CN110628807B

CN110628807B - Salicornia europaea SePSS protein and coding gene and application thereof

Info

Publication number: CN110628807B
Application number: CN201810538242.4A
Authority: CN
Inventors: 李银心; 郭杰; 台方; 吕素莲
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2018-05-30
Filing date: 2018-05-30
Publication date: 2021-06-18
Anticipated expiration: 2038-05-30
Also published as: CN110628807A

Abstract

The invention discloses a salicornia europaea SePSS protein, and a coding gene and application thereof. Experiments prove that the salicornia SePSS gene is subjected to salt-induced expression, and the expression level is the highest when the salicornia SePSS gene is treated by 200mM NaCl for 12 hours and the expression level is the highest when the salicornia SePSS gene is treated by 800mM NaCl for 24 hours. SePSS gene is used for complementing Arabidopsis AtPSS mutant, and phenotypes such as PS content, cytoplasmic membrane polarity, chloroplast content, fresh weight, root length and the like of a complementing strain are restored, and the restoring effect is the same as that of the AtPSS gene. The fresh weight of the SePSS over-expressed line was significantly higher than that of the wild type Arabidopsis thaliana under 150mM NaCl treatment. The results prove that SePSS has a PS synthesis function and is very important for maintaining the polarity of a cytoplasmic membrane, and the salt resistance of Arabidopsis can be improved by over-expressing the SePSS gene. The work provides theoretical basis and gene resources for genetic improvement of stress-resistant crops.

Description

Salicornia europaea SePSS protein and coding gene and application thereof

Technical Field

The invention relates to a salicornia serpens SePSS protein, and a coding gene and application thereof.

Background

The problem of soil salination is ubiquitous throughout the world. Saline-alkali soil poses serious threat to agricultural production and restricts economic development. Many measures are taken to improve saline-alkali soil, including traditional agricultural means such as water conservancy engineering and the like, but the effect is not obvious. With the development of biological science and technology, the improvement of crops by using genetic engineering technology and the improvement of the salt tolerance of crops become an effective method for utilizing saline-alkali soil.

The cytoplasmic membrane is a barrier to exchange of substances inside and outside the cell, and plays an important role in maintaining a stable intracellular environment, nutrient absorption, cell recognition, signal transmission and the like. The plasma membrane of a cell generally maintains a polarization state of internal negative and external positive. When the cells are salt stressed, the external Na⁺A large amount of Na flows into the cells⁺The charged positive charges neutralize the negative charges on the cytoplasmic side of the cytoplasmic membrane, causing depolarization of the cytoplasmic membrane. After the cell plasma membrane is depolarized, the ion channel on the membrane is opened, so that the specificity is lost, the ion concentration in the cell is changed, the ion balance in the cell is destroyed, and osmotic stress and ion poison are caused to the cell. The negatively charged polar lipids such as PS and PI on the plasma membrane can neutralize the influx of Na from the outside world⁺The positive charge can slow down the depolarization of the cell membrane to a certain extent and increase the capability of the plant to resist salt stress.

PS exists in bacterial, yeast, plant and mammalian cells, and the principles of PS synthase and PS synthesis in cells of four origins differ. PS accounts for a small proportion of plant membrane lipids. In mung bean (Vigna radiate L.), PS accounts for 3.1% and 4.3% of glyceroglycolipids on the plasma and vacuolar membranes, respectively. In many plants, PS contains a large proportion of long chain fatty acids, and these PS molecules containing long chain fatty acids ensure stability during vesicle translocation from the endoplasmic reticulum by binding to the vesicles. Under stress conditions, the PS content of plants changes, which may be one of the ways plants respond to stress. Under the conditions of low sugar and drought stress, the PS content in carrot (Daucus carota) suspension cells and oat (Avena sativa) roots is remarkably increased. In contrast, the maize (Zea mays) was treated with 150mM NaCl and found to have a 36% increase in PS content. The PS content of the halophyte fox rice grass (Spartina patents) is increased 4 times after being treated by 170mM and 340mM NaCl.

Salicornia europaea (Salicornia europaea) is a fleshy euhalophyte of chenopodiaceae, widely distributed near coastal and inland salt lakes, capable of accumulating up to 50% by dry weight of NaCl, and considered as a higher plant most tolerant to salt in the world.

Disclosure of Invention

The invention aims to provide a salicornia serpens SePSS protein, a coding gene and application thereof.

The invention provides a method for cultivating transgenic plants (method A), which is characterized in that encoding genes of phosphatidylserine synthase are introduced into target plants to obtain transgenic plants; the transgenic plant has higher salt tolerance than the target plant.

In the method A, the coding gene of the phosphatidylserine synthase can be introduced into a target plant through a recombinant expression vector. The recombinant expression vector can be transformed into a target plant by a conventional biological method such as a Ti plasmid, a Ri plasmid, a plant virus vector, direct DNA transformation, microinjection, conductance, agrobacterium mediation and the like.

The recombinant expression vector containing the gene can be constructed by using the existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. When the gene is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters can be added in front of the transcription initiation nucleotide, and can be used alone or combined with other plant promoters; in addition, when the gene is used to construct a recombinant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codons or adjacent regions initiation codons, 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 used may be processed, for example, by adding genes expressing color-changing enzymes or luminescent compounds in plants, antibiotic markers having resistance, or chemical-resistant marker genes, etc.

The recombinant expression vector can be specifically a recombinant plasmid obtained by inserting a coding gene of phosphatidylserine synthase into a multiple cloning site of a vector pCAMBIA1301 or pCAMBIA 1300-35S.

The recombinant expression vector can be specifically a recombinant plasmid obtained by inserting a coding gene of phosphatidylserine synthase between BglII enzyme cutting sites of a pCAMBIA1301 vector.

The recombinant expression vector can be specifically pCAMBIA1300-35S, wherein a coding gene of phosphatidylserine synthase is inserted between BamH I enzyme cutting sites of a GFP vector to obtain the recombinant plasmid.

The invention also protects the application of the method A in plant breeding.

The plant breeding is to breed plants with high salt tolerance.

The invention also provides a method (method B) for improving the salt tolerance of plants, which is used for improving the expression quantity and/or activity of the phosphatidylserine synthase in target plants and improving the salt tolerance of the plants.

The invention also protects the application of the method B in plant breeding.

The plant breeding is to breed plants with high salt tolerance.

The invention also protects the phosphoacyl serine synthase or the coding gene of the phosphoacyl serine synthase and the application of the coding gene in the regulation of the plant salt tolerance.

The invention also protects the use of phosphatidylserine synthases, or genes encoding phosphatidylserine synthases, in plant breeding.

The plant breeding is to breed plants with high salt tolerance.

Any of the above-described phosphatidylserine synthases is the phosphatidylserine synthase SePSS or the phosphatidylserine synthase AtPSS1 or the phosphatidylserine synthase AtPSS 2.

The phosphatidylserine synthase SePSS is (a1) or (a2) as follows:

(a1) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;

(a2) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1, is related to the salt tolerance of plants and is derived from the sequence 1.

In order to facilitate the purification and detection of the phosphatidylserine synthase SePSS of (a1), a tag as shown in Table 1 may be attached to the amino terminus or the carboxyl terminus of a protein consisting of the amino acid sequence shown in sequence 1 of the sequence listing.

TABLE 1 sequences of tags

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

The phosphatidylserine synthase SePSS of (a2) above may be synthesized artificially, or may be obtained by synthesizing the coding gene and then performing biological expression. The gene encoding the phosphatidylserine synthase SePSS of (a2) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence No. 2 of the sequence Listing, and/or by performing missense mutation of one or several base pairs, and/or by attaching the coding sequence of the tag shown in Table 1 above to its 5 'end and/or 3' end.

The phosphatidylserine synthase AtPSS1 is (b1) or (b2) as follows:

(b1) a protein consisting of an amino acid sequence shown in a sequence 5 in a sequence table;

(b2) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the sequence 5, is related to the salt tolerance of plants and is derived from the sequence 5.

In order to facilitate purification and detection of the phosphatidylserine synthase AtPSS1 in (b1), a tag as shown in Table 1 may be attached to the amino terminus or the carboxyl terminus of the protein consisting of the amino acid sequence shown in sequence 5 in the sequence listing.

The phosphatidylserine synthase AtPSS1 in (b2) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression. The coding gene of the phosphatidylserine synthase AtPSS1 in (b2) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence No. 4 in the sequence Listing, and/or by carrying out missense mutation of one or several base pairs, and/or by connecting the coding sequence of the tag shown in Table 1 at its 5 'end and/or 3' end.

The phosphatidylserine synthase AtPSS2 is (c1) or (c2) as follows:

(c1) a protein consisting of an amino acid sequence shown as a sequence 7 in a sequence table;

(c2) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 7, is related to the salt tolerance of plants and is derived from the sequence 7.

In order to facilitate purification and detection of the phosphatidylserine synthase AtPSS2 in (c1), a tag as shown in Table 1 may be attached to the amino terminus or the carboxyl terminus of the protein consisting of the amino acid sequence shown in sequence No. 7 in the sequence Listing.

The phosphatidylserine synthase AtPSS2 in (c2) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression. The coding gene of the phosphatidylserine synthase AtPSS2 in (c2) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence No. 6 in the sequence Listing, and/or by carrying out missense mutation of one or several base pairs, and/or by connecting the coding sequence of the tag shown in Table 1 at its 5 'end and/or 3' end.

The coding gene of any one of the phosphatidylserine synthases is SePSS gene or AtPSS1 gene or AtPSS2 gene.

The SePSS gene is a DNA molecule as described in any one of (d1) - (d 4):

(d1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

(d2) DNA molecule shown in sequence 2 in the sequence table;

(d3) a DNA molecule which is hybridized with the DNA sequence defined in (1) or (2) under strict conditions and encodes a protein related to the salt tolerance of plants;

(d4) and (3) DNA molecules which have more than 90% of homology with the DNA sequences defined in (1) or (2) or (3) and encode proteins related to plant salt tolerance.

The AtPSS1 gene is a DNA molecule as described in any one of (e1) - (e 4):

(e1) the coding region is a DNA molecule shown as a sequence 4 in the sequence table;

(e2) DNA molecule shown in sequence 4 in the sequence table;

(e3) a DNA molecule which is hybridized with the DNA sequence defined in (1) or (2) under strict conditions and encodes a protein related to the salt tolerance of plants;

(e4) and (3) DNA molecules which have more than 90% of homology with the DNA sequences defined in (1) or (2) or (3) and encode proteins related to plant salt tolerance.

The AtPSS2 gene is a DNA molecule as described in any one of (f1) - (f 4):

(f1) the coding region is a DNA molecule shown as a sequence 6 in a sequence table;

(f2) DNA molecule shown in sequence 6 in the sequence table;

(f3) a DNA molecule which is hybridized with the DNA sequence defined in (1) or (2) under strict conditions and encodes a protein related to the salt tolerance of plants;

(f4) and (3) DNA molecules which have more than 90% of homology with the DNA sequences defined in (1) or (2) or (3) and encode proteins related to plant salt tolerance.

The stringent conditions can be hybridization and washing with 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS solution at 65 ℃ in DNA or RNA hybridization experiments.

Any of the plants may specifically be a dicotyledonous plant or a monocotyledonous plant.

The dicotyledonous plant can be a plant of the order Capricorales. The plant of order Capparis can be a plant of the family Brassicaceae. The cruciferous plant may be a plant of the arabidopsis thaliana family. The Arabidopsis plant may be an Arabidopsis plant. The arabidopsis plant may specifically be arabidopsis thaliana, e.g. colombian ecotype arabidopsis thaliana.

The dicotyledonous plant may be a Chenopodiaceae plant. The Chenopodiaceae plant can be Salicornia plant. The Salicornia plant may be Salicornia herbacea (S.europeae).

Experiments prove that the salicornia SePSS gene is subjected to salt-induced expression, and the expression level is the highest when the salicornia SePSS gene is treated by 200mM NaCl for 12 hours and the expression level is the highest when the salicornia SePSS gene is treated by 800mM NaCl for 24 hours. SePSS gene is used for complementing Arabidopsis AtPSS mutant, and phenotypes such as PS content, cytoplasmic membrane polarity, chloroplast content, fresh weight, root length and the like of a complementing strain are restored, and the restoring effect is the same as that of the AtPSS gene. The fresh weight of the SePSS over-expressed line was significantly higher than that of the wild type Arabidopsis thaliana under 150mM NaCl treatment. These results suggest that SePSS has a function of PS synthesis, is very important for maintaining the polarity of the cytoplasmic membrane, and that overexpression of the SePSS gene can improve the salt resistance of Arabidopsis thaliana. The work provides theoretical basis and gene resources for genetic improvement of stress-resistant crops.

Drawings

FIG. 1 is an analysis of SePSS gene expression patterns under salt treatment.

FIG. 2 shows subcellular localization of SePSS protein.

FIG. 3 shows the relative PSS gene transcript content of Arabidopsis mutants and partially transgenic lines.

FIG. 4 shows the cell and plasma membrane PS contents of different lines of Arabidopsis.

FIG. 5 shows plasma membrane polarity assays of Arabidopsis wild type, mutant, over-expression and complementation lines under NaCl treatment.

FIG. 6 shows the NaCl tolerance of Arabidopsis wild type, mutant and complementation lines.

FIG. 7 shows the NaCl tolerance of Arabidopsis thaliana wild-type and overexpression lines.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Salicornia europaea (s. europeae) seeds: dafeng Jinglong ocean industry development Co., Ltd, Jiangsu province.

pCAMBIA1300-35S, GFP vector: reference documents: juanjuan Feng, Pengxiang Fan, Ping Jiang, Sulian Lv, Xianyang Chen, Yinxin Li (2013) Chloroplast-targeted Hsp90 plants in plant stages with displacement and organization in Arabidopsis sites linking with VIPP1.physiologia Plantarum,150(2) 292-; the public is available from the plant institute of Chinese academy of sciences.

pCAMBIA2300 vector: please provide a purchase route or literature. Changsha Yingrun Biotechnology Co., Ltd

C58 agrobacterium: reference documents: hongmiao Song, Pengxiang Fan, Yinxin Li (2009) Overexpression of Organllar and Cytosalic AtHSP90in Arabidopsis thaliana third Plant Toleranceo oxide stress.plant Mol Biol Rep,27: 342-349; the public is available from the plant institute of Chinese academy of sciences.

Bunsen tobacco (Nicotiana benthamiana): reference documents: xianyang Chen, Hexigeduleng Bao, Jie Guo, Weitao Jia, Fan Tai, Lingling Nie, Ping Jiang Juan, Juanjuan Feng, Sulian Lv, Yinxin Li (2014) Na⁺/H⁺exchanger 1 substrates in tobacaco disease depth with access to photophtora parasitica var. nicotiniae by affecting vacuolar pH and priming the alcoholic reactive system J Exp Bot,65:6107 + 6122; the public is available from the plant institute of Chinese academy of sciences.

pCAMBIA1301 vector: beijing Huayuyo Biometrics.

Wild type Arabidopsis thaliana (Col-0): reference documents: juanjuan Feng, Pengxiang Fan, Ping Jiang, Sulian Lv, Xianyang Chen, Yinxin Li (2013) Chloroplast-targeted Hsp90 plants in plant stages with displacement and organization in Arabidopsis sites linking with VIPP1.physiologia plant, 150(2) 292-; the public is available from the plant institute of Chinese academy of sciences.

Arabidopsis thaliana loss-of-function mutant atpss (SALK-128223): arabidopsis Biological Resource Center (ARBC). The arabidopsis function deletion mutant has the same sequence with Columbia ecological arabidopsis except atpss gene by whole genome sequencing.

Example 1 discovery of SePSS protein of Salicornia europaea and Gene encoding the same

The total RNA of the aerial parts of salicornia europaea is extracted and reverse transcribed into cDNA. Through a large amount of sequence analysis, expression analysis and functional verification, a DNA coding sequence of the phosphatidylserine synthase is found from the cDNA, as shown in a sequence 2 of a sequence table, and the coded protein is shown in a sequence 1 of the sequence table.

The protein shown in sequence 1 of the sequence table is named as SePSS protein and consists of 425 amino acid residues, and the protein has nine transmembrane domains and does not contain signal peptide. The gene encoding SePSS protein was named SePSS gene, and its CDS full length was 1728 bp.

Example 2 analysis of SePSS Gene expression

After sowing Salicornia europaea seeds for 30 days, seedlings with consistent growth are taken, and are respectively cultured for different times (0, 3, 6, 12, 24, 72 or 168 hours) by 1/2Hoagland nutrient solutions containing NaCl (0, 200 or 800mM) with different concentrations, and aerial parts are taken for gene expression detection.

Extracting total RNA of the aerial part, carrying out reverse transcription to obtain cDNA, detecting the expression condition of the SePSS gene by using a Real-time quantitative PCR (qRT-PCR) method by using the cDNA as a template (detecting by using a primer pair consisting of a primer SePSS-qPCR-Forward and a primer SePSS-qPCR-Reversed), and using the alpha-tubulin gene as an internal reference (detecting by using a primer pair consisting of a primer alpha-tubulin-qPCR-Forward and a primer alpha-tubulin-qPCR-Reversed).

α-tubulin-qPCR-Forward：5'-CGAAAGTTGGGGGCTCGAAG-3'；

α-tubulin-qPCR-Reversed：5'-CCCCGGAACCCAAAGACTTTG-3'；

SePSS-qPCR-Forward：5'-GCGATGATGCTCGGTTGTTC-3'；

SePSS-qPCR-Reversed：5'-TTCGTCAAAGACTGTGTCATAA-3'。

The results are shown in FIG. 1. The results showed that the SePSS gene expression levels in the aerial parts of Salicornia europaea exhibited a tendency to increase and then decrease when treated with 200 and 800mM NaCl. The SePSS expression level was highest when treated with 200mM NaCl for 12h and 800mM NaCl for 24 h.

Example 3 subcellular localization of SePSS proteins

1. pCAMBIA1300-35S, SePSS-GFP vector: inserting the double-stranded DNA molecule described in the sequence 2 of the sequence table between BamH I enzyme cutting sites of the pCAMBIA1300-35S and the GFP vector to obtain the pCAMBIA1300-35S and the SePSS-GFP vector (sequencing verification is performed).

2. pCAMBIA2300-35S, HDEL-mCherry vector: the double-stranded DNA molecule described in sequence 3(HDEL-mCherry fragment) of the sequence table is used for replacing the fragment between the Sal I and Hind III enzyme cutting sites of the pCAMBIA2300 vector to obtain the pCAMBIA2300-35S, namely, the HDEL-mCherry vector (sequencing verification is performed).

3. Introducing the pCAMBIA1300-35S vector SePSS-GFP vector into C58 agrobacterium to obtain recombinant strain 35S vector SePSS-GFP. The recombinant strain 35S was inoculated with SePSS-GFP and cultured with shaking (28 ℃ C., 180rpm) to OD in LB liquid medium containing 20. mu.M AS, 10mM MES, 100. mu.M Kan and 100. mu.M Rif₆₀₀1.8. The culture system was centrifuged to collect cells, which were then washed with 10mM MgCl solution containing 200. mu.M AS₂And 10mM MES distilled water to resuspend the cells to the bacterial solution OD_600nmStanding for 5-6h under the condition of 1.4-1.6 to obtain a recombinant strain 35S, namely SePSS-GFP bacterial liquid.

4. The pCAMBIA2300-35S vector HDEL-mCherry vector is introduced into C58 agrobacterium to obtain recombinant bacteria 35S HDEL-mCherry. The recombinant strain 35S, HDEL-mCherry, was inoculated into LB liquid medium containing 20. mu.M AS, 10mM MES, 100. mu.M Kan and 100. mu.M Rif and cultured with shaking (28 ℃, 180rpm) to OD₆₀₀1.8. The culture system was centrifuged to collect cells, which were then washed with 10mM MgCl solution containing 200. mu.M AS₂And 10mM MES distilled water to resuspend the cells to the bacterial solution OD_600nmStanding for 5-6h under the condition of 1.4-1.6 to obtain the recombinant bacterium 35S, namely HDEL-mCherry bacterium liquid.

5. Mixing 1 volume part of the recombinant bacterium 35S, the SePSS-GFP bacterium solution and 1 volume part of the recombinant bacterium 35S, the HDEL-mCherry bacterium solution and injecting the mixture into the leaf of the native tobacco, and observing protein subcellular localization by using a laser scanning confocal microscope after the tobacco is normally cultured for 2 days.

The results are shown in FIG. 2. The results show that the green fluorescence emitted by the GFP fusion protein overlaps with the red fluorescence emitted by the mCherry fusion protein, indicating that the SePSS protein is located on the endoplasmic reticulum.

Example 4 SePSS protein and use of the encoding Gene thereof

First, construction of expression vector

1. pCAMBIA1301-35S SePSS vector: double-stranded DNA molecules (SePSS1 gene) shown in sequence 2 of the sequence table are inserted between BamH I enzyme cutting sites of the pCAMBIA1301 vector to obtain pCAMBIA1301-35S, namely SePSS1 vector (sequencing verification is performed).

2. pCAMBIA1301-35S AtPSS1 vector: double-stranded DNA molecules (AtPSS1 gene) shown in sequence 4 of the sequence table are inserted between BglII enzyme cutting sites of pCAMBIA1301 vector to obtain pCAMBIA1301-35S, wherein the vector is AtPSS1 (sequencing verification).

3. pCAMBIA1301-35S AtPSS2 vector: double-stranded DNA molecules (AtPSS2 gene) shown in sequence 6 of the sequence table are inserted between BglII enzyme cutting sites of pCAMBIA1301 vector to obtain pCAMBIA1301-35S, wherein the vector is AtPSS2 (sequencing verification).

AtPSS1 and AtPSS2 are two transcripts of the phosphatidylserine synthase gene in Arabidopsis.

The double-stranded DNA molecule shown in sequence 4 of the sequence table encodes a protein (AtPSS1 protein) shown in sequence 5 of the sequence table.

The double-stranded DNA molecule shown in sequence 6 of the sequence table encodes a protein (AtPSS2 protein) shown in sequence 7 of the sequence table.

Second, construction of transgenic Arabidopsis thaliana

1. The recombinant strain 35S, SePSS, is obtained by introducing the pCAMBIA1301-35S, SePSS vector into C58 agrobacterium. SePSS is used for transforming wild arabidopsis thaliana (Col-0) by utilizing agrobacterium-mediated flower dipping method to obtain T₀SePSS Arabidopsis thaliana was transformed. Will T₀Seeds of SePSS Arabidopsis thaliana were surface sterilized and then uniformly sown on 1/2MS solid medium containing 50mg/L Hyg, first cultured in dark at 4 ℃ for 2 days, then cultured in light (16h light/8 h dark) at 22 ℃ for 5-10 days, and then the surviving seedlings were transferred to culture soil to grow until the seeds were mature. This step is repeated until T is obtained₃Transgenic homozygous lines (OES1, OES2 and OES3) were generated.

2. The recombinant strain 35S, SePSS, is obtained by introducing the pCAMBIA1301-35S, SePSS vector into C58 agrobacterium. SePSS utilizes an agrobacterium-mediated flower dipping method to transform an arabidopsis function deletion mutant atpss to obtain T₀SePSS generation complements Arabidopsis thaliana. Will T₀Seeds of SePSS-generation complementary Arabidopsis thaliana are sterilized on the surface and then uniformly sown in 1/2MS solid culture containing 50mg/L HygCulturing in dark at 4 deg.C for 2 days, culturing in light at 22 deg.C (16h light/8 h dark) for 5-10 days, and transferring the survived seedlings to culture soil to grow until the seeds are mature. This step is repeated until T is obtained₃Transgenic homozygous lines (CS) were generated.

3. The pCAMBIA1301-35S, AtPSS1 vector is introduced into C58 agrobacterium tumefaciens to obtain recombinant strain 35S, AtPSS 1. The recombinant strain 35S (AtPSS 1) is transformed into wild arabidopsis thaliana (Col-0) by an agrobacterium-mediated floral dip method to obtain T₀Transfer to AtPSS1 Arabidopsis thaliana. Will T₀Seeds transferred to AtPSS1 Arabidopsis thaliana are sterilized on the surface, uniformly sown on 1/2MS solid culture medium containing 50mg/L Hyg, firstly cultured in dark at 4 ℃ for 2 days, then cultured in light (16h light/8 h dark) at 22 ℃ for 5-10 days, and then the survival seedlings are transferred to culture soil to grow until the seeds are mature. This step is repeated until T is obtained₃Generation transgenic homozygous lines (OEA1-1, OEA1-2 and OEA 1-3).

4. The pCAMBIA1301-35S, AtPSS1 vector is introduced into C58 agrobacterium tumefaciens to obtain recombinant strain 35S, AtPSS 1. The recombinant strain 35S is characterized in that AtPSS1 is transformed into Arabidopsis thaliana function deletion mutant AtPSS by utilizing agrobacterium-mediated flower dipping method to obtain T₀The generation AtPSS1 complements arabidopsis. Will T₀Seeds of complementary arabidopsis thaliana of generation AtPSS1 are evenly sown on 1/2MS solid culture medium containing 50mg/L Hyg after surface sterilization, firstly cultured in dark at 4 ℃ for 2 days, then cultured in light (16h light/8 h dark) at 22 ℃ for 5-10 days, and then the survival seedlings are transferred to culture soil to grow until the seeds are mature. This step is repeated until T is obtained₃Transgenic homozygous lines (CA1) were generated.

5. The pCAMBIA1301-35S, AtPSS2 vector is introduced into C58 agrobacterium tumefaciens to obtain recombinant strain 35S, AtPSS 2. The recombinant strain 35S (AtPSS 2) is transformed into wild arabidopsis thaliana (Col-0) by an agrobacterium-mediated floral dip method to obtain T₀Transfer to AtPSS2 Arabidopsis thaliana. Will T₀Seeds transferred to AtPSS2 Arabidopsis thaliana are sterilized on the surface, uniformly sown on 1/2MS solid culture medium containing 50mg/L Hyg, firstly cultured in dark at 4 ℃ for 2 days, then cultured in light (16h light/8 h dark) at 22 ℃ for 5-10 days, and then the survival seedlings are transferred to culture soil to grow until the seeds are mature. This step is repeated until T is obtained₃Generation transgenic homozygous lines (OEA2-1, OEA2-2 and OEA 2-3).

6. The pCAMBIA1301-35S, AtPSS2 vector is introduced into C58 agrobacterium tumefaciens to obtain recombinant strain 35S, AtPSS 2. The recombinant strain 35S is characterized in that AtPSS2 is transformed into Arabidopsis thaliana function deletion mutant AtPSS by utilizing agrobacterium-mediated flower dipping method to obtain T₀The generation AtPSS2 complements arabidopsis. Will T₀Seeds of complementary arabidopsis thaliana of generation AtPSS2 are evenly sown on 1/2MS solid culture medium containing 50mg/L Hyg after surface sterilization, firstly cultured in dark at 4 ℃ for 2 days, then cultured in light (16h light/8 h dark) at 22 ℃ for 5-10 days, and then the survival seedlings are transferred to culture soil to grow until the seeds are mature. This step is repeated until T is obtained₃Transgenic homozygous lines (CA2) were generated.

7. The method adopts a pCAMBIA1301 vector to replace pCAMBIA1301-35S, wherein a SePSS vector is operated according to the

steps

1 and 2 respectively to obtain a transferred empty vector contrast 1 and a transferred empty vector contrast 2.

Third, gene expression detection of transgenic Arabidopsis

And (3) the plant to be detected: wild type Arabidopsis (Col-0), Arabidopsis functional deletion mutant atpss, T₃Generation transgenic homozygous lines (OES1, OES2 and OES3), T₃Generation transgenic homozygous lines (CS) and T₃Generation transgenic homozygous lines (OEA1-1, OEA1-2 and OEA1-3), T₃Generation transgenic homozygous line (CA1), T₃Generation transgenic homozygous lines (OEA2-1, OEA2-2 and OEA2-3), T₃Generation transgenic homozygous lines (CA2), and T₃Empty vector controls 1 and 2 were replaced.

The seed surface of the plant to be tested is sterilized, and then is sown on 1/2MS solid culture medium, and is cultured in dark at 4 ℃ for 2 days, and then is cultured in light at 22 ℃ (16h light/8 h dark) for three weeks. 10 strains (about 100ng) of each strain are taken, total RNA is extracted, the total RNA is reversely transcribed into cDNA, the cDNA is taken as a template, and a Real-time quantitative PCR (qRT-PCR) method is adopted to detect the expression condition of the SePSS gene (the detection is carried out by using a primer pair consisting of a primer SePSS-qPCR-Forward and a primer SePSS-qPCR-reverse) or the expression condition of the AtPSS1 gene (the detection is carried out by using a primer pair consisting of a primer AtPSS1-qPCR-Forward and a primer AtPSS 1-qPCR-reverse) or the expression condition of the AtPSS2 gene (the detection is carried out by using a primer AtPSS2-qPCR-Forward and a primer AtPSS 2-qPCR-Reve)Detection of primer pair composed of rsed), using α -tubulin gene as internal reference (detection using primer pair composed of α -tubulin-qPCR-Forward and α -tubulin-qPCR-Reversed), by 2^-ΔΔCtThe method of (3) relatively quantifies gene expression.

actin-qPCR-Forward：5'-ATATGCCTATCTACAGGGTT-3'；

actin-qPCR-Reversed：5'-ATACAATTTCCCGTTCTGCTGT-3'；

SePSS-qPCR-Forward：5'-GCGATGATGCTCGGTTGTTC-3'；

SePSS-qPCR-reversed：5'-TTCGTCAAAGACTGTGTCATAA-3'；

AtPSS1-qPCR-Forward：5'-TGAAGTTTCTCCACCCTGACC-3'；

AtPSS1-qPCR-reversed：5'-TTGAAAGCACCCAAAGAAGC-3'；

AtPSS2-qPCR-Forward：5'-GGCTCTTCCAGATGTTGTTAC-3'；

AtPSS2-qPCR-reversed：5'-CGCAGTGGCAGTTGAGTTAT-3'。

The results are shown in FIG. 3. The results show that the PSS gene expression levels of all transgenic lines are up-regulated to different degrees, which shows that SePSS, AtPSS1 and AtPSS2 genes are transferred into Arabidopsis thaliana and can be stably expressed, and atPSs is remarkably down-regulated, which shows that the T-DNA insertion causes the suppression of the PSS gene of Arabidopsis thaliana on the transcription level. The expression levels of SePSS, AtPSS1 and AtPSS2 genes in the transgenic empty vector control line 1 are unchanged compared with the wild type, and the expression levels of SePSS, AtPSS1 and AtPSS2 genes in the transgenic empty vector control line 2 are unchanged compared with the mutant AtPSS.

Fourthly, detecting the plasma membrane protein content, the PS content and the polarity of the transgenic arabidopsis thaliana

And (3) the plant to be detected: wild type Arabidopsis (WT), Arabidopsis functional deletion mutant atpss, T₃Generation transgenic homozygous lines (OES1, OES2 and OES3), T₃Generation transgenic homozygous lines (CS) and T₃Generation transgenic homozygous lines (OEA1-1, OEA1-2 and OEA1-3), T₃Generation transgenic homozygous line (CA1), T₃Generation transgenic homozygous lines (OEA2-1, OEA2-2 and OEA2-3) and T₃Generation transgenic homozygous lines (CA2) andT₃empty vector controls 1 and 2 were replaced.

1. The seed surface of the plant to be tested is sterilized, and then is sown on 1/2MS solid culture medium, and is cultured in dark at 4 ℃ for 2 days, and then is cultured in light at 22 ℃ (16h light/8 h dark) for three weeks. Reference is made to the following documents: the method of Tang W (2012) Quantitative analysis of plasma membrane Protein using two-dimensional difference electrophoresis. plant Signalling Networks, Springer,67-82, was used to extract plasma membrane and measure the Protein content of the plasma membrane using Modified Bradford Protein Assay Kit (Biotech). The cell and plasma membrane PS content was determined using Plant PS ELISA KIT (R & D _ Systems, USA). The results are shown in FIG. 4. The results show that the PS content of the over-expressed and complementation lines does not change much compared to wild type Arabidopsis, both in the cell and in the plasma membrane; the PS content in the mutant strain is significantly lower than that of the wild type. The detection result of the transgenic empty vector control strain 1 has no significant difference with the wild type, and the detection result of the transgenic empty vector control strain 2 has no significant difference with the mutant.

2. Leaves of 3-week-old seedlings of plants to be tested were stained with a solution containing NaCl (0 or 200mM) and 1. mu.M DiBAC4(3) (Molecular Probes, USA) for 10min, and then DiBAC4(3) fluorescence was observed with a Leica laser scanning confocal microscope with 488nm excitation light and 500-550nm emission light (see Konrad KR and Hedrich R (2008) The use of voltage-sensitive powers to monitor signal-induced changes in membrane potential-induced-polymerization cells. plant Journal,55(1): 161-73.).

The results are shown in FIG. 5. The results show that when salt treatment is not carried out, the plasma membranes of wild type seedlings, mutant seedlings, overexpression seedlings and complementary seedlings of the strain have no green fluorescence, and the phenomenon of obvious depolarization does not occur; under the treatment of 200mM NaCl, the plasma membranes of all the strain seedlings have green fluorescence in different degrees, which indicates that all the strain seedlings have depolarization in different degrees, especially the mutant seedlings have the strongest fluorescence, which indicates that the mutant seedlings have strong depolarization. The level of green fluorescence emitted by the over-expressed, complementation and wild type seedlings was consistent, indicating that their plasma membrane depolarization levels were similar. The detection result of the transgenic empty vector control strain 1 has no significant difference with the wild type, and the detection result of the transgenic empty vector control strain 2 has no significant difference with the mutant.

Fifth, salt tolerance detection of transgenic Arabidopsis

And (3) the plant to be detected: wild type Arabidopsis (WT), Arabidopsis functional deletion mutant atpss, T₃Generation transgenic homozygous lines (OES1, OES2 and OES3), T₃Generation transgenic homozygous lines (CS) and T₃Generation transgenic homozygous lines (OEA1-1, OEA1-2 and OEA1-3), T₃Generation transgenic homozygous line (CA1), T₃Generation transgenic homozygous lines (OEA2-1, OEA2-2 and OEA2-3) and T₃Generation transgenic homozygous line (CA2), T₃Empty vector controls 1 and 2 were replaced.

The seed surface of the plant to be tested is sterilized, and then is sown on 1/2MS solid culture medium, and is cultured in dark at 4 ℃ for 2 days, and then is cultured in light at 22 ℃ (16h light/8 h dark) for three weeks. Shoots with consistent growth were picked and transferred to 1/2MS medium containing 0 and 150mM NaCl for vertical culture. After 7 days of treatment, the length, fresh weight and chlorophyll content of the main root of the plant are measured.

The chlorophyll content detection method comprises the following steps: extracting chlorophyll from leaves by 80% acetone extraction method, respectively measuring D645 and D663 by spectrophotometer method, and calculating chlorophyll content according to the following formula: c_T＝20.29D₆₄₅+8.05D₆₆₃。)。

The results are shown in FIGS. 6 and 7. The results show that the growth conditions of the wild type, the mutant and the transgenic strain line are basically consistent on 1/2MS medium, and the growth conditions are greatly different under the 150mM NaCl stress condition. Under salt stress, wild type, mutant and transgenic seedling growth were inhibited. The complementary strain is consistent with the wild type phenotype in the aspects of total chlorophyll content, fresh weight of overground parts and root length, and has no significant difference; the mutant is obviously lower than the wild type and complementary strains in the aspects of total chlorophyll content, fresh weight of overground parts and root length. Compared with the wild type, the over-expression strain has no significant difference in the aspects of total chlorophyll content and root length, but the fresh weight of the overground part is significantly larger than that of the wild type, which indicates that the salt resistance of the over-expression Arabidopsis is enhanced to a certain extent. The detection result of the transgenic empty vector control strain 1 has no significant difference with the wild type, and the detection result of the transgenic empty vector control strain 2 has no significant difference with the mutant.

Sequence listing

<110> institute of plant of Chinese academy of sciences

<120> Salicornia europaea SePSS protein, coding gene and application thereof

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 425

<212> PRT

<213> Salicornia Herbacea (S. europeae)

<400> 1

Met Glu Pro Asp Ser Cys Arg Arg Ile Ser Arg Lys Asp Ile Asn His

1 5 10 15

Thr Asn Gly Asp Ala Ser Tyr Ser Cys Ala Asp Asp Glu Leu Asp Pro

20 25 30

Trp Thr Ala Trp Ala Tyr Lys Pro Arg Thr Ile Thr Leu Leu Phe Ile

35 40 45

Gly Ala Cys Phe Leu Ile Trp Ala Ser Gly Ala Leu Asp Pro Asp Ser

50 55 60

Ser Arg Ser Gly Asp Val Val Thr Ser Val Lys Arg Gly Val Trp Ala

65 70 75 80

Met Ile Ala Val Phe Leu Thr Tyr Cys Leu Leu Gln Ala Pro Ser Thr

85 90 95

Val Leu Ile Arg Pro His Pro Ala Ile Trp Arg Leu Val His Gly Ile

100 105 110

Ala Val Ile Tyr Leu Val Ala Leu Thr Phe Leu Leu Phe Gln Lys Arg

115 120 125

Asp Asp Ala Arg Leu Phe Met Lys Tyr Leu His Ser Asp Leu Gly Val

130 135 140

Glu Leu Pro Glu Arg Ser Tyr Gly Ser Asp Cys Arg Ile Tyr Val Pro

145 150 155 160

Glu Asp Pro Thr Asn Arg Phe Arg Asn Leu Tyr Asp Thr Val Phe Asp

165 170 175

Glu Phe Phe Leu Ala His Ile Leu Gly Trp Trp Gly Lys Ala Ile Met

180 185 190

Ile Arg Asn Gln Pro Leu Cys Trp Val Leu Ser Ile Gly Phe Glu Met

195 200 205

Met Glu Val Thr Phe Arg His Met Leu Pro Asn Phe Asn Glu Cys Trp

210 215 220

Trp Asp Ser Ile Ile Leu Asp Ile Leu Leu Cys Asn Trp Phe Gly Ile

225 230 235 240

Trp Thr Gly Met Arg Thr Val Lys Tyr Phe Asp Gly Lys Thr Tyr Glu

245 250 255

Trp Val Gly Ile Ser Arg Gln Pro Asn Ile Met Gly Lys Val Lys Arg

260 265 270

Thr Leu Gly Gln Phe Thr Pro Ala Arg Trp Asp Lys Asp Glu Trp His

275 280 285

Pro Leu Leu Gly Pro Trp Arg Phe Val Gln Val Leu Phe Leu Cys Leu

290 295 300

Val Phe Leu Thr Val Glu Leu Asn Thr Phe Phe Leu Lys Phe Cys Leu

305 310 315 320

Trp Ile Pro Pro Ser Asn Pro Ile Ile Thr Tyr Arg Leu Ile Leu Trp

325 330 335

Trp Leu Ile Ala Val Pro Thr Ile Arg Glu Tyr Asn Ser Tyr Leu Gln

340 345 350

Asp Arg Lys Gln Val Lys Lys Val Gly Ala Leu Cys Trp Leu Ala Leu

355 360 365

Ala Ile Cys Ile Val Glu Leu Leu Ile Cys Ile Lys Phe Gly His Gly

370 375 380

Leu Tyr Pro Asn Leu Met Pro Ile Trp Leu Val Ile Phe Trp Ser Ser

385 390 395 400

Phe Gly Val Val Leu Leu Met Phe Leu Val Ile Trp Thr Trp Lys Leu

405 410 415

His Lys Tyr Ser Glu Lys Lys Lys Gln

420 425

<210> 2

<211> 1278

<212> DNA

<213> Salicornia Herbacea (S. europeae)

<400> 2

atggaacctg atagttgtag gaggatcagc cgaaaggata tcaaccatac aaatggcgat 60

gcaagctact catgtgctga tgatgaactt gatccttgga ctgcttgggc ttataagcct 120

cgcacaataa ctttgttgtt tattggtgct tgtttcctta tctgggctag tggagctctt 180

gatcctgaca gcagcagatc tggtgatgtg gttacatcag tcaaaagggg tgtatgggca 240

atgattgcag tttttcttac ttactgcttg ttacaagcac catcaacggt ccttatacga 300

ccacatcctg caatttggcg cttagttcac ggaattgctg ttatttacct tgttgctctg 360

acgtttttgc ttttccagaa gcgcgatgat gctcggttgt tcatgaaata tctccattca 420

gatttaggtg ttgaacttcc tgaaagatct tatggctcag attgccgaat atatgttcct 480

gaagacccca ccaacaggtt tagaaacctt tatgacacag tctttgacga atttttccta 540

gctcatattc tcggatggtg gggcaaggca atcatgattc ggaatcaacc tttgtgctgg 600

gtattatcaa ttggttttga aatgatggag gtcaccttcc gtcatatgct accaaatttc 660

aacgagtgtt ggtgggacag tattattctg gacatcctgc tctgcaattg gtttggcatt 720

tggactggta tgcgtactgt gaagtatttc gatgggaaaa catatgagtg ggtaggaata 780

agtcgccagc caaatataat gggcaaggta aaacgtactt tggggcaatt cacacctgca 840

cgatgggata aagatgaatg gcatccgcta cttggtcctt ggcgttttgt ccaagttctg 900

ttcctttgcc ttgtgttttt gacagtggag ttgaatacat tttttctgaa gttttgtctc 960

tggattcctc ccagcaatcc tattatcact tataggttaa ttttatggtg gctaattgct 1020

gttcctacta tccgagaata taattcctat ctacaggaca gaaaacaagt aaagaaggta 1080

ggggcattat gttggcttgc cttggctatt tgcattgttg agctactcat ctgtattaag 1140

tttgggcatg gattataccc aaatctaatg cctatatggt tggtgatttt ttggtcatca 1200

tttggagtag tacttctgat gtttttggta atatggacat ggaaattgca caagtattca 1260

gaaaagaaga aacaatga 1278

<210> 3

<211> 734

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

catgatgagc ttatggtgag caagggcgag gaggataaca tggccatcat caaggagttc 60

atgcgcttca aggtgcatat ggagggctcc gtgaacggcc acgagttcga gatcgagggc 120

gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt 180

ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc 240

tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc 300

aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc 360

tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc 420

gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac 480

cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc 540

cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc 600

gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg 660

gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag 720

tagagatcca ctag 734

<210> 4

<211> 1362

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 4

atggaaccca atgggtacag gaaagaaaga agaaaggaac aacatttggg gagaatgaat 60

ggtggtggtg gtgatgttga gactgatctt gatccatgga ctgcatgggc ttacaagcct 120

cgcactatct ctcttctcct tattggcgct tgttttctca tttgggctag tggagctctt 180

gatcctgaca gcactacatc tgatgatctt gtcacttctg tcaaaagggg agtgtgggct 240

atgatcgctg tctttcttgc ttactctttg ctccaggccc cttcaacggt tctaatcagg 300

ccccatcctg caatctggcg cttagttcat ggaatggccg tcatttactt agttgcactt 360

acttttttgc tctttcagag acgtgatgat gcacggcagt tcatgaagtt tctccaccct 420

gaccttggaa tagaacttcc tgagaaatca tacggtgctg attgccgtat atatgtgcct 480

gatcacccaa caaacaggtt taagaatctt tatgacacag tatttgatga gtttttcttg 540

gctcacatct ttggttggtg ggggaaagca attctgatcc ggaaccagcc gcttctttgg 600

gtgctttcaa ttggttttga gttattggag gttacttttc ggcatatgtt accaaatttc 660

aacgagtgct ggtgggacag tattgttctt gatatcttga tatgcaactg gtttggtatc 720

tgggcaggaa tgtatacagt tcgatatttt gatggaaaaa catatgagtg ggttggcatt 780

agccgccagc ctaacattat tggcaaagtg aaaaggacac tggggcagtt cacaccagct 840

cactgggaca aagacgagtg gcatccgctg cagggacctt ggcgttttat tcaagtactc 900

actctttgca tcatattctt gacagtggag ctgaacacat tctttctcaa gtttagcctg 960

tggatccccc ctcgaaaccc agtgattctc tataggctga tcttgtggtg gctcatagcg 1020

ataccaacaa cacgggagta caattcatat cttcaagaca gaaaacctgt gaaaaaggtg 1080

ggagcattct gttggctgtc actggggata tgcatagtag aacttctcat ctgcatcaag 1140

tttggaagtg gtaggattgt acccaacaga aatgccattg tgggtagtga cactatgggg 1200

aagtgtggga cttggacttg tggccttctt gctgtcttgg acatggaaga tccagaagat 1260

cctcgcgcaa aagagacgtt aagcgcatca ttattgttgc agcagagacg agagagaaat 1320

agaggtgaga cagtgaaatg ccattgtggt ttttttttgt aa 1362

<210> 5

<211> 453

<212> PRT

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 5

Met Glu Pro Asn Gly Tyr Arg Lys Glu Arg Arg Lys Glu Gln His Leu

1 5 10 15

Gly Arg Met Asn Gly Gly Gly Gly Asp Val Glu Thr Asp Leu Asp Pro

20 25 30

Trp Thr Ala Trp Ala Tyr Lys Pro Arg Thr Ile Ser Leu Leu Leu Ile

35 40 45

Gly Ala Cys Phe Leu Ile Trp Ala Ser Gly Ala Leu Asp Pro Asp Ser

50 55 60

Thr Thr Ser Asp Asp Leu Val Thr Ser Val Lys Arg Gly Val Trp Ala

65 70 75 80

Met Ile Ala Val Phe Leu Ala Tyr Ser Leu Leu Gln Ala Pro Ser Thr

85 90 95

Val Leu Ile Arg Pro His Pro Ala Ile Trp Arg Leu Val His Gly Met

100 105 110

Ala Val Ile Tyr Leu Val Ala Leu Thr Phe Leu Leu Phe Gln Arg Arg

115 120 125

Asp Asp Ala Arg Gln Phe Met Lys Phe Leu His Pro Asp Leu Gly Ile

130 135 140

Glu Leu Pro Glu Lys Ser Tyr Gly Ala Asp Cys Arg Ile Tyr Val Pro

145 150 155 160

Asp His Pro Thr Asn Arg Phe Lys Asn Leu Tyr Asp Thr Val Phe Asp

165 170 175

Glu Phe Phe Leu Ala His Ile Phe Gly Trp Trp Gly Lys Ala Ile Leu

180 185 190

Ile Arg Asn Gln Pro Leu Leu Trp Val Leu Ser Ile Gly Phe Glu Leu

195 200 205

Leu Glu Val Thr Phe Arg His Met Leu Pro Asn Phe Asn Glu Cys Trp

210 215 220

Trp Asp Ser Ile Val Leu Asp Ile Leu Ile Cys Asn Trp Phe Gly Ile

225 230 235 240

Trp Ala Gly Met Tyr Thr Val Arg Tyr Phe Asp Gly Lys Thr Tyr Glu

245 250 255

Trp Val Gly Ile Ser Arg Gln Pro Asn Ile Ile Gly Lys Val Lys Arg

260 265 270

Thr Leu Gly Gln Phe Thr Pro Ala His Trp Asp Lys Asp Glu Trp His

275 280 285

Pro Leu Gln Gly Pro Trp Arg Phe Ile Gln Val Leu Thr Leu Cys Ile

290 295 300

Ile Phe Leu Thr Val Glu Leu Asn Thr Phe Phe Leu Lys Phe Ser Leu

305 310 315 320

Trp Ile Pro Pro Arg Asn Pro Val Ile Leu Tyr Arg Leu Ile Leu Trp

325 330 335

Trp Leu Ile Ala Ile Pro Thr Thr Arg Glu Tyr Asn Ser Tyr Leu Gln

340 345 350

Asp Arg Lys Pro Val Lys Lys Val Gly Ala Phe Cys Trp Leu Ser Leu

355 360 365

Gly Ile Cys Ile Val Glu Leu Leu Ile Cys Ile Lys Phe Gly Ser Gly

370 375 380

Arg Ile Val Pro Asn Arg Asn Ala Ile Val Gly Ser Asp Thr Met Gly

385 390 395 400

Lys Cys Gly Thr Trp Thr Cys Gly Leu Leu Ala Val Leu Asp Met Glu

405 410 415

Asp Pro Glu Asp Pro Arg Ala Lys Glu Thr Leu Ser Ala Ser Leu Leu

420 425 430

Leu Gln Gln Arg Arg Glu Arg Asn Arg Gly Glu Thr Val Lys Cys His

435 440 445

Cys Gly Phe Phe Leu

450

<210> 6

<211> 1419

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 6

atggcttcta caggtggtgg gaaagatgga tctaaaggtt ttgtgaagag ggttacatca 60

actttctcca ttaggaaaaa gaagaacaca acaagtgatc caaaactact tcttcctcga 120

tcgaaatcaa ccggtgctaa ctatgaatct atgaggctac ctcaggggaa aaaggctctt 180

ccagatgttg ttacaacaaa agacacaaag agaaccaaat ctgcaggtgt ttcgccacaa 240

ccaagacgtg aaaagattga tgaatccggt aaacagttta tgaaggtgag atgttttgat 300

gacagtgact ccatttggtt atcttcagat tgtgcctctc ctacgtctct tttagaggaa 360

cgtagattat ctgtctcgtt tcatttctca gtagacgaaa agatcgtctc gtggttgtcc 420

agtgtggcta actcttctct gtctttaaat caagaatcca ccagctcaaa caaagagaat 480

catcatcaaa aaagttcaaa gaacacaaaa acttctttag aaaacgttcg aaaagatgga 540

aaagtttgca actcatcagc tgggaaagct cgtggtactg gttctgcaaa gccgtcttta 600

ccagaaagca acaacaagac ttgtcctcag aaacaatgtg aagagtcatc tatttccaac 660

agatttgtga ctcttgaaga aaagaaagtt agcttctcag tagcaaaaac agagaagtct 720

ccttcaccag ataactcaac tgccactgcg acatcatcat taaagaagag tgcagagatt 780

ggggtcacaa agagtaagat tgttgtggag ccactttttt ggccatttga gcagaagttt 840

gattggacac cagaggatat tttaaagcat ttttcaatgt ctccgaggag aaagaagtcg 900

ctaggatcca agattgcagg tacctctcca agatcaatga gggcacaact ccaaacaaga 960

aagctagatc taaaagaagg gtgtaagaga aagctcatgt tcaacggtcc tggatcaaat 1020

tcaaaaccaa caagaatccc agaactaaac agaacaatca gcaatagcag caacaatagt 1080

agcatgaaga aaaccgagat cagcaagaac caacaaccta taaggaacag tgtgaagaga 1140

aacaaaagtt taccgtcgag gttgagaaaa tcgagcaaaa tatcttcaaa ggtggtacct 1200

attgaagctg cggaagagag tggagaaata gttaaagagc aaaaaacacc taagaagctc 1260

atcatgaccc gcaagtccag gacattctta gaagatgact ttgctttaat gaatgatttc 1320

tctatagaaa aggcggtcgg gctttgcgag tttaagggaa gagaaggcat agattcagat 1380

ttcaacactg atggtttctt gttcgacgat tctctatga 1419

<210> 7

<211> 472

<212> PRT

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 7

Met Ala Ser Thr Gly Gly Gly Lys Asp Gly Ser Lys Gly Phe Val Lys

1 5 10 15

Arg Val Thr Ser Thr Phe Ser Ile Arg Lys Lys Lys Asn Thr Thr Ser

20 25 30

Asp Pro Lys Leu Leu Leu Pro Arg Ser Lys Ser Thr Gly Ala Asn Tyr

35 40 45

Glu Ser Met Arg Leu Pro Gln Gly Lys Lys Ala Leu Pro Asp Val Val

50 55 60

Thr Thr Lys Asp Thr Lys Arg Thr Lys Ser Ala Gly Val Ser Pro Gln

65 70 75 80

Pro Arg Arg Glu Lys Ile Asp Glu Ser Gly Lys Gln Phe Met Lys Val

85 90 95

Arg Cys Phe Asp Asp Ser Asp Ser Ile Trp Leu Ser Ser Asp Cys Ala

100 105 110

Ser Pro Thr Ser Leu Leu Glu Glu Arg Arg Leu Ser Val Ser Phe His

115 120 125

Phe Ser Val Asp Glu Lys Ile Val Ser Trp Leu Ser Ser Val Ala Asn

130 135 140

Ser Ser Leu Ser Leu Asn Gln Glu Ser Thr Ser Ser Asn Lys Glu Asn

145 150 155 160

His His Gln Lys Ser Ser Lys Asn Thr Lys Thr Ser Leu Glu Asn Val

165 170 175

Arg Lys Asp Gly Lys Val Cys Asn Ser Ser Ala Gly Lys Ala Arg Gly

180 185 190

Thr Gly Ser Ala Lys Pro Ser Leu Pro Glu Ser Asn Asn Lys Thr Cys

195 200 205

Pro Gln Lys Gln Cys Glu Glu Ser Ser Ile Ser Asn Arg Phe Val Thr

210 215 220

Leu Glu Glu Lys Lys Val Ser Phe Ser Val Ala Lys Thr Glu Lys Ser

225 230 235 240

Pro Ser Pro Asp Asn Ser Thr Ala Thr Ala Thr Ser Ser Leu Lys Lys

245 250 255

Ser Ala Glu Ile Gly Val Thr Lys Ser Lys Ile Val Val Glu Pro Leu

260 265 270

Phe Trp Pro Phe Glu Gln Lys Phe Asp Trp Thr Pro Glu Asp Ile Leu

275 280 285

Lys His Phe Ser Met Ser Pro Arg Arg Lys Lys Ser Leu Gly Ser Lys

290 295 300

Ile Ala Gly Thr Ser Pro Arg Ser Met Arg Ala Gln Leu Gln Thr Arg

305 310 315 320

Lys Leu Asp Leu Lys Glu Gly Cys Lys Arg Lys Leu Met Phe Asn Gly

325 330 335

Pro Gly Ser Asn Ser Lys Pro Thr Arg Ile Pro Glu Leu Asn Arg Thr

340 345 350

Ile Ser Asn Ser Ser Asn Asn Ser Ser Met Lys Lys Thr Glu Ile Ser

355 360 365

Lys Asn Gln Gln Pro Ile Arg Asn Ser Val Lys Arg Asn Lys Ser Leu

370 375 380

Pro Ser Arg Leu Arg Lys Ser Ser Lys Ile Ser Ser Lys Val Val Pro

385 390 395 400

Ile Glu Ala Ala Glu Glu Ser Gly Glu Ile Val Lys Glu Gln Lys Thr

405 410 415

Pro Lys Lys Leu Ile Met Thr Arg Lys Ser Arg Thr Phe Leu Glu Asp

420 425 430

Asp Phe Ala Leu Met Asn Asp Phe Ser Ile Glu Lys Ala Val Gly Leu

435 440 445

Cys Glu Phe Lys Gly Arg Glu Gly Ile Asp Ser Asp Phe Asn Thr Asp

450 455 460

Gly Phe Leu Phe Asp Asp Ser Leu

465 470

Claims

1. A method for cultivating transgenic plants, which is to introduce the coding gene of phosphoacyl serine synthase SePSS into a target plant to obtain a transgenic plant; the salt tolerance of the transgenic plant is higher than that of a target plant;

the phosphoacyl serine synthase SePSS is a protein consisting of an amino acid sequence shown as a sequence 1 in a sequence table;

the plant is Arabidopsis thaliana.

2. The method of claim 1, wherein: the encoding gene of the phosphoacyl serine synthase SePSS is a DNA molecule shown as a sequence 2 in a sequence table.

3. A method for improving plant salt tolerance is to improve the expression level of the phosphoacyl serine synthase SePSS in a target plant and improve the plant salt tolerance;

the plant is Arabidopsis thaliana.

4. Use of the gene encoding the phosphatidylserine synthase SePSS of any of claims 1 to 3 or the phosphatidylserine synthase SePSS of claim 1 or 2 for modulating salt tolerance in plants; the plant is Arabidopsis thaliana.

5. Use of the method of any one of claims 1 to 3 or the phosphoserine synthase SePSS of any one of claims 1 to 3 or the gene encoding the phosphoserine synthase SePSS of claim 1 or 2 in plant breeding;

the breeding aims to breed plants with high salt tolerance;

the plant is Arabidopsis thaliana.