Protein LcSAT1 and application of related biological materials thereof

Abstract

The invention provides application of a protein LcSAT1 and a related biological material of the protein LcSAT1. The amino acid sequence of the LcSAT1 protein is as shown in SEQ ID No: 2. Wild leymus chinensis is used as a receptor material, and an LcSAT1 overexpression plant is obtained. Compared with a wild type, the stress resistance of an LcSAT1 overexpressed plant is remarkably improved. The LcSAT1 and the coding gene thereof have important theoretical and practical significance for cultivating leymus chinensis and other new plant varieties with improved stress resistance, can be applied to cultivation and identification of resistant plant varieties required by agriculture and animal husbandry and ecological environment management, and have relatively high practical application value. The method has a wide application prospect in the field of agriculture.

Description

Technical Field

[0001] The invention relates to a salt stress-related protein and its functional application, in particular to an application of a protein LcSAT1 and related biological materials. Background Art

[0002] Soil salinization seriously restricts agricultural development. Improving and restoring saline-alkali wasteland resources is of great significance to agricultural production development, land management, and ecological environment protection. It is an important reserve arable land resource in my country. The efficient utilization of saline-alkali land agriculture is of great significance to promoting sustainable agricultural development and ensuring food security. Salt stress is the main abiotic stress that affects crop seed germination, growth, and yield. The problem of land salinization has seriously endangered China's food security. It is urgent to breed salt-alkali-tolerant crops, improve the utilization rate of salinized land, and protect national food security. Mining key salt-tolerant genes in plants and deeply studying their molecular regulatory mechanisms can provide an important theoretical basis for the breeding of salt-alkali-tolerant crops. The role of salt-alkali-tolerant gene resources in breeding is mainly reflected in improving the salt-alkali tolerance of crops, so that they can grow on saline-alkali land and increase food yield and land utilization. Therefore, the discovery and application of salt-alkali-tolerant gene resources provide an important theoretical basis and practical guidance for the breeding of salt-alkali-tolerant crops. Leucine-rich repeat receptor protein kinases (LRR-RLKs) are the largest subfamily in the plant receptor protein kinase family, accounting for more than half of the entire family. Their main characteristics are similar to the structure of RLK, that is, they contain a transmembrane domain, a C-terminal intracellular kinase domain and an extracellular ligand domain. The extracellular ligand domain of this type of protein is mainly composed of 1-32 tandemly arranged leucine repeat units. As a receptor for signal recognition, LRR-RLKs recognize and bind to specific signal molecules to transmit signals, playing an important role in the recognition and defense of plants against pathogenic microorganisms. LRR receptor kinases are widely involved in the perception and transmission of plant developmental signals. They regulate plant growth and development processes, mediate the signal transduction of plant hormones, participate in the recognition and defense response of plants to pathogenic microorganisms, and respond to the signal transduction of abiotic stress in plants. They can regulate plant responses to adverse stress and improve stress resistance. Summary of the invention

[0003] The technical problem to be solved by the present invention is how to improve the stress resistance of plants, especially the salt tolerance.

[0004] In order to solve this problem, the present invention provides the use of a protein or a substance for regulating the expression of a gene encoding the protein or a substance for regulating the activity or content of the protein, wherein the protein is LcSAT1 protein, which is any one of the following: A1) a protein having an amino acid sequence of SEQ ID No: 2, A2) a protein obtained by replacing and / or deleting and / or adding amino acid residues of the protein of A1) and having an identity of 80% or more with the protein of A1) and being related to plant stress resistance, A3) A fusion protein obtained by connecting the N-terminus or / and C-terminus of A1) or A2) to a protein tag; The application may be any of the following: B1) Improve plant stress resistance, B2) Increase plant POD activity, B3) Increase the activity of plant SOD, B4) Reduce the malondialdehyde (MDA) content of plants under salt stress conditions.

[0005] The plant stress resistance may be salt resistance.

[0006] The protein consists of 438 amino acid residues and contains an LRR-RLK domain. The sequence between amino acids 68-324 is a 7-repeat Leucine-rich repeat domain (pfam: PF00560), and the sequences between amino acids 68-86, 91-111, 115-136, 237-255, 258-277, 281-305, and 308-324 together constitute an L domain-like. The sequence between amino acids 378-400 is a membrane-embedded protein domain, the sequence between amino acids 401-438 is an extracellular domain, and the sequences 58-156 are protein kinase EMS1 domains. In the above application, the protein may be derived from Leymus chinensis.

[0007] In the above application, protein identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be calculated.

[0008] The above-mentioned 80% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0009] The 80% or more identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 85% or more identity may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 90% or more identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 95% or more identity may be at least 95%, 96%, 97%, 98%, or 99% identity.

[0010] In the above application, the regulation may be at least one of the following six types of regulation: C1) regulation at the transcriptional level of the coding gene, C2) regulation after transcription of the coding gene, C3) regulation of the transport of RNA encoding the gene, C4) regulation of the translation of the coding gene, C5) regulation of the degradation of the mRNA encoding the gene, C6) Post-translational regulation of the gene.

[0011] In the above application, the substance may be any of the following biological materials: D1) a nucleic acid molecule encoding the above protein; D2) an expression cassette containing the nucleic acid molecule described in D1); D3) a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) a recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3); D5) a transgenic plant cell line containing the nucleic acid molecule described in D1), or a transgenic plant cell line containing the expression cassette described in D2); D6) transgenic plant tissue containing the nucleic acid molecule described in D1), or transgenic plant tissue containing the expression cassette described in D2); D7) A transgenic plant organ containing the nucleic acid molecule described in D1) or a transgenic plant organ containing the expression cassette described in D2).

[0012] Furthermore, in the biological material, the recombinant microorganism D4) can specifically be yeast, bacteria, algae and fungi.

[0013] Furthermore, the recombinant microorganism may be Agrobacterium, and the Agrobacterium is LBA4404.

[0014] Furthermore, in the biological material, D6) the plant tissue may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and / or anthers.

[0015] Furthermore, in the biological material, D7) the transgenic plant organ can be the root, stem, leaf, flower, fruit and seed of the transgenic plant.

[0016] Existing plant expression vectors can be used to construct LcSAT1 The plant expression vector includes a binary Agrobacterium vector and a vector that can be used for plant gene targeting, such as pCAMBIA3301, pCAMBIA1300, pBI121, pCAMBIA1301-Ubi, pS1300 or other derived plant expression vectors. LcSAT1 The plant expression vector can be transformed into plant cells or tissues through conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc. The transformed plant host can be monocots such as Leymus chinensis, barley, wheat, etc., or dicots such as Arabidopsis, tomato, soybean, and alfalfa.

[0017] use LcSAT1 When constructing a recombinant expression vector with a gene, any enhancing, constitutive, tissue-specific or inducible promoter can be added before the transcription initiation nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin gene Ubiquitin promoter (pUbi), etc., which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct a plant expression vector, an enhancer can also be used, including a translation enhancer or a transcription enhancer, and these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive, and can be natural or synthetic. The translation initiation region can come from the transcription initiation region or the structural gene.

[0018] 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 genes that can express enzymes or luminescent compounds that can produce color changes in plants (GUS gene, luciferase gene, etc.), antibiotic resistance markers (gentamicin marker, kanamycin marker, etc.) or chemical resistance marker genes (such as herbicide resistance genes), etc.

[0019] The recombinant expression vector can be specifically a recombinant expression vector obtained by inserting the coding gene of the above-mentioned salt stress specific expression protein of Leymus chinensis into the multiple cloning site of pCAMBIAsuper1300-GFP, such as pCAMBIA 1300- LcSAT1 -GFP.

[0020] The present invention also provides a method for improving plant stress resistance, which comprises improving plant stress resistance by regulating the expression of the coding gene of the protein in the target plant.

[0021] The present invention also provides a method for producing highly stress-resistant plants, which comprises the steps of regulating the expression of the coding gene of the above-mentioned protein in the target plant to obtain a plant with improved stress resistance; the plant with improved stress resistance has higher stress resistance than the target plant.

[0022] In the above, the regulation refers to upregulation, improvement or increase.

[0023] In the above method, the regulation can be at least one of the following 6 types of regulation: E1) regulation at the transcriptional level of the coding gene, E2) is a post-transcriptional regulation of the coding gene. E3) regulation of the transport of RNA encoding the gene, E4) regulation of the translation of the coding gene, E5) regulation of the degradation of the mRNA encoding the gene, E6) Post-translational regulation of the gene.

[0024] Regulating the expression of the gene encoding the protein in the target plant may be by introducing the gene encoding the protein into the target plant.

[0025] In the above method, the protein is derived from Leymus chinensis.

[0026] In the above, the plant may be any one of the following: F1) monocots, F2) Gramineae, F3) Poaceae, F4) Leymus, F5) Leymus chinensis.

[0027] The present invention also provides a product, which is the above protein or biological material.

[0028] The quantitative PCR method was used to detect the expression of the gene in different tissues of Leymus chinensis. The seedlings of Leymus chinensis were treated with salt and the quantitative PCR method was used to detect the expression of the gene before and after the stress. LcSAT1 The expression of genes.

[0029] In order to prove the function of LcSAT1 protein, the constructed LcSAT1 The gene overexpression vector was transferred into Leymus chinensis to construct LcSAT1 overexpression strains. After salt treatment of wild-type Leymus chinensis and LcSAT1 overexpression Leymus chinensis, the phenotypic differences between the two before and after salt treatment were observed, and the changes in SOD, POD, and MDA contents of the two before and after stress were measured. The results are as follows Figure 7 The results showed that LcSAT1 overexpression improved the resistance of Leymus chinensis to salt stress.

[0030] The present invention provides a key protein LcSAT1 related to salt stress of Leymus chinensis derived from the LRR-RLK family and its encoding gene. Experiments have shown that LcSAT1 is located on the plasma membrane; it is induced by salt stress, participates in the response of Leymus chinensis to salt stress, and improves the salt resistance of the plant. LcSAT1 and its encoding gene have important theoretical and practical significance for breeding new varieties of Leymus chinensis and other plants with improved stress resistance, can be used for the breeding and identification of resistant plant varieties required for agriculture, animal husbandry and ecological environment management, and have high practical application value. The present invention has broad application prospects in the fields of agriculture and economic energy crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 For PCR amplification LcSAT1 Agarose gel electrophoresis detection results of full-length cDNA.

[0032] Figure 2 Schematic diagram of the domain structure of LcSAT1 protein.

[0033] Figure 3 for LcSAT1 Gene expression results in different tissues of Leymus chinensis and after salt treatment.

[0034] Figure 4 for LcSAT1 Subcellular localization results of genes.

[0035] Figure 5 For PCR testing LcSAT1 The results of transforming positive plants of Leymus chinensis with gene overexpression vector.

[0036] Figure 6 for LcSAT1 Phenotypic differences between gene overexpressing plants and common Leymus chinensis after salt treatment.

[0037] Figure 7 for LcSAT1 Results of physiological index measurements of gene overexpression plants after salt treatment compared with common Leymus chinensis. DETAILED DESCRIPTION

[0038] 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.

[0039] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0040] The following examples were processed using SPSS 11.5 statistical software, and the experimental results were expressed as mean ± standard deviation and tested using One-way ANOVA.

[0041] Embodiment 1, LcSAT1 Obtaining gene overexpression plants 1. Genes specifically expressed in Leymus chinensis under salt stress LcSAT1 Sequence cloning 1.1 Plant material processing and total RNA extraction Total RNA was extracted from Leymus chinensis seedlings and tested by 1% agarose gel electrophoresis. The RNA concentration and integrity were determined by Scan Drop. The results showed that the extracted RNA was of good quality and of appropriate concentration.

[0042] 1.2 Genes specifically expressed in Leymus chinensis under salt stress LcSAT1 Full-length cDNA clone The total RNA extracted from the Leymus chinensis seedlings in step 1.1 was used as a template and PrimeScript TM 1st StrandcDNA Synthesized Kit (Takara) was used to reverse synthesize the first strand cDNA according to the instructions of the kit. The reaction system and reaction conditions are as follows: Oligo-dT (10 pmol / μl) 1 μl, Total RNA (≤ 1μg) 2μl, dNTP Mixture (10mmol / l each) 1.0μl, 5×Buffer 4.0μl, RNase Inhibitor (40U / μl) 0.5μl, PrimeScript RTase (200U / μl) 0.5μl, RNase-free distilled water 11 μl; 65℃ 5min, 42℃ 45min, 70℃ 15min. The synthesized first strand cDNA was stored at -20℃ for later use. Then, the first-strand cDNA was used as a template, and primers were designed referring to the CDS sequence of the LcSAT homologous gene Lc5Xm00620 in the sequenced genome sequence of Leymus chinensis. The primer LcSAT-F: 5′-ATGCCCTTTTCTCAAGCACTGG -3′ was paired with the primer LcSAT-R: 5′-TTATTCTGCGGCTGTGTTCTTTGCGAA -3′ for PCR amplification; the PCR reaction system was: 2 μl each of cDNA template, primer LcSAT-F and primer LcSAT-R, 25 μl of 2*Phanta Max Master Mix (Nanjing Novozyme Biotechnology Co., Ltd.), and 19 μl of ddH2O; the reaction conditions were: pre-denaturation at 95°C for 5 min; then 95°C for 15 s, 58°C for 15 s, and 72°C for 3 min, for a total of 35 cycles; and finally extension at 72°C for 5 min.

[0043] After the reaction, the PCR amplification products were detected by 1% agarose gel electrophoresis. Figure 1 As shown. Lane M is the DNA molecular weight standard of TRANS2000 DNA molecular weight standard (Beijing Quanshijin Biotechnology Co., Ltd.), and lane 1 is the PCR amplification product. The results show that the target fragment with a length of about 1400bp was obtained by PCR amplification. The amplified product was recovered and purified, and connected to the Blunt Zero vector (Beijing Quanshijin Biotechnology Co., Ltd., CB501-01). The ligated product was transformed into Escherichia coli DH5α competent cells, and the positive clones were screened for bacterial liquid PCR identification. The plasmids of the positive clones were extracted for sequencing, and the sequencing results were compared and analyzed. The results showed that the length of the fragment was 1314bp, and its deoxyribonucleotide sequence was shown in sequence 1 in the sequence table, which had a high homology with the Lc5Xm00620 gene in Leymus chinensis, indicating that the fragment was the LcSAT1 gene sequence of Leymus chinensis.

[0044] Sequence 1 (SEQ ID NO: 1, 1314 bp) Leymus chinensis ) LcSAT1 The CDS sequence is as follows: 5′-ATGCCCTTTTCTCAAGCACTGGAAACACTTGCCCCAGGCCTTGCGGAGGAGGAGCGCCTTGAGCTGCGAG

[0045] 2. LcSAT1 Sequence analysis of genes and prediction of the structure and function of their encoded proteins DNAMAN and OMIGA software were used to analyze the LcSAT1 The full-length cDNA sequence of LcSAT1 was analyzed by bioinformatics. The sequence was 1314 bp in length and encoded a protein consisting of 438 amino acid residues (SEQ ID NO: 2, 438aa). The domain structure of LcSAT1 was analyzed by Pfam (http: / / pfam.xfam.org / ) server. The results showed that the protein contained a typical LRR-RLK domain and a protein kinase domain. The sequences between amino acids 68-86, 91-111, 115-136, 237-255, 258-277, 281-305, and 308-324 of the protein were conserved Leucine-rich repeat domains (pfam: PF00560), which together formed L domain-like; the sequences between amino acids 378-400 of the protein were membrane mosaic protein domains, the sequences between amino acids 401-438 of the protein were extracellular domains, and the sequences between amino acids 58-156 were protein kinase EMS1 domains. The above analysis results show that LcSAT1 contains a protein kinase domain and belongs to the LRR-RLK protein family ( Figure 2 ).

[0046] Sequence 2 is Leymus chinensis ( Leymus chinensis ) LcSAT1 protein sequence (438aa), as follows: MPFSQALETLAPGLAEEERLELRGWRRLAMSRWLWRPVISSRPSSLAHQLPPTAINAIRISICKSQQLVYLDLSNNFLEGEIPQCFDIKKMQYLLLSNNSLSGKFPEFL QNNTSMEFLDLTWNKLSGRLPTWIGDLGKLRFVLLSHNAFFDNIPIDITRLLRDLQYSDLSGNSFSGAIPCLSNLTLMTEVQNEFMDKDDLQIGGVIRTGADYLGQILSVI TKGQQLLYGRTLVYFVGIDLSGNSLTGEIPTDITSLAALMNLNLSSNKLSGHIPNMVGAMQSLVSLDLSQNKNKLSGERPSSLSNLASLAALNLSYNNFSGRIPSGRQLD TLNADNPSLMYIGNSELCGPPLHNNCLGNDSFIIHGDPGSSKQEFDPLTFYFGLVLGLVVGLWAVFCALLFKRTWRIAYFQLFDEAYDEVYVFMVVKWASFAKNTAAEW.

[0047] 3. LcSAT1 Expression pattern analysis 3.1 LcSAT1 Analysis of expression patterns in different tissues of Leymus chinensis Total RNA was extracted from the root apical bud, leaf, flag leaf, spike, root and rhizome of Leymus chinensis seedlings grown normally for 8 weeks, and analyzed by fluorescence quantitative PCR. LcSAT1 Variations in gene expression patterns in different tissues.

[0048] use LqCy The gene is used as the internal reference for the reaction and the primers are designed. LqCy -F: 5′-CCGTGCTCAATGGGATACTTC-3′, LqCy -R: 5′-CCCTCGTCTGTGACAATGGTAC-3′.

[0049] According to Leymus chinensis LcSAT1 Design specific primers for cDNA sequences: QF: 5′-CTGGTGAAAGGCCGTCAAGTTTG-3′ QR: 5′-CCCACTTTACAACCATGAATACA-3′ was used for Q-PCR amplification. The reaction system was: SYBR GreenMix 10µL, QF0.4µL, QR0.4µL, ddH2O7.2µL, cDNA template 2µL (the reverse transcription product was diluted 10 times as a template), total volume: 20µL. Real-time quantitative PCR reaction was completed in two steps, and the reaction program was: 95℃60s; 95℃15s, 65℃45s; 40 cycles. The obtained data and Ct value analysis were performed using Mx3000p software. The results are as follows Figure 3 As shown in A. The results show that LcSAT1 The gene expression level in leaves was higher than that in stems, and the expression level in stems was significantly higher than that in roots.

[0050] 3.2 Leymus chinensis LcSAT1 Analysis of gene expression patterns after salt treatment After the 8-week-old Leymus chinensis seedlings were subjected to salt stress (4°C) for different time periods (0, 1, 3, 6, 12, 24 and 48 h), the total RNA of the Leymus chinensis seedlings under the above different treatments was extracted and analyzed by fluorescence quantitative PCR. LcSAT1 Changes in gene expression patterns under salt stress conditions. The primers and reaction systems used refer to the above-mentioned analysis process of expression patterns in different tissues.

[0051] The results are as follows Figure 3 As shown in B. The results show that LcSAT1 After 1 hour of salt treatment, the expression level began to rise slowly, reaching a maximum of 2.5 times that of untreated at 6 hours, and began to decline at 12 hours. LcSAT1 The expression level of the gene increased after salt stress, but not significantly.

[0052] 4. Subcellular localization of LcSAT1 protein The amplified product from step 1.2 above LcSAT1 gene as a template, using the upstream primer 5′- cggTCTAGA ATGCCCTTTTCTCAAGCACTGG-3′( Xgf Restriction site and downstream primer 5′- tctGGTACC TTCTGCGGCTGTGTTCTTTGCGAA-3′( KqI The amplification product was amplified by double enzyme digestion ( Xgf and KqI ) was inserted into the multiple cloning site of the plant binary expression vector pCAMBIAsuper1300-GFP ( Xgf and Between KpnI ) (Changsha Aibiwei Biotechnology Co., Ltd., HG-VZH1330), and the recombinant expression vector was named pCAMBIA 1300- LcSAT1- GFP. pHG-VZH13301300-LcSAT1- GFP can be expressed in plants LcSAT1 gene, and can be fused with EGFP for subcellular localization observation. 1 μg of the above recombinant expression vector was transferred into Agrobacterium EHA105 strain and transformed into tobacco epidermal cells for transient expression. pCAMBIA 1300-GFP tobacco epidermal cells were used as a control, and the vector construction was as follows Figure 4 In Figure A, after 24-48 hours of culture, the cells were observed and photographed under a laser confocal scanning microscope (Bio-Rad MRC 1024). Figure 4 As shown in B. As can be seen, the empty vector was transferred pCAMBIA The protein expressed in 1300-GFP transgenic cells is distributed throughout the cell, while LcSAT1 Recombinant expression vector p1300-LcSAT1- The protein expressed in GFP transgenic cells is localized on the plasma membrane. p1300- LcSAT1- GFP vector co-expression, the results are as follows Figure 4 Middle C. The results showed that LcSAT1 was localized on the plasma membrane and co-expressed with PIP2.

[0053] 5. LcSAT1 Construction of gene overexpression plants The p1300-LcSAT1- The GFP recombinant plasmid was transferred into the Agrobacterium tumefaciens LBA4404 strain, and the successfully transformed strain was identified and infected with the leaves of Leymus chinensis using the disc method. The specific steps are: 5.1 Take young and healthy leaves, rinse them with distilled water, wash them with 70% ethanol for 45 seconds, disinfect them with 0.1% mercuric chloride for 6-8 minutes, rinse them with sterile water for 5 times, and absorb the moisture with sterile filter paper.

[0054] 5.2 Cut the sterile leaves into small pieces of 0.5cm×0.5cm, with the adaxial side of the leaves facing downward, and inoculate them on the callus induction medium for pre-cultivation for 2-3 days.

[0055] 5.3 Pick a single colony of Agrobacterium tumefaciens and inoculate it into 20 ml YEB culture medium supplemented with corresponding antibiotics, and culture it on a constant temperature shaker at 27℃ until the OD value reaches 0.6-0.8. Take 1%-2% of the above culture and transfer it into fresh YEB culture medium without antibiotics, and continue to culture for 6 hours. When the OD value reaches 0.2-0.5, it can be used for transformation.

[0056] 5.4 On the clean bench, pour the bacterial solution into a sterile small culture dish, take out the pre-cultured explants from the culture solution, and soak them in the bacterial solution for 1-5 minutes. Take out the explants and absorb the attached bacterial solution on sterile filter paper. Then inoculate them on callus induction or differentiation medium and culture them in the dark at 28℃ for 2-4 days.

[0057] 5.5 The co-cultivated explants were transferred to a sterilized differentiation and callus induction medium with selective pressure. The selective culture was carried out at 25°C under light conditions.

[0058] 5.6 After 2-3 weeks of selective culture, the transformed cells of the explants will differentiate into resistant adventitious buds or produce resistant callus. These resistant materials are transferred to the corresponding subculture selection medium to allow them to grow and induce differentiation.

[0059] 5.7 When the adventitious buds grow to more than 1 cm, cut them off and insert them into the rooting medium containing selective pressure for root culture. Adventitious roots will grow in about two weeks.

[0060] The induction medium formula is: MS medium, supplemented with 5 mg / L 2,4-D, 0.15 mg / L 6-BA, 30 g / L sucrose, and 4 g / L agar powder.

[0061] The formula of YEB culture medium is: beef extract 5g / L, yeast extract 1g / L, peptone 5g / L, sucrose 5g / L, magnesium sulfate (MgSO4.7H2O) 4g / l.

[0062] The differentiation medium formula is: MS medium supplemented with 1 mg / L 2,4-D, 1 mg / L zeatin, 4 g / L agar powder, 400 mg / L carbenicillin and 30 mg / L hygromycin.

[0063] The formula of the subculture selection medium is: MS medium, supplemented with 1 mg / L 2,4-D, 300 mg / L hydrolyzed casein, 500 mg / L proline, 100 mg / L inositol, 40 g / L sucrose, and 4 g / L agar powder.

[0064] The rooting medium formula was: MS medium supplemented with 0.05 mg / L NAA, 4 g / L agar powder, 400 mg / L carbenicillin, and 30 mg / L hygromycin.

[0065] The positive buds were identified at the DNA level. Genomic DNA of 12 T0 plants was extracted and the upstream identification primer F: 5'-ctcggatacttacgtcacgtcttgc-3' (corresponding to pCAMBIA1300- -LcSAT1-GFP MAS promoter position 340 to 365) and downstream identification primer R: 5'-AGAGCTGGAAATAAGCGATTCTCCATG -3' (corresponding to nucleotides 1208-1234 of SEQ ID NO: 2) were used to perform PCR amplification of the plants and identify the insertion of foreign genes at the DNA level. M is a marker; lanes 1-10 are 10 T0 generation plants respectively. The identification results are shown in Figure 5 As shown in Figure 2, the DNA electrophoresis bands of the proposed LcSAT1 overexpression plants 1-10 were consistent with the expected amplification electrophoresis bands of the LcSAT1 overexpression recombinant plasmid, which were about 1200 bp, indicating that the T0 generation plants 1-10 were all LcSAT1 overexpression positive plants (also known as LcSAT1 overexpression plants). Figure 5 The results showed that a total of 10 LcSAT1 overexpression lines were obtained, which were T0 generation plants. T0 generation seeds were harvested, T0 generation seeds were self-pollinated to obtain T1 generation seeds, T1 generation seeds were planted to obtain T2 generation seedlings and the following tests were performed. Total RNA was extracted from the T2 generation of LcSAT1 overexpressing plants and their recipient wild-type Leymus chinensis. After reverse transcription, real-time fluorescence quantitative PCR was performed on the transgenic plants using quantitative primers F: 5'-CTGGTGAAAGGCCGTCAAGTTTG-3' and R: 5'-CCCACTTTACAACCATGAATACA-3'. The LcGADPH gene was used as the internal reference gene (upstream internal reference primer: 5'-CCGTGCTCAATGGGATACTTC-3'; downstream internal reference primer: 5'-CCCTCGTCTGTGACAATGGTAC-3'). -△△CT The data were processed by the method to detect the expression of LcSAT1 gene in each transgenic line. Compared with the recipient material wild-type Leymus chinensis, the expression of LcSAT1 gene was significantly upregulated in 3 of the 10 transgenic lines, named L1, L2 and L3. The expression levels of LcSAT1 gene in transgenic lines L1, L2 and L3 were 56.5, 62.5 and 68.5 times that of wild-type Leymus chinensis, respectively. Example 2: Phenotypic analysis of LcSAT1 overexpressing plants 1. Analysis of salt phenotypes in LcSAT1 overexpressing plants The experiment was repeated three times, and the experimental method was as follows: seeds of LcSAT1 overexpression lines L1, L2 and L3 and wild-type Leymus chinensis were sown in pots filled with soil, and rooted seedlings were cultivated and cultured together under normal growth conditions (in the experimental greenhouse of the Institute of Botany, Chinese Academy of Sciences, with a daytime temperature of 30±2℃ and a nighttime temperature of 20±2℃) for 3 months. Then, 10 strains of each line were selected for 400mMNaCl treatment (500ml of treatment solution was poured into the pot), and normal culture was resumed after 7 days for 20 days. The phenotypic data were observed and recorded, and the changes in its physiological indicators were measured. The results are as follows Figure 6 The results showed that under normal growth conditions, there was no significant difference in the growth status of LcSAT1 overexpression lines L1, L2 and L3 and wild-type Leymus chinensis W1, W2 and W3. After salt treatment, ordinary Leymus chinensis died (survival rate was 0), while the survival rates of LcSAT1 overexpression lines L1, L2 and L3 were 76.5%, 72.5% and 78.5% respectively, and they were able to grow normally, which was also confirmed by the content values ​​of their physiological indicators. This shows that overexpression of LcSAT1 can improve the salt tolerance of plants.

[0066] 2. Results of physiological index measurements of LcSAT1 overexpressing plants after salt treatment In order to further verify the salt stress tolerance of LcSAT1 overexpressing plants, a series of physiological and biochemical indices such as POD and SOD activities and MDA content of the above-mentioned 400mMNaCl treatment materials were determined. The detection kits and methods were: POD activity detection peroxidase test kit (visible spectrophotometry, Shanghai Zeye Biotechnology Co., Ltd., ZY-6059S), total SOD activity detection kit (NBT method, Shanghai Zeye Biotechnology Co., Ltd., ZY68533FA), plant malondialdehyde (MDA) detection kit (TBA colorimetric method, Shanghai Zeye Biotechnology Co., Ltd., ZY603102B). The results are as follows: Figure 7 The results showed that before salt treatment, the physiological indicators of LcSAT1 overexpression plants and common Leymus chinensis plants did not differ much, but after salt treatment, the POD and SOD contents in LcSAT1 overexpression plants were significantly higher than those in common Leymus chinensis, while the MDA content was significantly lower than that in common Leymus chinensis, proving that the LcSAT1 overexpression strain did have a higher tolerance to salt.

[0067] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein, characterized in that: The protein is LcSAT1 protein, which is any one of the following: A1) a protein having an amino acid sequence of SEQ ID No: 2, A2) a protein obtained by replacing and / or deleting and / or adding amino acid residues of the protein of A1) and having an identity of 80% or more with the protein of A1) and being related to plant stress resistance, A3) A fusion protein obtained by connecting the N-terminus or / and C-terminus of A1) or A2) to a protein tag; The application is any of the following: B1) Improve plant stress resistance, B2) Increase plant POD activity, B3) Increase the activity of plant SOD, B4) Reduce the malondialdehyde (MDA) content of plants under salt stress conditions.

2. The use according to claim 1, characterized in that: The protein is derived from Leymus chinensis.

3. The use according to claim 1 or 2, characterized in that: The regulation is at least one of the following 6 types of regulation: C1) regulation at the transcriptional level of the coding gene, C2) regulation after transcription of the coding gene, C3) regulation of the transport of RNA encoding the gene, C4) regulation of the translation of the coding gene, C5) regulation of the degradation of the mRNA encoding the gene, C6) Post-translational regulation of the gene.

4. The use according to any one of claims 1 to 3, characterized in that: The substance is any of the following biological materials: D1) A nucleic acid molecule encoding the protein according to claim 1 or 2; D2) an expression cassette containing the nucleic acid molecule described in D1); D3) a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) a recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3); D5) a transgenic plant cell line containing the nucleic acid molecule described in D1), or a transgenic plant cell line containing the expression cassette described in D2); D6) transgenic plant tissue containing the nucleic acid molecule described in D1), or transgenic plant tissue containing the expression cassette described in D2); D7) A transgenic plant organ containing the nucleic acid molecule described in D1) or a transgenic plant organ containing the expression cassette described in D2).

5. A method for improving plant stress resistance, characterized in that: The method comprises improving plant stress resistance by regulating the expression of the gene encoding the protein of claim 1 or 2 in the target plant.

6. A method for producing a plant with high stress resistance, characterized in that: The method comprises the steps of regulating the expression of the gene encoding the protein of claim 1 or 2 in the target plant to obtain a plant with improved stress resistance; the plant with improved stress resistance has higher stress resistance than the target plant; the regulation is up-regulation, improvement or increase.

7. The method according to claim 5 or 6, characterized in that: The regulation is at least one of the following 6 types of regulation: E1) regulation at the transcriptional level of the coding gene, E2) is a post-transcriptional regulation of the coding gene. E3) regulation of the transport of RNA encoding the gene, E4) regulation of the translation of the coding gene, E5) regulation of the degradation of the mRNA encoding the gene, E6) Post-translational regulation of the gene.

8. The method according to claim 5, 6 or 7, characterized in that: The protein is derived from Leymus chinensis.

9. The use according to claims 1 to 4 or the method according to claims 5 to 8, characterized in that: The plant is any of the following: F1) Monocots, F2) Gramineae, F3) Gramineae, F4) Leymus plants, F5) Leymus chinensis.

10. A product, characterized in that The product is the protein described in claim 1 or the biological material described in claim 4.

Images