagain et al, 2013). Therefore, the research on improving the nitrogen utilization efficiency of plants in a saline environment and cultivating nitrogen-efficient crops has guiding significance for popularizing the crop planting range and improving the yield.

Plant plasma membrane is the key structure of cell perception external signal, and the composition and content of lipid substance in plant plasma membrane are changed under the stress condition, and the structural characteristics of plasma membrane are changed simultaneously, namely plasma membrane remodeling occurs (Chalbi et al, 2014). Plasma membrane remodeling causes a corresponding change in the physical structure of the plasma membrane, affecting the lateral flow rate of the membrane, the function of effector molecules on the membrane, and the achievement of normal function of membrane proteins (Meer et al, 2008; Zauber et al, 2014), while plant uptake and utilization of nitrogen depends on the integrity of the plasma membrane and the transport proteins located thereon. Thus, the plasma membrane remodeling pattern of a plant is closely related to its salt tolerance and efficiency of nitrogen uptake and utilization.

Unlike sweet soil plants, halophytes can complete the whole life cycle in saline and alkaline land and accumulate larger biomass, which indicates that halophytes are more adaptive to the saline environment than the sweet soil plants, and have different plasma membrane remodeling forms to enable the absorption and utilization of nitrogen to be more efficient. Compared with sweet plants, the halophyte plant membranes have higher phytosterol abundance. Sterols, which are important components of biological membrane systems, can regulate the fluidity and permeability of plasma membranes, and are also important components for the formation of lipid rafts. In addition, sterols are also precursors of hormones such as brassinolide, and play an important role in plant growth and development and in response to stress (Chilobrachys, et al, 2013).

Phytosterols can be classified as 24-methyl sterols (campesterols) or 24-ethyl sterols (sitosterol, stigmasterol) based on the presence of a methyl or ethyl side chain group at the 24-carbon atom. The formation of side chain groups is mainly catalyzed by Sterol Methyltransferases (SMT) (EC2.1.1.41) which are dependent on S-adenosyl-L-methionine (SAM) (Valiova et al, 2016). SMT includes two families, SMT1 and SMT2, where SMT 2is able to catalyze 24-methylene cholestenol, adding a second alkyl group to the 24-carbon to start the synthesis of 24-ethyl phytosterols, separate from the branch of the synthesis of 24-methyl phytosterols, to regulate the ratio of 24-methyl sterols to 24-ethyl sterols (Bouvier-Nave et al, 1998). Due to the longer side chain of 24-ethyl sterol, plasma membrane rigidity is better maintained than 24-methyl sterol (Grunwald, 1971; Schuler et al, 1991). Accordingly, 24-ethyl sterols have also been shown to be widely involved in plant response to biotic and abiotic stress.

Salicornia europaea L is a true halophyte of Chenopodiaceae, can tolerate 1000mM NaCl, has huge biomass and fruiting amount, is rich in protein, and is an excellent material for researching salt tolerance of plants and a high-efficiency utilization mechanism of nutrient elements (Venturia and Sagi, 2012).

Disclosure of Invention

An object of the present invention is to provide a protein.

The protein provided by the invention is SeSMT2 protein and is (1) or (2) as follows:

(1) a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table;

(2) the amino acid sequence shown in the sequence 2 in the sequence table is substituted and/or deleted and/or added by one or more amino acid residues and is derived from the protein which is derived from the (1) and has the same function as the salicornia europaea.

Nucleic acid molecules encoding the above proteins are also within the scope of the present invention.

The nucleic acid molecule is a DNA molecule according to any one of the following 1) to 4):

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

2) the coding region is a DNA molecule shown in 17 th to 1119 th sites of a sequence 4 in a sequence table;

3) DNA molecules which hybridize under stringent conditions with the DNA sequences defined in 1) or 2) and which code for proteins having the same function;

4) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the DNA sequence defined in 1) or 2) and derived from Salicornia europaea encoding a DNA molecule having the same functional protein.

Recombinant vectors, expression cassettes or recombinant bacteria comprising the above-described nucleic acid molecules are also within the scope of the present invention.

In the embodiment of the invention, the recombinant vector is pGWB651-35S: SeSMT2 and pGWB502 omega-35S: SeSMT 2;

pGWB 651-35S-SeSMT 2 fusion vector was obtained by replacing the DNA fragment shown in SEQ ID No. 17-1116 of pGWB651 vector with the fragment between the attR1 and attR2 sites, and SeSMT2 expression was driven by CaMV35S promoter.

SeSMT2 fusion vector is obtained by replacing the DNA fragment shown in 17 th to 1119 th positions of the sequence 4 with the fragment between attR1 and attR2 sites of pGWB502 omega vector, and the CaMV35S promoter drives SeSMT2 expression.

The application of the protein, the nucleic acid molecule or the recombinant vector, the expression cassette or the recombinant bacterium in regulating and controlling the stress tolerance of plants is also within the protection scope of the invention;

or, the application of the protein, the nucleic acid molecule or the recombinant vector, the expression cassette or the recombinant bacterium in improving the efficiency of the nitrogen utilization of plants in a saline environment is also within the protection scope of the invention;

alternatively, the application of the protein, the nucleic acid molecule or the recombinant vector, the expression cassette or the recombinant bacterium in the cultivation of plants with high stress tolerance is also within the protection scope of the invention.

The application of the AtSMT2 protein or the coding nucleic acid molecule thereof or the recombinant vector, the expression cassette or the recombinant bacterium containing the nucleic acid molecule in regulating and controlling the stress tolerance of plants is also within the protection scope of the invention;

or, the application of the AtSMT2 protein or the coding nucleic acid molecule thereof or the recombinant vector, the expression cassette or the recombinant bacterium containing the nucleic acid molecule in the cultivation of plants with high stress tolerance is also within the protection scope of the invention;

or, the application of the AtSMT2 protein or the coding nucleic acid molecule thereof or the recombinant vector, the expression cassette or the recombinant bacterium containing the nucleic acid molecule in improving the efficiency of the nitrogen utilization of plants in the saline environment is also within the protection scope of the invention;

or, the application of the AtSMT3 protein or the coding nucleic acid molecule thereof or the recombinant vector, the expression cassette or the recombinant bacterium containing the nucleic acid molecule in regulating and controlling the stress tolerance of plants is also within the protection scope of the invention;

or, the application of the AtSMT3 protein or the coding nucleic acid molecule thereof or the recombinant vector, the expression cassette or the recombinant bacterium containing the nucleic acid molecule in improving the efficiency of the nitrogen utilization of plants in the saline environment is also within the protection scope of the invention;

or, the application of the AtSMT3 protein or the coding nucleic acid molecule thereof or the recombinant vector, the expression cassette or the recombinant bacterium containing the nucleic acid molecule in the cultivation of plants with high stress tolerance is also within the protection scope of the invention;

the amino acid sequence of the AtSMT2 protein is as follows:

(1) a sequence consisting of amino acid residues coded at 27 th to 1112 th positions of a sequence 5 in a sequence table;

(2) the protein derived from the protein (1) through substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the (1) and having the same function in Arabidopsis thaliana.

The amino acid sequence of the AtSMT3 protein is as follows:

(3) a sequence consisting of amino acid residues coded at 27 th to 1106 th positions of the sequence 6 in the sequence table;

(4) the amino acid sequence shown in (3) is substituted and/or deleted and/or added by one or more amino acid residues, and is derived from the protein which is derived from (1) and has the same function in Arabidopsis thaliana.

In the above application, the stress tolerance is salt tolerance or low nitrogen tolerance. Specifically, the low nitrogen concentration is 1mM or less.

Another object of the present invention is to provide a method for breeding transgenic plants having improved stress tolerance.

The method provided by the invention is 1) or 2):

1) the method comprises the following steps: improving the content and/or activity of the protein in the target plant to obtain a transgenic plant;

2) the method comprises the following steps: improving the expression of nucleic acid molecules encoding the protein in a target plant to obtain a transgenic plant;

the transgenic plant has higher tolerance than the target plant.

In the above method, the increase in the content and/or activity of the protein in the target plant or the increase in the expression of the nucleic acid molecule encoding the protein in the target plant is achieved by introducing the nucleic acid molecule into the target plant.

In the above method, the stress tolerance is salt tolerance or low nitrogen tolerance.

The work clones a gene (named as SeSMT2) for coding C-24 sterol methyl transferase 2 from salicornia europaea for the first time, researches the function of improving the plant salt adaptation and nitrogen utilization efficiency for the first time, and has guiding significance for screening and cultivating salt-tolerant crops with high nitrogen efficiency and improving productivity. In addition, in view of the effects of phytosterol in medical care aspects such as blood pressure and blood fat reduction, immunity improvement, cancer prevention or alleviation, Alzheimer's disease and the like, the cloning and function analysis of key genes in the salicornia europaea sterol synthetic pathway can also lay an important theoretical basis for the large-scale production of phytosterol by using a halophyte reactor.

SMT 2is a key enzyme for the synthesis of 24-ethyl sterols and plays an important role in plant growth and development and in response to stress. The gene SeSMT2 for coding sterol methyl transferase 2is obtained by cloning from salicornia europaea for the first time, and the amino acid sequence analysis shows that the gene has higher homology with spinach SoSMT 2. The subcellular localization of the SeSMT2 protein was observed through a tobacco transient expression system, and the co-localization of the fusion protein of the SeSMT2 protein and GFP and an endoplasmic reticulum Marker was found, which is consistent with the report of sterol synthesis in the endoplasmic reticulum. When the salt stress treatment is carried out for 3d, the expression level of SeSMT2 on the aerial part of salicornia europaea is obviously up-regulated, and the expression level is opposite to the expression level change of homologous genes in arabidopsis thaliana. Compared with wild arabidopsis, the overexpression of SeSMT2 can reduce the sensitivity of transgenic arabidopsis to salt, is more suitable for low-nitrogen conditions, and can absorb and utilize nitrogen more efficiently in a salt environment, and is particularly represented by the remarkable increase of the fresh weight, the lateral root number and the main root length of the overground part. Particularly, under the condition of simultaneous treatment of low nitrogen and salt, an Arabidopsis plant line over-expressing SeSMT2 also grows better than a plant line over-expressing Arabidopsis homologous genes, and the lateral root number and the main root length are higher, so that the gene has the function of efficiently utilizing nitrogen in a salinized environment compared with the homologous genes in Arabidopsis.

The present work found that salicornia serrulata SeSMT2 was tolerant to low nitrogen, which was also the first time that SMT2 was found to function under low nitrogen conditions. Considering that the natural habitat of salicornia europaea is mostly on coastal beaches and can realize seawater cultivation, the salicornia europaea not only can grow on saline-alkali land with effective nitrogen content and realize high biomass, but also can possibly grow in a water area with environmental problems of water eutrophication and the like caused by applying excessive nitrogen fertilizer and is used for restoring ecological environment. Meanwhile, the abundance of phytosterol and the like in the bristled salicornia is high, and the bristled salicornia also has the potential of being used as raw materials for extracting compounds with health care effects such as sterol and the like.

In conclusion, salicornia serrulata SeSMT2 can absorb more nutrients to supply the overground part through the development of a root system development network, and can simultaneously respond to low nitrogen and salt stress, so that the nitrogen is efficiently utilized in the salinized soil. The salicornia europaea SeSMT2 gene can provide theoretical guidance for genetic engineering modification for creating salt-tolerant crop varieties with high nitrogen efficiency, and further prompts excellent growth conditions of the salicornia europaea in saline-alkali lands with insufficient effective nitrogen content and great potential of the salicornia europaea SeSMT2 gene as a bioreactor for extracting phytosterol.

Drawings

FIG. 1 shows the expression patterns of Salicornia europaea and Arabidopsis SMT2 under NaCl treatment.

FIG. 2is a subcellular localization study of SeSMT2 protein.

FIG. 3 shows the expression of transcripts of SMT2 in each transgenic Arabidopsis line.

FIG. 4 shows the tolerance of Arabidopsis wild type and SMT2 transgenic lines to NaCl and low nitrogen.

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.

Example 1 cloning of SeSMT2 Gene

One, SeSMT2 gene full length CDS cloning and bioinformatics analysis

Total RNA from aerial parts of Salicornia Herbacea (Salicornia Herbacea seeds from Hippocampus Crystal industries, Ltd. of Jiangsu province) was extracted in small amounts using Trizol reagent (Takala corporation, 9109), and first strand cDNA was obtained using reverse transcriptase from all-type gold.

The forward primer SeSMT2-F (5'-ATGGATTCCATGGCATTGTACTG-3') and the reverse primer SeSMT2-R (5'-TTAAGACTTATCATGATGATCAATAGGC-3') were designed, respectively.

Using Salicornia europaea cDNA as a template, the full-length cDNA was amplified by two rounds of PCR using Easypfu DNA Polymerase (all-purpose gold, Beijing). The specific reaction conditions are as follows: the first round of PCR reaction is carried out at 95 ℃ for 5 min; 4 cycles: 95 deg.C, 30sec, 51 deg.C, 30sec, 72 deg.C, 1min, 30 sec; 5 cycles: 95 deg.C, 30sec, 50 deg.C, 30sec, 72 deg.C, 1min, 30 sec; 5 cycles: 95 deg.C, 30sec, 49 deg.C, 30sec, 72 deg.C, 1min, 30 sec; 18 cycles: 95 deg.C, 30sec, 48 deg.C, 30sec, 72 deg.C, 1min, 30 sec; 72 ℃ for 10 min. Taking the first round PCR product as a template to carry out second round PCR, wherein the reaction conditions are the same as the first round.

The obtained PCR product was ligated

T1 Simple Cloning Vector (all-purpose gold, Beijing) to obtain pEASY-SeSMT2 Vector, which was confirmed by sequencing.

Sequencing results show that the full length of the amplified CDS sequence is 1086bp, 361 amino acids are coded and named as SeSMT2 gene, and the coded protein is marked as SeSMT2 protein.

The nucleotide sequence of the SeSMT2 gene is a sequence 1 in a sequence table; the amino acid sequence of the encoded protein SeSMT 2is a sequence 2 in a sequence table; the vector containing the SeSMT2 gene was named pEASY-SeSMT2 vector.

Second, analysis of Gene expression under salt treatment

Salt treatment: salicornia europaea seeds were sown in pots (nutrient soil: vermiculite, 1:1) 7cm × 7cm and after one week of germination, watered with 1/2 Hoagland's nutrient solution once a week. After germination, the seeds are irrigated by 1/2Hoagland nutrient solution containing 0, 200 and 600mM NaCl, and overground parts and root materials treated for 0, 6h, 12h, 24h and 3d are respectively taken for gene expression detection.

Extracting total RNA with Trizol reagent, reverse transcribing with reverse transcription kit (gold, Beijing), and collecting the RNA with THUNDERBIRD

Fluorescent quantitative qRT-PCR assay for qPCR mix (Toyobo, Japan)And (6) measuring. The salicornia alpha-tubulin gene is used as an internal reference and is processed by 2^-ΔΔCtThe method of (3) relatively quantifies gene expression. The Arabidopsis thaliana AtSMT2 and AtSMT3 genes were also searched in the database of Arabidopsis eFP (http:// bar. utoronto. ca/eFP/cgi-bin/efpWeb. cgi) for expression patterns for comparison under 150mM NaCl treatment.

The primer sequences used in the fluorescent quantitative qRT-PCR were as follows:

tubulin primer in salicornia europaea interior label:

SeTubulin-qPCR-F：5'-CAGTGCCTTTGAGCCATCTTC-3'

SeTubulin-qPCR-R：5'-CTGAATGGTTCGCTTGGTCTT-3'

salicornia europaea SeSMT2 gene primers:

SeSMT2-qPCR-F：5'-CTGTACAGCCGGTCTCCTTTTCGCC-3'

SeSMT2-qPCR-R：5'-GGAAGCTCTGACCCCAGCCCCACTC-3'

the fluorescent quantitative qRT-PCR detection result is shown in figure 1, the expression mode of the A. salicornia SeSMT2 gene under NaCl treatment is the average value +/-SE, n is 3, and different letters on an error line indicate that the expression quantity of the same part is obviously different when P is less than 0.05; B. the expression patterns of Arabidopsis AtSMT2 and AtSMT3 genes under NaCl treatment, and the original data are derived from Arabidopsis eFP database. The expression level of the SMT2 gene in roots of salicornia europaea treated under the optimal conditions (200mM NaCl) and the stress conditions (600mM NaCl) is reduced overall, while the expression level of SeSMT2 in aerial parts is increased remarkably after fluctuating up and down (FIG. 1A). Under the stress condition (150mM NaCl), the expression levels of AtSMT2 and AtSMT3 in the aerial parts and roots of Arabidopsis thaliana are reduced (FIG. 1B). The results show that at 3d, the expression level of SMT2 in the aerial parts of Salicornia europaea under the optimal salt condition and salt stress is remarkably increased compared with the control, and the expression level of the aerial parts of SMT2 and SMT3 in Arabidopsis under the salt stress is remarkably reduced. This indicates that SeSMT2, the aerial part of Salicornia europaea, was up-regulated by salt induction at 3d, and that Arabidopsis AtSMT2 and AtSMT3 were down-regulated by salt induction.

Third, SeSMT2 protein subcellular localization

1. Construction of protein subcellular localization vector

Selecting pENTRY^TMDirectional TOPO (Invitrogen, K240020) Entry vector, primers Not I-SeSMT2-651-Entry-F and Asc I-SeSMT2-651-Entry-R were designed.

Salicornia europaea SeSMT2 gene primers:

NotI-SeSMT2-651-Entry-F：

5'-ATAAGAATGCGGCCGCCCCCTTCACCATGGATTCCATGGCATTG-3'

AscI-SeSMT2-651-Entry-R：

5'-AGGCGCGCCCACCCTTAGACTTATCATGATGATCAATA-3'

using pEASY-SeSMT2 plasmid as template, Not I-SeSMT2-651-Entry-F and Asc I-SeSMT2-651-Entry-R as primers, Easypfu DNA Polymerase (all-type gold, Beijing) was used for PCR amplification to obtain 1125bp PCR product.

The nucleotide sequence of the amplification product of the pEASY-SeSMT2 plasmid is sequence 3, wherein the 27 th to 1109 th position of the sequence 3 is a SeSMT2 nucleotide sequence with a stop codon removed, the 17 th to 26 th positions of the sequence 3 are upstream homology arms, and the 1110 th and 1116 th positions are downstream homology arms;

the recovered PCR product was ligated with the entry vector pENTRY^TMThe Directional TOPO was ligated by T4 ligase (all-grass Kabushiki Kaisha, FL101-01) to construct pENTRY^TMD-TOPO-SeSMT2 vector, for sequencing confirmation. Mixing pENTRY^TMD-TOPO-SeSMT2 plasmid was ligated with GFP vector pGWB651 (ref "Nakamura S, Mano S, Tanaka Y, et al (2010) Gateway vectors with the biochemical resistance gene, bar, as a selection marker for plant transformation. bioscience, Biotechnology, and Biochemistry,74:1315-^TM LR Clonase^TMII Enzyme Mix (Invitrogen,11791-020) to construct pGWB 651-35S:SeSMT 2 fusion vector.

And carrying out enzyme digestion verification on the fusion expression vector. The digestion with Sma I and Xba I gave 443bp and 11267bp products, and the digestion with SacI and SalI gave 1403bp and 10307bp products. 2, the correct insertion of the gene fragment into the vector is verified by the enzyme digestion results.

pGWB 651-35S-SeSMT 2 fusion vector was obtained by replacing the DNA fragment shown in SEQ ID No. 17-1116 with the fragment between the attR1 and attR2 sites of pGWB651 vector, and expression of SeSMT1a was driven by CaMV35S promoter.

2. Protein subcellular localization

The pGWB651-35S:: SeSMT2 fusion vector was transferred into C58 Agrobacterium (Beijing Huayuyo Biometrics, huayueyayang 1728S) to obtain recombinant Agrobacterium C58/pGWB651-35S:: SeSMT 2.

An endoplasmic reticulum Marker (RFP-HDEL, reference "Hu S, Ye H, Cui Y, Jiang L. (2020) AtSec62is criterion for plant degradation and is involved in ER-phase in Arabidopsis thaliana. journal of integrated plant biology,62:181 @ 200.") was transferred into C58 Agrobacterium (Beijing Huayuyo Bio Inc., huauyeyueyang 1728S) to obtain a control Agrobacterium C58/Marker (RFP-HDEL).

Recombinant Agrobacterium and control Agrobacterium were cultured overnight (28 ℃, 180rpm) to OD in LB liquid medium containing 20. mu.M AS (Acetosyringone ), 10mM MES, 100. mu.M Kan (Kanamycin ) and 100. mu.M Rif (Rifamicin, Rifampicin), respectively, with shaking₆₀₀2.0. After collecting the cells by centrifugation, the cells were washed with 10mM MgCl solution containing 200. mu.M AS₂10mM MES solution resuspended to OD₆₀₀Standing for 6h when the temperature is 2.0.

The two resuspended bacterial solutions were mixed at a ratio of 1: 1. The bacterial suspension was injected into native tobacco (described in Xianyang, incorporated herein by reference)Chen,Hexigeduleng Bao,Jie Guo,Weitao Jia,Fang Tai,Lingling Nie,Ping Jiang,Juanjuan Feng,Sulian Lv,Yinxin Li.(2014)Na⁺/H⁺exchanger 1 substrates in a nanobioc dis nature discovery vacuolar pH and priming the alcoholic system of journal of Experimental Botany,65: 6107-.

The results are shown in FIG. 2, subcellular localization of SeSMT 2. The scale bar is 10 μm. The green fluorescence emitted by Salicornia europaea SeSMT2 and GFP fusion proteins all overlapped with the red fluorescence emitted by endoplasmic reticulum marker (RFP-HDEL), indicating that these proteins are predominantly localized on the endoplasmic reticulum. This is consistent with the report of studies on phytosterols transported to the plasma membrane after synthesis in the endoplasmic reticulum.

Example 2 study of SeSMT2 in plant stress resistance

Construction of gene overexpression vector

Selecting pENTRY^TMA directive TOPO Entry vector was designed with primers Not I-SeSMT2-502-Entry-F/Asc I-SeSMT 2-502-Entry-R. Salicornia europaea SeSMT2 gene primers:

salicornia europaea SeSMT2 gene primers:

Not I-SeSMT2-502-Entry-F：

5'-ATAAGAATGCGGCCGCCCCCTTCACCATGGATTCCATGGCATTG-3'

Asc I-SeSMT2-502-Entry-R：

5'-AGGCGCGCCCACCCTTTTAAGACTTATCATGATGATCAATA-3'

using pEASY-SeSMT2 as a template and Not I-SeSMT2-502-Entry-F and Asc I-SeSMT2-502-Entry-R as primers, Easypfu DNA Polymerase (all-type gold, Beijing) was used for PCR amplification to obtain 1128bp PCR product.

The nucleotide sequence of the amplification product of the pEASY-SeSMT2 plasmid is sequence 4 (containing homology arms), wherein, the 27 th to 1112 th positions of the sequence 4 are SeSMT2 gene sequences, the 17 th to 26 th positions of the sequence 4 are upstream homology arms, and the 1113 th and 1119 th positions are downstream homology arms;

the recovered PCR products are respectively led to a portal vector through T₄Construction of pENTRY by ligase (all-grass of King Ltd., FL101-01)^TMD-TOPO-SeSMT2 vector, for sequencing confirmation.

Then adopting Gateway^TMLR Clonase^TMII Enzyme Mix (Invitrogen,11791-020) reaction of pENTR^TMthe/D-TOPO-SeSMT 2 intermediate vector plasmid is connected with a pGWB502 omega target vector (Addgene,74844) through a homologous recombination technology to construct a pGWB502 omega-35S fusion expression vector of SeSMT 2.

Enzyme digestion verification is carried out on the fusion expression vector. The enzyme digestion is carried out by SmaI to obtain 2652bp and 8802bp products, and the enzyme digestion is carried out by SalI and XbaI to obtain 277bp and 11177bp products. 2, the correct insertion of the gene fragment into the vector is verified by the enzyme digestion results.

SeSMT2 fusion vector is obtained by replacing the DNA fragment shown in 17 th to 1119 th positions of the sequence 4 with the fragment between attR1 and attR2 sites of pGWB502 omega vector, and the CaMV35S promoter drives SeSMT2 gene expression.

The pGWB502 omega-35S:: the AtSMT2 fusion vector is obtained by replacing a DNA fragment shown in the 17 th to 1119 th positions of a sequence 5 (a fragment of an Arabidopsis AtSMT2 sequence containing a homologous arm, wherein the nucleotide sequence of AtSMT 2is 27 th to 1112 th positions of the sequence 5) with a fragment between attR1 and attR2 sites of the pGWB502 omega vector, and the expression of AtSMT2 gene is driven by a CaMV35S promoter.

The pGWB502 omega-35S:: AtSMT3 fusion vector is obtained by replacing a DNA fragment (a fragment of Arabidopsis AtSMT3 sequence containing a homologous arm, wherein the nucleotide sequence of AtSMT 2is 27 th-1106 th sequence) shown in 17 th-1113 th position of a sequence 6 with a fragment between attR1 and attR2 sites of a pGWB502 omega vector, and the expression of AtSMT3 gene is driven by a CaMV35S promoter.

Second, obtaining and screening transgenic arabidopsis

1. Acquisition of transgenic Arabidopsis

The constructed plant expression vector pGWB502 omega-35S is used for transforming agrobacterium tumefaciens C58 strain by SeSMT2 to obtain recombinant strain C58/pGWB502 omega-35S, and SeSMT 2.

Recombinant strain C58/pGWB502 Ω -35S:SeSMT 2 was transformed into wild type Arabidopsis thaliana (Columbia-0, WT, described in the literature "Sulia Lv^*,Ping Jiang^*,Lingling Nie,Xianyang Chen,Fang Tai,Duoliya Wang,Pengxiang Fan,Juanjuan Feng,Hexigeduleng Bao,Jinhui Wang,Yinxin Li.(2015)H⁺-pyrophosphatase from Salicornia europaea confers tolerance to simultaneously occurring salt stress and nitrogen deficiency in Arabidopsis and wheat.Plant Cell&Environment,38(11), 2433-₀Transfer SeSMT2 Arabidopsis thaliana.

The above transfer method wild type Arabidopsis thaliana was transformed by Agrobacterium-mediated flower dipping method, with reference to the method of Zhang et al (2006) (reference "Zhang XR, Henriques R, Lin SS, et al (2006) Agrobacterium-mediated transformation of Arabidopsis thaliana used in Nature Protocols,1: 641-646.").

2. Screening of transgenic Arabidopsis homozygous lines

Will T₀SeSMT2 transferred Arabidopsis seeds were surface sterilized and then spread evenly on 1/2MS solid medium containing 25mg/L Hygromycin (Hygromycin, Hyg), first placed 2 days at 4 ℃ in the dark and then cultured for 7 days at 25 ℃ under the light. And selecting positive seedlings, transferring the positive seedlings into culture soil, and growing until the seeds are mature. Collecting T by dividing individual plant₁And (5) seed generation.

Reference T₀Generation of seed, for T₁The seeds are used for surface disinfection and sowing. After the seeds germinate for 7 days, counting the segregation ratio of the offspring, selecting a strain with the segregation ratio of 3:1, transferring the positive seedlings to nutrient soil for growth until the seeds are mature, and collecting T by separating the strains₂And (5) seed generation.

Reference T₀Generation of seed, for T₂The seeds are used for surface disinfection and sowing. After seeds germinate for 7d, selecting a strain with inseparable offspring (namely a homozygote), transferring and culturing positive seedlings until T is collected₃And (5) seed generation.

The same method is adopted to transfer pGWB502 omega-35S:: AtSMT2 and pGWB502 omega-35S:: AtSMT3 into wild type Arabidopsis thaliana respectively, and T is obtained by cultivation₃Transfer AtSMT2 Arabidopsis thaliana and T₃Transfer to AtSMT3 Arabidopsis thaliana.

3. Gene expression quantity determination of transgenic arabidopsis homozygous line SeSMT2

For wild type Arabidopsis thaliana and T₃Transformation of SeSMT2 Arabidopsis thaliana (SeSMT 2-OElines), T₃Generation of AtSMT2 Arabidopsis thaliana (AtSMT 2-OElines) and T₃Sterilizing the surface of seeds of AtSMT3 Arabidopsis thaliana (AtSMT 3-OElines), sowing on 1/2MS culture medium, dark culturing at 4 deg.C for 2 days, culturing at 25 deg.C under illumination for two weeks, collecting 10 strains (about 100ng), extracting total RNA, digesting with DNase, reverse transcribing to obtain cDNA as template, and collecting the cDNA

qPCR mix (Toyobo, Japan) was subjected to fluorescent quantitative RT-PCR detection.

Taking an Arabidopsis thaliana Actin gene asInternal reference, passing through 2^-ΔΔCtThe method of (3) relatively quantifies gene expression.

The primer sequences used in the fluorescent quantitative RT-PCR were as follows:

an arabidopsis internal standard Actin gene primer:

AtActin-qPCR-F：5'-ATATGCCTATCTACAGGGTT-3'

AtActin-qPCR-R：5'-ATACAATTTCCCGTTCTGCTGT-3'

salicornia europaea SeSMT2 gene specific primers:

SeSMT2-qPCR-F：5'-CTGTACAGCCGGTCTCCTTTTCGCC-3'

SeSMT2-qPCR-R：5'-GGAAGCTCTGACCCCAGCCCCACTC-3'

arabidopsis AtSMT2 gene specific primer:

AtSMT2-qPCR-F：5'-CTACAATCTCGTCACCGACATA-3'

AtSMT2-qPCR-R：5'-GGATCTTTTGACCAGGTTTGAC-3'

arabidopsis AtSMT3 gene specific primers:

AtSMT3-qPCR-F：5'-AAGCTCGAAGAAGTATACTCGG-3'

AtSMT3-qPCR-R：5'-GAATCACGTCCTTGTGTTCTTC-3'

results are shown in fig. 3, CK, control; SeSMT2-OE lines, transgenic lines overexpressing SeSMT 2; AtSMT2-OE lines, transgenic lines overexpressing AtSMT 2; AtSMT3-OE lines, transgenic lines overexpressing AtSMT 3. Indicates that under the same treatment conditions, the values achieved significant differences in P <0.05, P <0.01 and P <0.001 levels, respectively, compared to the control. It can be seen that the transcript levels of SMT2 in salicornia europaea and arabidopsis SMT2 overexpression homozygous lines were significantly improved over wild type. This indicates that SeSMT2, AtSMT2, and AtSMT3 have been transferred into Arabidopsis thaliana and are stably expressed.

Second, salt resistance detection of transgenic arabidopsis

Wild type Arabidopsis thaliana (WT), T, were treated with 70% ethanol and 1% sodium hypochlorite (purchased from Beijing chemical Co., Ltd.)₃SeSMT2 Arabidopsis thaliana (SS2-1, SS2-2) and T are substituted₃Generation AtSMT2 Arabidopsis thaliana (AS2-1, AS2-2) and T₃Replacing AtSMT3 Arabidopsis thaliana (AS3-1, AS3-2) seeds for surface disinfection, and cleaning with sterilized waterWashed 3-5 times and then sown on 1/2MS culture medium. Dark processing at 4 deg.C for 2d, transferring to artificial climate chamber (temperature 24-25 deg.C, relative humidity 60-70%, illumination condition 16h light/8 h dark), and culturing. After 5d of seed germination, shoots with consistent growth were picked and transferred to 1/2MS medium (normal CK condition), 1/2MS medium containing 150mM NaCl (150mM NaCl was added to 1/2MS medium), 100. mu.M KNO ₃1/2MS low nitrogen salt-added medium (KNO₃Adding into 1/2MS culture medium, and adding KNO₃The concentration of (2) is 100. mu.M; LN) and a catalyst containing 100. mu.M KNO ₃1/2MS Low Nitrogen salt supplemented Medium with 150. mu.M NaCl (NaCl and KNO)₃Added to 1/2MS medium at a NaCl concentration of 150mM and KNO₃The concentration of (2) is 100. mu.M; LN + NaCl) and then subjected to vertical salt stress culture under light (16 h light/8 h dark light conditions). After 7 days of salt stress culture, the fresh weight, the number of lateral roots, the length of main roots and the content of chlorophyll of the overground parts of the plants are measured.

The chlorophyll content determination method comprises the following steps: after weighing the plants, soaking in 95% ethanol for 3 days, the absorbances of the leachate at 665, 649 and 470nm wavelengths were measured, respectively, and the chlorophyll content and chlorophyll a/chlorophyll b ratio were further calculated (reference: Lightendaler HK. (1987) chlorophenyls and carotenoids: Pigments of photosynthetic biomembranes. method in Enzymology,148: 350-.

Phenotypic results are shown in FIG. 4, A. Normal growth Conditions (CK) and 100. mu.M KNO₃(LN)、150mM NaCl(CK)、100μM KNO₃Phenotype of Arabidopsis thaliana wild type and SMT2 transgenic lines treated with +150mM NaCl (LN + NaCl). WT, wild type; AS2-1, AS2-2, T₃Transferring an AtSMT2 Arabidopsis strain; AS3-1, AS3-2, T₃Transferring an AtSMT3 Arabidopsis strain; SS2-1, SS2-2, T₃Transferring SeSMT2 Arabidopsis; B. fresh weight of aerial parts; C. the number of lateral roots; D. root length; E. chlorophyll; F. chlorophyll a/chlorophyll b ratio. The scale is 1 cm. Data are mean ± SE (n-18), and indicate that under the same treatment conditions, the values are in P compared to the wild type, respectively<0.05 and P<Significant differences were achieved at the 0.01 level. It can be seen that in the normal growth of the stripUnder the circumstances, the lateral roots of the transgenic line are more than that of the wild type. At low nitrogen (100. mu.M KNO)₃) At 7 days of treatment, Arabidopsis lateral root growth was promoted. Growth of Arabidopsis thaliana was inhibited at 7 days of salt stress (150mM NaCl).

The statistics of physiological indexes show that T is obtained after low-nitrogen treatment for 7 days₃The fresh weight, the lateral root number and the main root length of the overground part of a transgenic SeSMT2 Arabidopsis (SS2-1 and SS2-2) strain are all higher than those of a wild type strain, wherein the phenotypes of SeSMT2 and AtSMT3 overexpression strains are more remarkable; when the salt treatment is carried out for 7 days, the fresh weight and the main root length content of the overground part of the over-expression strain are higher than those of the wild type; the fresh weight of the aerial parts of the over-expressed strains was significantly higher than that of the wild type at 7 days of low nitrogen salt treatment, the main root length of the SeSMT2 and AtSMT3 over-expressed strains was significantly higher than that of the other strains, and the lateral root number of the SeSMT2 over-expressed strain was also significantly higher than that of the other strains (FIGS. 4B-F).

These results demonstrate that overexpression of SeSMT2 improves salt resistance and low nitrogen tolerance of Arabidopsis at the seedling stage, and that overexpression of AtSMT2 and AtSMT3 can improve tolerance of Arabidopsis in salt stress and low nitrogen habitats for the first time.

SEQUENCE LISTING

<110> institute of plant of Chinese academy of sciences

<120> salicornia serrulata SeSMT2 protein and coding gene and application thereof

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 1086

<212> DNA

<213> Artificial sequence

<400> 1

atggattcca tggcattgta ctgcacagcc ggtctccttt tcgccggcct ttactggttt 60

gtcttcgtcc acggtcccgc cgaacaaaag ggcaaacgcg ccgtcgatct ttccggcggt 120

tctatctccg ccgagaaagt ccaagacaag taccgcgact actggtcctt cttccgccgt 180

cctaaggaga ttgaaacggc ggagaaagtc cccgatttcg tcgacacctt ctacaacctc 240

gtcaccgata tctacgagtg gggctggggt cagagcttcc acttctcacc ctccatccgc 300

ggcaaatcga acgccgacgc cactcgtatt cacgaacaaa tggccgtcga tctcatcaat 360

gtcgccccgg ggcagaagat cttagacgtc ggctgcggcg tgggcgggcc aatgcgggcc 420

attgcggccc attctagggc taaagtcaca ggaatcacca ttaacgaata ccaggttaaa 480

agagctaagc tccacaataa gaaagcaggg cttgattcac tgtgcgaggt cgtatgcggt 540

aatttcctcg agatgccgtt cgatgacaac acctttgacg gcgcgtacgc catcgaagcc 600

acgtgtcacg cgccgaagct tcaagaagtt tacgccgaga tctaccgcgt aatgaagcct 660

ggaacgctgt tcatctcata cgagtgggtc accactgaca agtacgacaa cgataacaag 720

gagcaccgtg acattatcca ggggatcgag cagggggacg cgctcccagg gctgaggaac 780

tacacagaca ttcctacggt ggcgaaagcg gttggattcg aggtggtttc cgaaaaagac 840

ctagcggcgc caccggcgga gccgtggtgg agccggttga agatgggaag gattgcgtac 900

tggagaaacc atatcgtcgt aactgtgctc gcatatctag ggattgcacc acaaggaacg 960

gtggatgttc atgaaatgct gtttaaaacc gctgatttcc ttacacgtgg cggtgattat 1020

ggtatattta gccctatgca tatgatcctc tgcagaaagc ctattgatca tcatgataag 1080

tcttaa 1086

<210> 2

<211> 361

<212> PRT

<213> Artificial sequence

<400> 2

Met Asp Ser Met Ala Leu Tyr Cys Thr Ala Gly Leu Leu Phe Ala Gly

1 5 10 15

Leu Tyr Trp Phe Val Phe Val His Gly Pro Ala Glu Gln Lys Gly Lys

20 25 30

Arg Ala Val Asp Leu Ser Gly Gly Ser Ile Ser Ala Glu Lys Val Gln

35 40 45

Asp Lys Tyr Arg Asp Tyr Trp Ser Phe Phe Arg Arg Pro Lys Glu Ile

50 55 60

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

65 70 75 80

Val Thr Asp Ile Tyr Glu Trp Gly Trp Gly Gln Ser Phe His Phe Ser

85 90 95

Pro Ser Ile Arg Gly Lys Ser Asn Ala Asp Ala Thr Arg Ile His Glu

100 105 110

Gln Met Ala Val Asp Leu Ile Asn Val Ala Pro Gly Gln Lys Ile Leu

115 120 125

Asp Val Gly Cys Gly Val Gly Gly Pro Met Arg Ala Ile Ala Ala His

130 135 140

Ser Arg Ala Lys Val Thr Gly Ile Thr Ile Asn Glu Tyr Gln Val Lys

145 150 155 160

Arg Ala Lys Leu His Asn Lys Lys Ala Gly Leu Asp Ser Leu Cys Glu

165 170 175

Val Val Cys Gly Asn Phe Leu Glu Met Pro Phe Asp Asp Asn Thr Phe

180 185 190

Asp Gly Ala Tyr Ala Ile Glu Ala Thr Cys His Ala Pro Lys Leu Gln

195 200 205

Glu Val Tyr Ala Glu Ile Tyr Arg Val Met Lys Pro Gly Thr Leu Phe

210 215 220

Ile Ser Tyr Glu Trp Val Thr Thr Asp Lys Tyr Asp Asn Asp Asn Lys

225 230 235 240

Glu His Arg Asp Ile Ile Gln Gly Ile Glu Gln Gly Asp Ala Leu Pro

245 250 255

Gly Leu Arg Asn Tyr Thr Asp Ile Pro Thr Val Ala Lys Ala Val Gly

260 265 270

Phe Glu Val Val Ser Glu Lys Asp Leu Ala Ala Pro Pro Ala Glu Pro

275 280 285

Trp Trp Ser Arg Leu Lys Met Gly Arg Ile Ala Tyr Trp Arg Asn His

290 295 300

Ile Val Val Thr Val Leu Ala Tyr Leu Gly Ile Ala Pro Gln Gly Thr

305 310 315 320

Val Asp Val His Glu Met Leu Phe Lys Thr Ala Asp Phe Leu Thr Arg

325 330 335

Gly Gly Asp Tyr Gly Ile Phe Ser Pro Met His Met Ile Leu Cys Arg

340 345 350

Lys Pro Ile Asp His His Asp Lys Ser

355 360

<210> 3

<211> 1125

<212> DNA

<213> Artificial sequence

<400> 3

ataagaatgc ggccgccccc ttcaccatgg attccatggc attgtactgc acagccggtc 60

tccttttcgc cggcctttac tggtttgtct tcgtccacgg tcccgccgaa caaaagggca 120

aacgcgccgt cgatctttcc ggcggttcta tctccgccga gaaagtccaa gacaagtacc 180

gcgactactg gtccttcttc cgccgtccta aggagattga aacggcggag aaagtccccg 240

atttcgtcga caccttctac aacctcgtca ccgatatcta cgagtggggc tggggtcaga 300

gcttccactt ctcaccctcc atccgcggca aatcgaacgc cgacgccact cgtattcacg 360

aacaaatggc cgtcgatctc atcaatgtcg ccccggggca gaagatctta gacgtcggct 420

gcggcgtggg cgggccaatg cgggccattg cggcccattc tagggctaaa gtcacaggaa 480

tcaccattaa cgaataccag gttaaaagag ctaagctcca caataagaaa gcagggcttg 540

attcactgtg cgaggtcgta tgcggtaatt tcctcgagat gccgttcgat gacaacacct 600

ttgacggcgc gtacgccatc gaagccacgt gtcacgcgcc gaagcttcaa gaagtttacg 660

ccgagatcta ccgcgtaatg aagcctggaa cgctgttcat ctcatacgag tgggtcacca 720

ctgacaagta cgacaacgat aacaaggagc accgtgacat tatccagggg atcgagcagg 780

gggacgcgct cccagggctg aggaactaca cagacattcc tacggtggcg aaagcggttg 840

gattcgaggt ggtttccgaa aaagacctag cggcgccacc ggcggagccg tggtggagcc 900

ggttgaagat gggaaggatt gcgtactgga gaaaccatat cgtcgtaact gtgctcgcat 960

atctagggat tgcaccacaa ggaacggtgg atgttcatga aatgctgttt aaaaccgctg 1020

atttccttac acgtggcggt gattatggta tatttagccc tatgcatatg atcctctgca 1080

gaaagcctat tgatcatcat gataagtcta agggtgggcg cgcct 1125

<210> 4

<211> 1128

<212> DNA

<213> Artificial sequence

<400> 4

ataagaatgc ggccgccccc ttcaccatgg attccatggc attgtactgc acagccggtc 60

tccttttcgc cggcctttac tggtttgtct tcgtccacgg tcccgccgaa caaaagggca 120

aacgcgccgt cgatctttcc ggcggttcta tctccgccga gaaagtccaa gacaagtacc 180

gcgactactg gtccttcttc cgccgtccta aggagattga aacggcggag aaagtccccg 240

atttcgtcga caccttctac aacctcgtca ccgatatcta cgagtggggc tggggtcaga 300

gcttccactt ctcaccctcc atccgcggca aatcgaacgc cgacgccact cgtattcacg 360

aacaaatggc cgtcgatctc atcaatgtcg ccccggggca gaagatctta gacgtcggct 420

gcggcgtggg cgggccaatg cgggccattg cggcccattc tagggctaaa gtcacaggaa 480

tcaccattaa cgaataccag gttaaaagag ctaagctcca caataagaaa gcagggcttg 540

attcactgtg cgaggtcgta tgcggtaatt tcctcgagat gccgttcgat gacaacacct 600

ttgacggcgc gtacgccatc gaagccacgt gtcacgcgcc gaagcttcaa gaagtttacg 660

ccgagatcta ccgcgtaatg aagcctggaa cgctgttcat ctcatacgag tgggtcacca 720

ctgacaagta cgacaacgat aacaaggagc accgtgacat tatccagggg atcgagcagg 780

gggacgcgct cccagggctg aggaactaca cagacattcc tacggtggcg aaagcggttg 840

gattcgaggt ggtttccgaa aaagacctag cggcgccacc ggcggagccg tggtggagcc 900

ggttgaagat gggaaggatt gcgtactgga gaaaccatat cgtcgtaact gtgctcgcat 960

atctagggat tgcaccacaa ggaacggtgg atgttcatga aatgctgttt aaaaccgctg 1020

atttccttac acgtggcggt gattatggta tatttagccc tatgcatatg atcctctgca 1080

gaaagcctat tgatcatcat gataagtctt aaaagggtgg gcgcgcct 1128

<210> 5

<211> 1128

<212> DNA

<213> Artificial sequence

<400> 5

ataagaatgc ggccgccccc ttcaccatgg actctttaac actcttcttc accggtgcac 60

tcgtcgccgt cggtatctac tggttcctct gcgttctcgg tccagcagag cgtaaaggca 120

aacgagccgt agatctctct ggtggctcaa tctccgccga gaaagtccaa gacaactaca 180

aacagtactg gtctttcttc cgccgtccaa aagaaatcga aaccgccgag aaagttccag 240

acttcgtcga cacattctac aatctcgtca ccgacatata cgagtgggga tggggacaat 300

ccttccactt ctcaccatca atccccggaa aatctcacaa agacgccacg cgcctccacg 360

aagagatggc cgtagatctg atccaagtca aacctggtca aaagatccta gacgtcggat 420

gcggtgtcgg cggtccgatg cgagcgattg catctcactc gcgagctaac gtagtcggga 480

ttacaataaa cgagtatcag gtgaacagag ctcgtctcca caataagaaa gctggtctcg 540

acgcgctttg cgaggtcgtg tgtggtaact tcctccagat gccgttcgat gacaacagtt 600

tcgacggtgc ttattccatc gaagccacgt gtcacgcgcc gaagctggaa gaagtgtacg 660

cagagatcta cagggtgttg aaacccggat ctatgtatgt gtcgtacgag tgggttacga 720

cggagaaatt taaggcggag gatgacgaac acgtggaggt aatccaaggg attgagagag 780

gcgatgcgtt accagggctt agggcttacg tggatatagc tgagacggct aaaaaggttg 840

ggtttgagat agtgaaggag aaggatctgg cgagtccacc ggctgagccg tggtggacta 900

ggcttaagat gggtaggctt gcttattgga ggaatcacat tgtggttcag attttgtcag 960

cggttggagt tgctcctaaa ggaactgttg atgttcatga gatgttgttt aagactgctg 1020

attatttgac cagaggaggt gaaaccggaa tattctctcc gatgcatatg attctctgca 1080

gaaaaccgga gtcaccggag gagagttctt gaaagggtgg gcgcgcct 1128

<210> 6

<211> 1122

<212> DNA

<213> Artificial sequence

<400> 6

ataagaatgc ggccgccccc ttcaccatgg actcggtggc tctctactgc accgctggtc 60

tcattgccgg cgccgtctac tggttcatat gcgtcctagg tccagcagaa cgaaaaggca 120

aacgagcctc tgatctctcc ggcggctcaa tctccgcaga aaaagtcaaa gacaactata 180

accaatactg gtctttcttc cgcaaaccaa aagagatcga atcagccgag aaagtacctg 240

acttcgtcga cacgttctac aatcttgtca ctgatatcta cgagtgggga tggggacaat 300

ctttccattt ctctcctcat gtccctggaa aatccgacaa agacgccaca agaatccacg 360

aagaaatggc cgtcgatctc atcaaagtga aaccgggaca aaagattctt gacgctggtt 420

gcggcgtggg tgggccgatg agagccatcg cggcccattc caaggcccaa gtcactggaa 480

tcactatcaa cgagtaccaa gtgcaacgag ccaagcttca caacaagaaa gctggacttg 540

attctctctg caacgtcgtt tgtggtaact ttttaaagat gccgttcgat gaaaacacgt 600

ttgacggagc ttactcgatc gaagctacgt gtcacgctcc taagctcgaa gaagtatact 660

cggagatctt cagagtgatg aaaccaggat ctttgttcgt gtcctacgaa tgggtcacca 720

ctgaaaaata cagagacgat gacgaagaac acaaggacgt gattcaaggg atcgagagag 780

gagacgcact tcctggacta agaagctacg ctgatatagc cgtgacggcg aagaaagttg 840

ggtttgaggt agtgaaggag aaagatttgg ctaaaccacc gtctaaaccg tggtggaacc 900

ggttaaagat gggaaggatt gcttattgga gaaaccatgt tgtggttgtg attctttctg 960

ctattggggt tgctcctaaa ggaactgttg atgttcataa gatgttgttt aagactgctg 1020

attatttgac cagaggtggt gagactggaa tcttctctcc gatgcatatg attctctgta 1080

gaaaaccaga gaaagcttct gaatgaaagg gtgggcgcgc ct 1122

Claims (7)

1. A protein is a protein consisting of an amino acid sequence shown as a sequence 2 in a sequence table.

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein:

the nucleic acid molecule is a DNA molecule of any one of the following 1) -2):

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

2) the coding region is a DNA molecule shown in 27 th-1112 th site of a sequence 4 in a sequence table.

4. A recombinant vector, expression cassette or recombinant bacterium comprising the nucleic acid molecule of claim 2 or 3.

5. Use of the protein of claim 1, the nucleic acid molecule of claim 2 or 3, or the recombinant vector, expression cassette or recombinant bacterium of claim 4 for modulating stress tolerance in a plant;

or, the use of the protein of claim 1, the nucleic acid molecule of claim 2 or 3, or the recombinant vector, expression cassette or recombinant bacterium of claim 4 to increase the efficiency of nitrogen utilization by plants in a saline environment;

or, the use of the protein of claim 1, the nucleic acid molecule of claim 2 or 3, or the recombinant vector, expression cassette or recombinant bacterium of claim 4 for growing plants with high stress tolerance;

the stress tolerance is salt tolerance or low nitrogen tolerance.

6. A method for breeding transgenic plants with improved stress tolerance, which comprises the following steps 1) or 2):

1) the method comprises the following steps: increasing the content and/or activity of the protein of claim 1 in a target plant to obtain a transgenic plant;

2) the method comprises the following steps: increasing expression in a plant of interest of a nucleic acid molecule encoding the protein of claim 1, resulting in a transgenic plant;

the transgenic plant has higher stress tolerance than the target plant;

the stress tolerance is salt tolerance or low nitrogen tolerance.

7. The method of claim 6, wherein:

the increase in the content and/or activity of the protein of claim 1 in a plant of interest, or the increase in the expression of a nucleic acid molecule encoding the protein of claim 1 in a plant of interest, is achieved by introducing the nucleic acid molecule of claim 2 or 3 into the plant of interest.

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