Application of protein SeCYP710A and related biological materials thereof in regulation and control of plant salt tolerance

Abstract

The invention discloses a protein SeCYP710A. The protein SeCYP710A is a protein shown as A1), A2) or A3): A1) a protein with an amino acid sequence shown as SEQ ID No.2; a2) a protein which is obtained by substitution and / or deletion and / or addition of amino acid residues on the amino acid sequence shown in A1), has the same function, is derived from A1) or has 80% or more of identity with the protein shown in A1) and has the same function; and A3) a fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of A1) or A2). The invention also discloses a protein SeCYP710A related biological material and application thereof in improving the salt tolerance of plants. The protein SeCYP710A related biological material can be used for regulating and controlling the salt tolerance of the plants.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to the application of protein and related biological materials in regulating plant salt tolerance. Background Art

[0002] Soil salinization is one of the important reasons for the degradation of the agricultural ecological environment and is also the main environmental factor limiting crop yields (Sharma et al., 2016). According to the latest data released by the Food and Agriculture Organization of the United Nations, the global saline soil area accounts for 8.7% of the earth's area, and 20%-50% of the cultivated land in all continents has high salinity (FAO, 2021). Therefore, studying the salt tolerance mechanism of plants, developing and utilizing salt-tolerant plant resources, and effectively utilizing saline-alkali land are of great significance to global agricultural development and ecological environment improvement.

[0003] The plant plasma membrane is a key structure for cells to sense external salt signals. Salt stress can cause changes in the composition and content of lipid substances in the plant plasma membrane, and at the same time change the structural characteristics of the plasma membrane, that is, plasma membrane remodeling occurs (Chalbi et al., 2014). As an important membrane lipid component of the plasma membrane, phytosterols can regulate the fluidity and permeability of the plasma membrane, and play an important role in plant growth and development and response to stress. In plants, phytosterols mainly include three categories: campesterol, sitosterol and stigmasterol. Among them, stigmasterol belongs to 24-ethyl sterol, which plays a more important role in maintaining the stability and integrity of plant cell membranes. In addition, stigmasterol also has a double bond at the 22nd carbon, and has strong antioxidant activity.

[0004] Stigmasterol is the main phytosterol located at the most downstream position of the phytosterol synthesis pathway. It is catalyzed by sterol C22-desaturase (CYP710A) to dehydrogenate the carbon 22 of sitosterol to form a double bond to generate stigmasterol. As a key enzyme in the phytosterol synthesis pathway, CYP710A is the only member of the CYP710 family and belongs to the cytochrome P450 monooxygenase (P450) family (Nelson & Werck-Reichhart, 2011). Although there are many studies on P450, few studies focus on CYP710. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to improve the salt tolerance of plants.

[0006] In order to solve the above technical problems, the present invention first provides a protein named SeCYP710A, wherein the protein SeCYP710A is the following protein A1), A2) or A3):

[0007] A1) a protein whose amino acid sequence is SEQ ID No. 2;

[0008] A2) a protein derived from A1) or having more than 80% identity with the protein shown in A1) and having the same function, obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in A1);

[0009] A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2).

[0010] The protein SeCYP710A may be derived from plants, and specifically may be derived from Salicornia europaea L.

[0011] The present invention also provides biological materials related to the protein SeCYP710A; the biological material is any one of the following B1) to B5):

[0012] B1) a nucleic acid molecule encoding the protein;

[0013] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0014] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0015] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0016] B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3).

[0017] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0018] In the above biological material, the coding sequence of the nucleic acid molecule described in B1) is shown as SEQ ID No. 1 in the sequence listing.

[0019] In order to solve the above technical problems, the present invention also provides a substance for increasing the activity or content of protein SeCYP710A, or an application of a substance for promoting or increasing the expression of a gene encoding the protein, wherein the application is any of the following:

[0020] P1. Use in improving the salt tolerance of Salicornia herba or in preparing a product for improving the salt tolerance of Salicornia herba;

[0021] P2. Application in increasing the total content of salicifolia sterols;

[0022] P3. Application in increasing the content of sitosterol and / or stigmasterol in Salicornia herba;

[0023] P4, application in improving the balance of salsa sterol;

[0024] P5. Application in promoting the synthesis of salsa sterol;

[0025] P6. Application in increasing the expression of SeSMT1 gene and / or SeSMT2 gene of Salicornia herba;

[0026] P7, application in promoting the synthesis of glycoside sterols of Salicornia herba;

[0027] P8, application in increasing the expression of SeUGT80A2 gene and / or SeUGT80B1 gene of Salicornia herba;

[0028] P9. Application in plant breeding.

[0029] In the above application, the purpose of plant breeding includes breeding varieties with high salt tolerance.

[0030] In the above application, SEQ ID No. 2 in the sequence listing consists of 511 amino acid residues.

[0031] In the above application, the substance that increases the expression of the gene encoding the protein can be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcription level of the gene; 2) regulation after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of the RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) regulation of the gene after translation (that is, regulation of the activity of the protein translated from the gene).

[0032] In the above application, the substance that promotes or increases the expression of the gene encoding the protein is a biological material related to the protein SeCYP710A protein; the biological material is any one of the following B1) to B5):

[0033] B1) a nucleic acid molecule encoding the protein;

[0034] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0035] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0036] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0037] B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3).

[0038] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0039] In the above application, the coding sequence of the nucleic acid molecule described in B1) is shown as SEQ ID No. 1 in the sequence listing.

[0040] In the above application, the expression cassette containing the nucleic acid molecule described in the biological material B2) refers to a nucleic acid molecule capable of expressing protein SeCYP710A in a host cell. The nucleic acid molecule may include not only a promoter for initiating transcription of protein SeCYP710A gene, but also a terminator for terminating transcription of protein SeCYP710A. Further, the expression cassette may also include an enhancer sequence.

[0041] Available existing plant expression vector construction contains the recombinant expression vector of the protein SeCYP710A gene expression cassette. The plant expression vector includes a binary Agrobacterium vector and a vector that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, pCAMBIA1391-Xb (CAMBIA company), etc. The plant expression vector can also include the 3' end non-translated region of the foreign gene, i.e., include polyadenylic acid signal and any other DNA fragments that participate in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as the non-translated region transcribed at the 3' end of the Agrobacterium crown gall induction (Ti) plasmid gene (such as the nopaline synthase gene Nos) and the plant gene (such as the soybean storage protein gene) all have similar functions. When using the gene of the present invention to construct a plant expression vector, an enhancer can also be used. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding an antibiotic marker gene (such as a gene that confers resistance to the antibiotic hygromycin. Considering the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened by adversity.

[0042] In the above applications, the recombinant microorganism in the biological material can specifically be yeast, bacteria, algae and fungi.

[0043] In order to solve the above technical problems, the present invention also provides a plant agent, which has the following functions:

[0044] W1. Improve plant salt tolerance;

[0045] W2, increase the total content of phytosterols;

[0046] W3. increasing the content of phytosterol and / or stigmasterol;

[0047] W4. Improve the balance of phytosterols;

[0048] W5, promote the synthesis of phytosterols;

[0049] W6. Increase the expression level of SeSMT1 gene and / or SeSMT2 gene in plants;

[0050] W7, promote the synthesis of plant glycoside sterols;

[0051] W8. Increase the expression level of Salicornia herb SeUGT80A2 gene and / or SeUGT80B1 gene.

[0052] The active ingredients of the plant reagent provided by the present invention contain the protein SeCYP710A protein or / and biological materials related to the protein SeCYP710A protein.

[0053] The active ingredients of the above-mentioned botanical agents may also contain other biological ingredients and / or non-biological ingredients. Those skilled in the art may determine the other active ingredients of the above-mentioned botanical agents based on the effect of improving the salt tolerance of plants.

[0054] In order to solve the above technical problems, the present invention also provides a method for cultivating plants with strong salt tolerance. The method for cultivating plants with strong salt tolerance provided by the present invention comprises introducing the coding gene of the protein SeCYP710A into the target plant to obtain the plant with strong salt tolerance; the salt tolerance of the plant with strong salt tolerance is stronger than that of the target plant.

[0055] The present invention also provides a method for improving plant salt tolerance, which comprises enhancing, increasing or up-regulating the activity and / or content of the protein SeCYP710A in the target plant, or / and enhancing, increasing or up-regulating the expression level of the gene encoding the protein SeCYP710A, so as to improve the plant salt tolerance.

[0056] In the above method, the coding sequence of the gene of the protein SeCYP710A is shown as SEQ ID No. 1 in the sequence listing.

[0057] In the above method, the plant may be a plant of the Amaranthaceae family, further may be a plant of the genus Salicornia, and more specifically may be Salicornia europaea L.

[0058] The protein SeCYP710A gene can be introduced into plant cells through conventional biotechnology methods such as Agrobacterium transformation.

[0059] The above-mentioned plants with strong salt tolerance may be transgenic plants or plants obtained through conventional breeding techniques such as hybridization.

[0060] In the above method, the transgenic plant is understood to include not only the first to second generation transgenic plants, but also their progeny. For transgenic plants, the gene can be propagated in the species, and the gene can also be transferred into other varieties of the same species using conventional breeding techniques, especially including commercial varieties. The transgenic plant includes seeds, callus, complete plants and cells.

[0061] The present invention cloned a gene encoding sterol C22-desaturase from the true halophyte Salicornia herba for the first time, and named it SeCYP710A. The full length of CDS is 1536bp, encoding 511 amino acids. The amino acid sequence has a conserved domain of P450 protein. Evolutionary analysis prediction shows that SeCYP710A is closely related to beets, quinoa and spinach, which are also Amaranthaceae plants. Protein subcellular localization shows that SeCYP710A is localized in the endoplasmic reticulum. Under salt deficiency and salt treatment for 12h, the expression level of the SeCYP710A gene in Salicornia herba at the transcriptional level was significantly increased, which was opposite to the expression trend of AtCYP710A1 and AtCYP710A2 in Arabidopsis thaliana under salt. The SeCYP710A gene was overexpressed and silenced in Salicornia in vitro cultured cells. The sterol content and salt resistance analysis of transgenic Salicornia cultured cells showed that compared with the control, under salt stress, the sterol content of overexpressed cells increased and the survival rate was significantly improved under salt; the sterol content of silenced cells decreased and the survival rate was significantly reduced. In particular, SeCYP710A can promote the synthesis of glycosidic sterols. These results show that overexpression of the SeCYP710A gene can improve the salt tolerance of plants, providing theoretical references and gene resources for the cultivation of salt-resistant crops, and also providing ideas and references for the large-scale production of plant sterols using halophyte reactors. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The protein sequence and bioinformatics analysis results of SeCYP710A in Example 1 of the present invention. Figure 1 (a) shows the amino acid sequence alignment of Salicornia SeCYP710A with Arabidopsis AtCYP710A1, AtCYP710A2, AtCYP710A3 and AtCYP710A4. The position marked by the horizontal line is the conserved domain of P450: FGA(W / I / N)GA(P / S)HQCVG. Figure 1Figure (b) shows the results of the evolutionary analysis of the amino acid sequence of SeCYP710A, the amino acid sequence comes from: Salicornia europea (SeCYP710A, MW052511), yeast (Saccharomyces cerevisiae, ScCYP61, P54781), Chlorella sorokiniana (Chlorella sorokiniana, CsCYP450, PRW58849.1), Physcomitrella patens (PpCYP710A1-1 like, XP_024383042.1; PpCYP710A1-2 like, XP_024365247.1), Taxus chinensis (CYP710A78, A0A291FAW7), Zea mays (Zea mays, Zea mays, ZmCYP710A11, A0A3L6FJ92), barley (Hordeum vulgare, HvCYP710A8, G5EKM8), Arabidopsis thaliana (Arabidopsisthaliana, AtCYP710A1, O64697; AtCYP710A2, O64698; AtCYP710A3, Q9ZV29; AtCYP710A4, Q9ZV28), soybean (Glycine max, GmCYP710A1, I1MF42; GmCYP710A2, I1M1E5), tomato (Solanum lycopersicum, SlCYP710A11, Q1T7C2), spinach (Spinacia oleracea, SoCYP701A11-like, XP_021839046.1), sugar beet (Beta vulgaris, BvCYP710A1, XP_010666372.1) and Chenopodiumquinoa, CqCYP710A11-like, XP_021765671.1. Figure 1 Figure (c) is the predicted map of the transmembrane domain of SeCYP710A. Figure 1 Figure (d) shows the subcellular localization of SeCYP710A, where GFP is green fluorescent protein (Green Fluorescent Protein), RFP-HDEL is the fusion expression protein of the endoplasmic reticulum localization marker HDEL (His-Asp-Glu-Leu) and red fluorescent protein (Red Fluorescent Protein), Merge is the combined image of green fluorescent protein and red fluorescent protein, and the scale bar is 10μm.

[0063] Figure 2This is the expression pattern of Salicornia herb and Arabidopsis CYP710A under treatment with different concentrations of NaCl in Example 1 of the present invention. Figure 2 (a) shows the expression changes of SeCYP710A gene of Salicornia herba after treatment with 200 mM NaCl as control (CK), 0 mM and 800 mM NaCl. Figure 2 (b) shows the expression changes of Arabidopsis thaliana AtCYP710A1 gene after treatment with 0 mM NaCl as control (CK) and 150 mM NaCl. Figure 2 (c) The expression changes of Arabidopsis thaliana AtCYP710A2 gene after treatment with 0 mM NaCl as control (CK) and 150 mM NaCl. The data are mean ± SE (n = 4), and different letters on the error bar indicate significant differences compared with the control (CK) at the p < 0.05 level.

[0064] Figure 3 It is the expression level of Salicornia herba SeCYP710A overexpressing cells SeCYP710A OE and silenced cells SeCYP710A RNAi in Example 1 of the present invention. Figure 3 (a) is the expression level of SeCYP710A in the in vitro cultured SeCYP710A overexpressing Salicornia herba cells SeCYP710AOE relative to the in vitro cultured control cells OE-EV overexpressing the empty vector pGWB502Ω. Figure 3 (b) is the expression level of SeCYP710A in the in vitro cultured Salicornia cells SeCYP710A RNAi relative to the in vitro cultured control cells RNAi-EV transfected with the RNAi empty vector PU1301. Among them, the internal reference is the Salicornia SeTubulin gene, the data are mean ± SE (n = 4), and the * on the error line indicates a significant difference compared with the control at the p < 0.05 level.

[0065] Figure 4 The sterol content and other related gene expression levels of Salicornia herba SeCYP710A overexpressing cells SeCYP710A OE and silenced cells SeCYP710A RNAi in Example 1 of the present invention. Figure 4(a) is the phytosterol content in SeCYP710A overexpression cells SeCYP710A OE and silenced cells SeCYP710A RNAi in vitro culture, OE-EV is the empty control of the overexpression vector; RNAi-EV is the empty control of the silenced vector. campesterol, campesterol; sitosterol, sitosterol; stigmasterol, stigmasterol; SG, steryl glycosides; ASG, acylsteryl glycosides; FS, free sterols; SE, steryl esters; data are mean ± SE (n = 3), different letters on the error bar indicate significant differences compared with the control at the p < 0.05 level. Figure 4 (b) is the expression levels of SeSMT1, SeSMT2, SeUGT80A2 and SeUGT80B1 in SeCYP710A OE cells and SeCYP710A RNAi silenced cells cultured in vitro. EV, in vitro cultured overexpression or silenced empty vector cell control, where the gene expression levels in the overexpression empty vector and silenced empty vector have been normalized. The internal reference is Salicornia SeTubulin, data are mean ± SE (n = 4), and the * on the error bar indicates a significant difference at the p < 0.05 level compared with the control.

[0066] Figure 5 The survival rates of SeCYP710A overexpressing cells SeCYP710A OE and silenced cells SeCYP710A RNAi in Example 1 of the present invention at different NaCl concentrations, OE-EV is an empty control for the overexpression vector; RNAi-EV is an empty control for the silenced vector. Figure 5 (a) is the survival rate at 0 mM NaCl concentration, Figure 5 (b) is the survival rate at 200 mM NaCl concentration, Figure 5 (c) is the survival rate at a concentration of 800 mM NaCl, where FDA means that cells are stained with fluorescein diacetate, and cells filled with green fluorescence are living cells; PI means that cells are stained with propidium iodide, and cells with red nuclei are dead cells; Merge means the combined image of red fluorescence and green fluorescence. Figure 5 (d) is the quantitative in vitro culture cell survival rate, the scale bar is 50 μm, the data are mean ± SE, n ≥ 20, and the different letters on the error bar represent that the survival rates of different cells under the same concentration of NaCl treatment are significantly different at the p < 0.05 level. DETAILED DESCRIPTION

[0067] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0068] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are conventional biochemical reagents and can be obtained from commercial sources unless otherwise specified.

[0069] 2 Plant strains

[0070] In the following examples, the Salicornia europaea L. seeds used were purchased from Jiangsu Jinglong Marine Industry Development Co., Ltd., and the harvest site was located in Dafeng City, Jiangsu Province.

[0071] In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

[0072] Example 1

[0073] This application cloned the gene encoding sterol C22-desaturase (SeCYP710A gene, encoding protein SeCYP710A) from Salicornia herba for the first time:

[0074] 1. Full-length CDS cloning of SeCYP710A gene

[0075] Trizol was used to extract a small amount of total RNA from the aboveground part of Salicornia herba, and the first-strand cDNA was obtained using a reverse transcription kit (Quanshijin, Beijing). The Unigene2485_SALfmcTARAAPEI-3 sequence in the Salicornia herba transcriptome was homologously compared on the NCBI website to determine that the known sequence contained a complete open reading frame (ORF). The forward primer SeCYP710A-F and the reverse primer SeCYP710A-R were designed respectively, and the primer sequences are as follows:

[0076] SeCYP710A-F: 5'-ATGAGCTTTGTTTTATCCGAAATTTGGG-3';

[0077] SeCYP710A-R: 5'-TCAAGGGAGAGAGAAGGACGGGAAT-3'.

[0078] The full length was amplified by one round of PCR using PrimeSTAR (Takara) with the cDNA of Salicornia herba as a template and the forward primer and reverse primer mentioned above.

[0079] The reaction system is: dNTP 4μL, 5× PrimeSTAR Buffer (Mg 2+ plus) 10 μL, PrimeSTAR HS DNA Polymerase 0.5 μL, SeCYP710A-F 1 μL, SeCYP710A-R 1 μL, cDNA 2-5 μL, add water to make up to 50 μL.

[0080] The reaction conditions are as follows: PCR reaction at 98°C, 5 min; 4 cycles: 98°C, 10 sec, 50°C, 15 sec, 72°C, 1 min; 4 cycles: 98°C, 10 sec, 51°C, 15 sec, 72°C, 1 min; 4 cycles: 98°C, 10 sec, 52°C, 15 sec, 72°C, 1 min; 18 cycles: 98°C, 10 sec, 53°C, 15 sec, 72°C, 1 min; 72°C, 10 min.

[0081] The obtained full-length cDNA was ligated into -T1 Simple Cloning vector (Quanshijin, Beijing), and obtained pEASY-SeCYP710A vector, which was sequenced and confirmed.

[0082] The sequencing results showed that the amplified CDS sequence contained a 1536 bp ORF, which was labeled as SeCYP710A (the nucleotide sequence is shown in SEQ ID No. 1), encoding a 511 amino acid protein, the encoded protein is labeled as SeCYP710A (the amino acid sequence is shown in SEQ ID No. 2), see Figure 1 (a).

[0083] The results of evolutionary analysis are shown in Figure 1 (b) found that SeCYP710A is more closely related to beets, quinoa and spinach, which are also Amaranthaceae plants. SeCYP710A is a sterol C-22 desaturase with characteristics of the cytochrome P450 superfamily. There is a highly conserved cysteine ​​residue in the fifth coordination site that is involved in binding heme iron, which is conserved in the C-terminal region of P450 proteins. The protein it encodes is predicted to have a transmembrane domain of 7-29aa, see Figure 1 (c).

[0084] 2. Gene expression analysis under salt treatment

[0085] 2.1 Analysis of SeCYP710A gene expression in Salicornia herba under salt treatment

[0086] Wild-type Salicornia seeds were purchased from Jiangsu Jinglong Ocean Industry Development Co., Ltd., harvested in Dafeng City, Jiangsu Province, and sown and cultivated in the greenhouse of the Institute of Botany, Chinese Academy of Sciences. Salicornia seeds were sown in 7 cm × 7 cm pots (nutrient soil: vermiculite, 1:1), and irrigated with 1 / 2 Hoagland nutrient solution one week after germination. After two weeks of growth, they were watered with 1 / 2 Hoagland nutrient solution containing 200 mM NaCl (the optimal growth environment for Salicornia) and treated with 1 / 2 Hoagland nutrient solution containing 0 mM, 200 mM and 800 mM NaCl three weeks later. The aboveground materials were taken for 0, 6 h, and 12 h treatment, respectively, for gene expression detection.

[0087] Total RNA was extracted using Trizol reagent and reverse transcribed using a reverse transcription kit (Quanshijin, Beijing) and THUNDERBIRD qPCR mix (Toyobo, Japan) was used for fluorescence quantitative qRT-PCR detection. -ΔΔCt The gene expression was relatively quantified by this method.

[0088] The primer sequences used in fluorescent quantitative qRT-PCR are as follows:

[0089] Salicornia internal standard Tubulin primers:

[0090] SeTubulin-qPCR-F: 5'-CAGTGCCTTTGAGCCATCTTC-3';

[0091] SeTubulin-qPCR-R: 5'-CTGAATGGTTCGCTTGGTCTT-3'.

[0092] Salicornia SeCYP710A primers:

[0093] SeCYP710A-qPCR-F: 5'-GTTTGGGAAGAAGTTGTTTGGCGAG-3';

[0094] SeCYP710A-qPCR-R: 5'-CTTTACTAAGATAAGGACCTGCGAA-3'.

[0095] The results of fluorescence quantitative qRT-PCR detection are shown in Figure 2Figure (a) shows the expression level of SeCYP710A gene in the aerial part of Salicornia herba cultivated under optimal conditions under salt deficiency stress (0mM NaCl) and salt stress (800mM NaCl): Under salt deficiency treatment (0mM NaCl), SeCYP710A gene was significantly upregulated at 6h and 12h, which were 138 times and 50 times that of the control (200mMNaCl), respectively; after 12h of salt stress treatment (800mM NaCl), the expression level of SeCYP710A increased to 44 times that of the control (200mMNaCl).

[0096] 2.2 Expression analysis of AtCYP710A1 and AtCYP710A2 genes in Arabidopsis thaliana under salt treatment

[0097] The wild type Arabidopsis thaliana was the Columbia wild type Col-0, which is recorded in the non-patent literature "Nordborg M, Hu TT, Ishino Y, et al. The pattern of polymorphism in Arabidopsis thaliana. PLoS biology, 2005, 3, e196.", and was harvested in the artificial climate chamber of the Institute of Botany, Chinese Academy of Sciences. The harvested and dried Arabidopsis seeds were soaked in 70% ethanol for 1 min, and then soaked in 1% sodium hypochlorite for 15 min after the ethanol was sucked out. After the sodium hypochlorite was sucked out, it was washed 3-5 times with deionized distilled water, and then the seeds were spread on 1 / 2MS solid culture medium (Murashige and Skoog, 1962). After vernalization at 4°C for 3 days in the dark, they were placed in a 22°C culture room and cultured under the light conditions of 16h light / 8h dark. One week after germination, the plants were treated with 1 / 2 Hoagland nutrient solution containing 0 mM and 150 mM NaCl, and the aboveground materials were taken at 0, 6 h, and 12 h of treatment for gene expression detection.

[0098] Total RNA was extracted using Trizol reagent and reverse transcribed using a reverse transcription kit (Quanshijin, Beijing) and THUNDERBIRD qPCR mix (Toyobo, Japan) was used for fluorescence quantitative qRT-PCR detection. Arabidopsis thaliana AtActin gene was used as an internal reference. -ΔΔCt The gene expression was relatively quantified by this method.

[0099] The primer sequences used in fluorescent quantitative qRT-PCR are as follows:

[0100] Arabidopsis thaliana internal standard AtActin primers:

[0101] AtActin-qPCR-F: 5'-ATATGCCTATCTACGAGGGTT-3'

[0102] AtActin-qPCR-R: 5'-ATACAATTTCCCGTTCTGCTGT-3'

[0103] Arabidopsis thaliana AtCYP710A1 Primers:

[0104] AtCYP710A1-qPCR-F: 5'-TCTATGGCCGCTCCCGATCAACCTGC-3'

[0105] AtCYP710A1-qPCR-R: 5'-TTCGGAGTGAAGCGGCGGTGGATTA-3'

[0106] Arabidopsis thaliana AtCYP710A2 Primers:

[0107] AtCYP710A2-qPCR-F: 5'-GCCTCTTCGGTGACCACAACCTTATA-3'

[0108] AtCYP710A2-qPCR-R: 5'-AGGTAGGGTCCAACAAAAACCGTCT-3'

[0109] The results of fluorescence quantitative qRT-PCR showed that under salt stress (150 mM NaCl), AtCYP710A1 (see Figure 2 (b)) and AtCYP710A2 (see Figure 2 (c)) The expression levels in the aerial part were reduced to 0.57 and 0.4 times of the control (0 mM NaCl) after 12 h.

[0110] This indicates that Arabidopsis AtCYP710A1 and AtCYP710A2 are downregulated after being induced by salt, while Salicornia SeCYP710A is upregulated after being induced by salt deficiency and salt treatment.

[0111] 3. Protein subcellular localization

[0112] 3.1 Construction of subcellular localization vector

[0113] Choose pENTRY TM Directional TOPO entry vector (Invitrogen, K240020), design primer NotI-SeCYP710A-651-Entry-F:

[0114] 5'-ATAAGAAT GCGGCCGCCCCCTTCACCATGAGCTTTGTTTTATCCGAAATTTGGG-3' (the underline indicates the Not I recognition sequence);

[0115] Asc I-SeCYP710A-651-Entry-R:

[0116] 5'-A GGCGCGCC CACCCTTTCAAGGGAGAGAGAAGGACGGGAAT-3′ (the underlined sequence indicates the Asc I recognition sequence).

[0117] PCR amplification was performed using the pEASY-SeCYP710A plasmid in step 1 as a template, Not I-SeCYP710A-651-Entry-F and Asc I-SeCYP710A-651-Entry-R as primers, and EasyPfu DNA Polymerase (Quanshijin, Beijing).

[0118] The recovered PCR products were mixed with pENTRY TM Directional TOPO entry vector ligation, construction of pENTRY TM D-TOPO-SeCYP710A vector was sequenced and confirmed.

[0119] PENTRY TM The D-TOPO-SeCYP710A plasmid was recombined with the GFP vector pGWB651 (Nakagawa et al., 2007) by homologous recombination enzyme Gateway TM LR Clonase TM ⅡEnzyme Mix and constructed the pGWB651-35S::SeCYP710A fusion vector, which expresses SeCYP710A and GFP fusion protein.

[0120] At least two pairs of restriction sites were selected by SnapGene Viewer software to verify the restriction digestion of the fusion vector. Products of 11858 bp and 303 bp were obtained by digestion with Sal I and BamH I, and products of 10762 bp and 1399 bp were obtained by digestion with EcoR V and BamH I. The results verified that the gene fragment was correctly inserted into the vector.

[0121] 3.2 Subcellular localization of SeCYP710A protein

[0122] The pGWB651-35S::SeCYP710A vector and endoplasmic reticulum marker (RFP-HDEL) were transformed into C58 Agrobacterium (Beijing Huayueyang Biological Company, huayueyang1728s). Agrobacterium was cultured overnight in LB liquid medium containing 20 μM acetosyringone, 10 mM MES, 100 μM kanamycin and 100 μM rifampicin with shaking (28°C, 200 rpm) until OD 600 =1.8~2.0. After centrifugation, the cells were collected and resuspended in distilled water containing 200μM AS, 10mM MgCl2, and 10mM MES to OD 600 =1.5, let stand for 6h. Mix the two resuspended bacterial solutions in a ratio of 1:1. Use a syringe to inject the bacterial solution into the leaves of Nicotiana benthamiana (recorded in the non-patent literature "Nicotiana benthamiana, Chen X, Bao H, Guo J, et al. Na+ / H+exchanger1participates in tobacco disease defence against Phytophthora parasitica var. nicotianae by affecting vacuolar pH and priming the antioxidative system. Journal of experimental botany, 2014, 65: 6107-6122."). After the tobacco is cultured normally for 2d to 3d, the subcellular localization of the protein is observed using a laser scanning confocal microscope. The GFP excitation light wavelength in the protein subcellular localization vector is 488nm, and the emission light is 500-580nm. The mCherry excitation light wavelength on the endoplasmic reticulum marker vector is 543nm, and the emission light is 580-630nm.

[0123] Protein subcellular localization results Figure 1 As shown in (d), the green fluorescence of the fusion protein of Salicornia herb SeCYP710A and GFP overlaps with the red fluorescence of the endoplasmic reticulum marker protein (HDEL-mCherry), indicating that SeCYP710A is localized in the endoplasmic reticulum.

[0124] 4 Obtaining transgenic Salicornia in vitro cultured cells

[0125] 4.1 Construction of gene expression vector (OE vector)

[0126] Choose pENTRY TM Directional TOPO entry vector (Invitrogen, K240020), designed primers as follows:

[0127] Not I-SeCYP710A-502-Entry-F:

[0128] 5'-ATAAGAAT GCGGCCGC CCCCTTCACCATGAGCTTTGTTTTATCCGAAATTTGGG-3' (the underline indicates the Not I recognition sequence);

[0129] Asc I-SeCYP710A-502-Entry-R:

[0130] 5'-A GGCGCGCC CACCCTTAGGGAGAGAGAAGGACGGGAAT-3' (the underlined sequence indicates the Asc I recognition sequence).

[0131] PCR amplification was performed using the pEASY-SeCYP710A plasmid in step 1 as a template, Not I-SeCYP710A-502-Entry-F and Asc I-SeCYP710A-502-Entry-R as primers, and EasyPfu DNA Polymerase (Quanshijin, Beijing).

[0132] The recovered PCR products were mixed with pENTRY TM Directional TOPO entry vector ligation, construction of pENTRY TM D-TOPO-SeCYP710A vector was sequenced and confirmed.

[0133] PENTRY TM The D-TOPO-SeCYP710A plasmid was recombined with the expression vector pGWB502Ω (Nakagawa et al., 2007) by homologous recombination enzyme Gateway TM LR Clonase TM ⅡEnzyme Mix was connected to construct the pGWB502Ω-35S::SeCYP710A fusion vector. At least two pairs of restriction sites were selected by SnapGene Viewer software to verify the restriction digestion of the fusion vector. The products of 9655bp and 2249bp were obtained by Sma I and BamH I, and the products of 8159bp and 3745bp were obtained by BsaΙ and HindШ. The results verified that the gene fragment was correctly inserted into the vector.

[0134] 4.2 Construction of gene silencing vector (RNAi vector)

[0135] Forward primer of Salicornia herba SeCYP710A gene:

[0136] KpnΙ-SeCYP710A-forward-F:

[0137] 5'-CGGGGTACCCCCGTTTGGGAAGAAGTTGTTTGGC-3'

[0138] HindШ-SeCYP710A-forward-R:

[0139] 5'-CCCAAGCTTTCATGTCACGAGCTAGTAACCGCAG-3'

[0140] Reverse primer of Salicornia herba SeCYP710A gene:

[0141] EcoRΙ-SeCYP710A-reverse-F:

[0142] 5'-CCGGAATTCTCATGTCACGAGCTAGTAACCGCAG-3';

[0143] XbaΙ-SeCYP710A-reverse-R:

[0144] 5'-GCTCTAGACCCGTTTGGGAAGAAGTTGTTTGGC-3'.

[0145] Using the pEASY-SeCYP710A plasmid in step 1 as a template, KpnΙ-SeCYP710A-forward-F and HindШ-SeCYP710A-forward-R were used as primers for cloning the forward fragment, EcoRΙ-SeCYP710A-reverse-F and XbaΙ-SeCYP710A-reverse-R were used as primers for cloning the reverse fragment, and EasyPfu DNA Polymerase (Quanshijin, Beijing) was used to PCR amplify the forward and reverse fragments of SeCYP710A, and KpnΙ and HindШ restriction sites were introduced at both ends of the forward target fragment, and EcoRΙ and XbaΙ restriction sites were introduced at both ends of the reverse target fragment.

[0146] The plasmid of the forward fragment gel recovery product and the empty vector pBIntron (recorded in the non-patent literature "Liu Y, Cui S, Wu F, et al. Functional conservation of MIKC*-Type MADS box genes in Arabidopsis and rice pollen maturation. Plant Cell, 2013, 25: 1288-1303.") was double-digested with Kpn Ι and HindШ (NEB, New England). The double-digested product was electrophoresed, gel-recovered, and the vector and the forward fragment were connected with T4 DNA Ligase (Quanshijin, Beijing) to obtain the pBIntron vector containing the forward fragment. It was gel-recovered with the reverse fragment and double-digested with EcoRΙ and XbaΙ (NEB, New England). The double-digested product was electrophoresed, gel-recovered, and the vector and the reverse fragment were connected with T4 DNA Ligase (Quanshijin, Beijing) to obtain the pBIntron vector with the forward and reverse fragments connected on both sides of Intron. It and the pU1301 empty vector (recorded in the non-patent literature "Lv S, Tai F, Guo J, et al. Phosphatidylserine synthase from Salicornia europaea is involved in plantsalt tolerance by regulating plasma membrane stability. Plant and Cell Physiology, 2021, 62: 66-79.") were double-digested with KpnΙ and SacΙ (NEB, New England). The double-digested product was electrophoresed, gel recovered, and T4 DNA Ligase (Quanshijin, Beijing) was used to connect the PU1301 vector and the Intron fragment with forward and reverse fragments to construct a silencing vector for SeCYP710A. At least two pairs of restriction sites were selected by SnapGene Viewer software, and the fusion vector was verified by restriction digestion. The products of 10200 bp, 2025 bp and 1094 bp were obtained by digestion with Xho I and BamH I, and the products of 12597 bp, 1530 bp and 252 bp were obtained by digestion with BamH I and HindШ. The results verified that the gene fragment was correctly inserted into the vector.

[0147] 4.3 Obtaining and screening transgenic Salicornia in vitro cultured cells

[0148] The pGWB502Ω-35S::SeCYP710A vector and the silencing vector of SeCYP710A were respectively transformed into C58 Agrobacterium (Beijing Huayueyang Biological Company, huayueyang1728s). Agrobacterium was cultured overnight with shaking in LB liquid medium containing 10 mM magnesium chloride, 200 μM acetosyringone, 10 mM MES (pH=5.7), 100 μM kanamycin and 100 μM rifampicin until OD600=1-1.5.

[0149] Take fresh wild type (WT) callus of Salicornia herba, crush it into small pieces of 0.01-0.02g with tweezers, put it into a 250mL conical flask, pour in Agrobacterium shaken overnight, shake the conical flask to completely immerse the callus, and then put it into a vacuum pump to evacuate to -0.8kg / cm 2 After that, restore the normal atmospheric pressure and repeat the pumping 7-8 times. Filter the mixture of bacterial solution and callus with sterilized gauze, and transfer the filtered callus to callus induction medium for co-cultivation. After culturing at 26°C in the dark for 3 days, transfer the callus on the medium to callus induction medium containing 100 mg / L timentin and culture at 26°C in the dark for 7 days to inhibit the growth of Agrobacterium.

[0150] The callus tissue after inhibiting the growth of Agrobacterium was transferred to a callus tissue culture medium supplemented with 100 mg / L timentin and 6.25 mg / L hygromycin for preliminary screening. After 30 days, it was transferred to a callus tissue culture medium supplemented with 100 mg / L timentin and 12.5 mg / L hygromycin for formal screening and cultured in a 26°C culture room away from light. Among them: the in vitro cultured OE cells of SeCYP710A obtained by transferring the pGWB502Ω-35S::SeCYP710A vector were called SeCYP710A OE, and the control was the in vitro cultured Salicornia herba cells obtained by transferring the empty vector pGWB502Ω, called OE-EV; the in vitro cultured RNAi cells of SeCYP710A obtained by transferring the SeCYP710A silencing vector were called SeCYP710A RNAi, and the control was the in vitro cultured Salicornia herba cells obtained by transferring the empty vector PU1301, called RNAi-EV. The above four in vitro cultured cells were transferred to new screening medium every 30 days or so, and the gene expression levels of callus tissue were detected about 3 months later to confirm the success of gene transformation. The gene expression detection of transgenic Salicornia in vitro cultured cells was as follows:

[0151] SeCYP710A OE, its control OE-EV, SeCYP710A RNAi, and its control RNAi-EV were taken for gene expression measurement. A small amount (about 100 ng) of each cell was taken to extract total RNA, which was reverse transcribed and analyzed using THUNDERBIRD qPCR mix (Toyobo, Japan) was used for fluorescence quantitative RT-PCR detection. -ΔΔCt The gene expression was relatively quantified by the method. The primer sequences used in the fluorescence quantitative RT-PCR were the same as those in step 2.1. The primers for internal standard Tubulin of Salicornia herba consisted of SeTubulin-qPCR-F and SeTubulin-qPCR-R, and the primers for SeCYP710A of Salicornia herba consisted of SeCYP710A-qPCR-F and SeCYP710A-qPCR-R.

[0152] The expression level of the transformed gene in the in vitro cultured OE cells (SeCYP710A OE) of SeCYP710A obtained after screening was 22 times that of the control with no OE vector (OE-EV). Figure 3 (a), which indicates that the SeCYP710A gene in the Salicornia cells SeCYP710A OE has been transferred into the Salicornia cells cultured in vitro and can be stably expressed.

[0153] In the in vitro cultured RNAi cells of SeCYP710A (SeCYP710A RNAi), the expression level of SeCYP710A (the silenced gene) also decreased to 14% of the RNAi empty vector control (RNAi-EV), as shown in Figure 3 (b), which indicates that the SeCYP710A gene has been successfully silenced in the SeCYP710A RNAi of Salicornia cells.

[0154] 5. Determination of sterol content in transgenic Salicornia herba cells cultured in vitro

[0155] In plants, sterols exist in free and conjugated forms. Among the types of conjugated sterols, in addition to the steryl esters (SE) also found in animal sterols, plants also have two types of glycosidic sterols, steryl glycosides (SG) and acyl steryl glycosides (ASG). However, in previous studies on the sterol content of plants, only the changes in the content of FS and SE were generally focused on, which may also be caused by the low content of glycosidic sterols in most plants. SG and ASG only account for a small part of the sterols in most plants, but they are similar to the sterol types of FS. In most plants, the sterol types are mainly sitosterol, and they mainly exist in free form. The inventors previously determined the sterol content of Salicornia europaea L. and found that it contained a large amount of sitosterol and stigmasterol at the same time, and they mainly existed in glycosidic form.

[0156] Take transgenic Salicornia cells (SeCYP710A OE obtained in step 4, its control OE-EV, SeCYP710ARNAi, its control RNAi-EV), add chloroform containing 0.01% butylated hydroxytoluene: methanol = 2:1 to extract lipids, ice-bath the centrifuge tube for 10 minutes, vortex mix again, and centrifuge for 15 minutes. Take the supernatant to a new tube and repeat the above steps, combine the two supernatants. Add 0.75% KCl solution, vortex mix, and centrifuge for 15 minutes. Pipette the chloroform phase below into a new tube and blow dry with nitrogen. Dissolve it with a small amount of chloroform and methanol (chloroform: methanol = 2:1), spot it on a high-performance thin layer chromatography silica gel plate (HSGF254, Yantai Jiangyou Silica Gel Development Co., Ltd.), and put it into a chromatography cylinder with an extender (dichloromethane: methanol: water = 85:15:0.5). After the sample run, the silica gel plate was sprayed with primulin (R054922, RHAWN / Shanghai Yien Chemical Technology Co., Ltd.) and the color was developed under a UV lamp with a UV wavelength of 340 nm. The silica gel powder of steryl glycoside (SG, Rf 0.35), acylated steryl glycoside (ASG, Rf 0.55), free sterol (FS, Rf 0.7) and steryl ester (SE, Rf 0.9) was scraped off, and FS and SE were eluted from the silica gel powder with dichloromethane, and SG and ASG were eluted with a solution of dichloromethane: methanol = 4:1.

[0157] 5α-cholestane (C8003, Sigma) was added to each eluate as an internal standard, and β-sitosterol (SS8580, Solarbio), stigmasterol (SS8710, Solarbio) and campesterol (SC8960, Solarbio) were used as the standards for sitosterol, stigmasterol and campesterol. The eluted SE was added with potassium hydroxide-ethanol solution and reacted at 70℃ for 2h; the eluted SG was added with hydrochloric acid-ethanol solution and reacted at 80℃ for 1h; the eluted ASG was first added with hydrochloric acid-ethanol solution and reacted at 80℃ for 1h, and then potassium hydroxide-ethanol solution was added and reacted at 70℃ for 2h. The above reaction products were extracted three times with equal volumes of n-hexane, and the extract and the eluate of FS were dried with a nitrogen blower and then added with N,O-bis(trimethylsilyl)trifluoroacetamide (containing trimethylchlorosilane) (15238, Sigma) for derivatization reaction at 80℃ for 1h. After the product was concentrated, it was subjected to GC-MS analysis. The instrument was Agilent 7890A / 8000B triple quadrupole gas chromatograph (GC-QQQ / MS), the chromatographic column was DB-5MS, the injection port temperature was 250°C, the injection volume was 1 μL, the split ratio was 10:1, the carrier gas was helium (He), and the flow rate was 1.2 mL / min.

[0158] The test results are detailed in Figure 4(a): The total content of sterols in various chemical forms in the in vitro cultured cells SeCYP710AOE overexpressing Salicornia SeCYP710A was 3.39 times that of the overexpressed empty cell control OE-EV, the total content of campesterol was reduced to nearly half of the control, and sitosterol and stigmasterol increased to 3 and 5 times of the control. The total content of sterols in the in vitro cultured SeCYP710A silenced cells SeCYP710A RNAi was reduced to 28.3% of the silenced empty control cells RNAi-EV, and campesterol, sitosterol and stigmasterol were reduced to 42%, 38% and 11% of the control, respectively.

[0159] In particular, Figure 4 As shown in (a), in Salicornia cells, glycosidic sterols (SG+ASG) have more significant changes when SeCYP710A is overexpressed and silenced than the sterols in most plants, which are in the free and ester forms (FS+SE). In connection with the changes in sterol content, this shows that when sterols are excessive, the content of glycosidic sterols increases more; when sterols are missing, the content of glycosidic sterols also decreases more. This suggests that in Salicornia, the balance of plant sterols mainly depends on glycosidic sterols.

[0160] At the same time, in order to further verify that SeCYP710A affects the main types of plant sterols other than stigmasterol and the content of glycosidic sterols, the inventors detected the gene expression of SeSMT1 and SeSMT2 in SeCYP710A overexpressing cells SeCYP710A OE and silenced cells SeCYP710A RNAi, and also detected the expression levels of gene members SeUGT80A2 and SeUGT80B1 encoding sterol glycosyltransferase SGT in Salicornia herba. SeSMT1 and SeSMT2 are members of SeSMT (sterol methyltransferase), a key gene in the sterol synthesis pathway of Salicornia herba. SeUGT80A2 and SeUGT80B1 are members of SeSGT (sterol glycosyltransferase), a key gene in Salicornia herba that catalyzes the synthesis of glycosidic sterols. Using Salicornia herba SeTubulin as an internal reference, the expression levels of each gene were detected. The primers used are as follows:

[0161] SeTubulin-qPCR-F:

[0162] 5'-CAGTGCCTTTGAGCCATCTTC-3'

[0163] SeTubulin-qPCR-R:

[0164] 5'-CTGAATGGTTCGCTTGGTCTT-3'

[0165] SeSMT1-qPCR-F:

[0166] 5'-CTGGTGTTGGCGGAAAGATCGAGAA-3'

[0167] SeSMT1-qPCR-R:

[0168] 5'-GTGCTCATGGCGTTTGATGCTCTCA-3'

[0169] SeSMT2-qPCR-F:

[0170] 5'-CTGTACAGCCGGTCTCCTTTTCGCC-3'

[0171] SeSMT2-qPCR-R:

[0172] 5'-GGAAGCTCTGACCCCAGCCCCACTC-3'

[0173] SeUGT80A2-qPCR-F:

[0174] 5'-TCCACATCCCCTCTCGCGTGTTAAG-3'

[0175] SeUGT80A2-qPCR-R:

[0176] 5'-CCCCAATCTTTAGGTTTAGGAA-3'

[0177] SeUGT80B1-qPCR-F:

[0178] 5'-AAGATTGGGGTCCTGAAGTTGATGT-3'

[0179] SeUGT80B1-qPCR-R:

[0180] 5'-AACACCAAGGTCTCCCCAACCACGA-3'

[0181] Results Figure 4 (b) SeCYP710A overexpression significantly upregulated the expression of other key genes involved in phytosterol biosynthesis and phytosterol glycosylation. Correspondingly, SeCYP710A silencing also significantly inhibited the expression of other genes. This indicates that SeCYP710A and other key genes in the sterol synthesis pathway work synergistically and can affect the expression changes of genes that catalyze the conversion of sterols from free state to glycoside state. This result also verifies for the first time that SeCYP710A can affect the synthesis of glycoside sterols.

[0182] 6. Salt tolerance test of transgenic Salicornia in vitro cultured cells

[0183] Salt tolerance is expressed by the survival rate of cells under salt. Through fluoresceindiacetate (FDA)-propidium iodide (PI) staining, FDA can be dispersed on the cell membrane of living cells and pass through the cell membrane to enter the cell interior to emit green fluorescence, while PI can penetrate the damaged cell membrane and form a bright red fluorescent complex on the cell nucleus, thereby marking living cells and dead cells respectively (Jones and Senft, 1985).

[0184] SeCYP710A OE cells, its control OE-EV, SeCYP710A RNAi, and its control RNAi-EV were treated with cell culture medium containing 0mM, 200mM, and 800mM NaCl for 12h, and then 5μM FDA and 10μM PI were added to stain the cells at room temperature in the dark for 40min. The cell survival status was observed under a 20x objective lens of a two-photon laser confocal microscope (Olympus, FV1000), and counted using ImageJ. The excitation and emission wavelengths of FDA were 488 and 500-530nm, and those of PI were 543 and 600-750nm.

[0185] The results are as follows Figure 5 As shown, the cell survival rate decreased with the increase of NaCl concentration, and at different NaCl concentrations, the survival rate of SeCYP710A overexpressing cells SeCYP710A OE was significantly higher than that of the overexpressing empty control OE-EV, and the silenced cells SeCYP710A RNAi were significantly lower than those of the silenced empty control RNAi-EV. Especially at 800mM NaCl, the survival rate of SeCYP710A overexpressing cells SeCYP710A OE was 89.83%, and the survival rate of silenced cells SeCYP710A RNAi was 26.74%. This result shows that the overexpression of the SeCYP710A gene improves the salt tolerance of Salicornia in vitro cultured cells, proving for the first time that SeCYP710A is involved in the salt tolerance response of Salicornia.

[0186] Stigmasterol is generally considered a "stress sterol", but its content in most plants is not high. This application cloned the gene SeCYP710A encoding sterol C22 desaturase from Salicornia for the first time. Amino acid sequence analysis of the protein SeCYP710A encoded by it found that it is more closely related to beets, quinoa and spinach, which are also Amaranthaceae plants. Subcellular localization observations using the tobacco transient expression system proved that the SeCYP710A protein was localized in the endoplasmic reticulum, which is consistent with reports that sterols are synthesized in the endoplasmic reticulum and then transported to the plasma membrane. Under salt deficiency and salt stress treatments, the expression level of SeCYP710A in the aboveground part of Salicornia increased significantly, which is opposite to the expression trend of the homologous gene induced by salt in Arabidopsis. The SeCYP710A gene was overexpressed in the Salicornia callus transformation system, and it was found that the sterol content of the transgenic Salicornia increased, especially the synthesis of glycosidic sterols was promoted. It is worth noting that in previous research reports, sterols in most plants exist mainly in free and ester forms, with very few glycosidic sterols. After the gene catalyzing glycosidic sterol synthase in Arabidopsis thaliana mutated, the plant became sensitive to temperature, and overexpression increased resistance, indicating that glycosidic sterols are helpful for plants to respond to adversity. This also verifies the findings of this study that overexpressing SeCYP710A Salicornia herba with increased glycosidic sterol content increased the survival rate in saline environments.

[0187] In summary, Salicornia herba SeCYP710A may maintain the balance of sterol content through the storage pool of glycosidic sterols: when the sterol content is too high, Salicornia herba converts free sterols into glycosidic sterols as a sterol storage pool; when the sterol content is too low, Salicornia herba hydrolyzes the glycosidic sterols in the storage pool. At the same time, by maintaining the balance of sterol content in the glycosidic sterol storage pool, SeCYP710A helps Salicornia herba resist saline environments. The gene and functional analysis of Salicornia herba SeCYP710A are expected to play a role in crop stress resistance genetic breeding, providing genetic references and theoretical support for the cultivation of salt-resistant crops; more importantly, as a key gene in the plant sterol synthesis pathway, its expression can be regulated through genetic engineering to regulate the synthesis and accumulation of plant sterols, promote the synthesis of glycosidic sterols, and provide guidance for the production and extraction of sterols using bioreactors.

[0188] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

[0189] sequence:

[0190] SEQ ID No.1:

[0191]

[0192]

[0193] SEQ ID No.2:

[0194] MSFVLSEIWVILKFGAPYLATLLALLVLIEQIKYLRRKSVVPGPTLVFPFLGNGIGLIR

[0195] NPTKFWDDQSDYAKSSSLGISVNYIIGKFILFIRSELSHKIFSNVRPDAFQLVGHP

[0196] FGKKLFGEHNMIYMYGQEHKDLRRRIVPNFTPKALATYCEIQQHVIVKHLKQWCE

[0197] NGSNPIKLRLLARDMNLETSQNVFAGPYLSKEARERFKVDYNLFNVGVLALPIDLP

[0198] GFAFRNARMAVDRLVETLTGCVEESKMKMSGSVEEEPKCLTDDFWMQELIREEK

[0199] ESGSETHAPPPNCSNRQIGGHVFDFLFAAQDASTSSLLWAVTLLDAHPDVLDKV

[0200] RHEVESVWDPESDSMITAEQLARMKYTQAVAREVVRVRAPATLVPHVAMEDFPL

[0201] TQDYTIQKGTIVFPSVYESSFQGFSDPERFDPDRFYSEERREEQLYKRNFLVFGA

[0202] GAHQCVGQRYALNLLVLFIAMFATLVDFKRHRSEGCDDLAFCPTICPKDDCVVSL

[0203] SSRTIKFPSFSLP

Claims

1. Protein, characterized in that: The protein is protein SeCYP710A, and the protein SeCYP710A is the following protein A1), A2) or A3): A1) a protein whose amino acid sequence is SEQ ID No. 2; A2) a protein derived from A1) or having more than 80% identity with the protein shown in A1) and having the same function, obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in A1); A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2).

2. The use according to claim 1, characterized in that: The protein SeCYP710A is derived from Salicornia europaea L.

3. Biomaterial, characterized in that: The biological material is any one of the following B1) to B5): B1) a nucleic acid molecule encoding the protein SeCYP710A according to claim 1 or 2; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3).

4. The biomaterial according to claim 3, characterized in that: B1) The coding sequence of the nucleic acid molecule is shown in SEQ ID No. 1 in the sequence listing.

5. A plant agent, characterized in that: The plant agent contains the protein SeCYP710A according to claim 1 or 2 and / or the biological material according to claim 3 or 4.

6. A method for cultivating plants with strong salt tolerance, characterized in that: The method comprises introducing the gene encoding the protein SeCYP710A according to claim 1 or 2 into a target plant to obtain a plant with strong salt tolerance; the salt tolerance of the plant with strong salt tolerance is stronger than that of the target plant.

7. A method for improving plant salt tolerance, characterized in that: The method comprises enhancing, increasing or up-regulating the activity and / or content of the protein according to claim 1 or 2 in the target plant, or / and enhancing, increasing or up-regulating the expression level of the gene encoding the protein according to claim 1 or 2 to improve the salt tolerance of the plant.

8. The method according to claim 6 or 7, characterized in that: The coding sequence of the coding gene is SEQ ID No. 1 in the sequence list.

9. The method according to any one of claims 6 to 8, characterized in that: The plant is a plant of the genus Salicornia.

10. The method according to claim 9, characterized in that: The plant is a Salicornia europaea L. plant.

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