Use of lc sfc6 protein and related biological materials

Abstract

The application provides application of LcSFC6 protein and related biological materials thereof. The amino acid sequence of the LcSFC6 protein is shown as SEQ ID No: 2. The application takes wild Leymus chinensis as a receptor material to obtain LcSFC6 The overexpression plant of the gene. Compared with the wild type, LcSFC6 The stress resistance of the overexpression plant is significantly improved. The LcSFC6 and the coding gene thereof have important theoretical and practical significance for cultivating new varieties of Leymus chinensis and other plants with improved stress resistance, and can be applied to cultivation and identification of plant varieties with resistance required by agriculture, animal husbandry and ecological environment management, and have high practical application value. The application has wide application prospect in the field of agriculture.

Description

Technical Field

[0001] This invention relates to the field of mutation or genetic engineering, and in particular to the application of LcSFC6 protein and related biomaterials. Background Technology

[0002] In recent years, my country has faced an annual shortage of up to 50 million tons of high-quality forage. Meanwhile, farmland protection and ecological protection red lines have restricted the large-scale planting and promotion of forage on arable land. Saline-alkali land is an important reserve resource in my country. By breeding salt-tolerant forage grasses, forage can be planted on saline-alkali land, effectively utilizing these previously unusable land resources. This not only increases land utilization but also provides a rich source of feed for animal husbandry, which is of great significance in alleviating my country's shortage of high-quality forage. Furthermore, breeding salt-tolerant forage grasses is crucial for promoting sustainable agricultural development. By breeding and promoting salt-tolerant forage grasses, the productivity of saline-alkali land can be improved, dependence on chemical fertilizers and pesticides can be reduced, agricultural non-point source pollution can be decreased, and the development of green and ecological agriculture can be promoted. Therefore, research on salt-tolerant plants and the breeding of new salt-tolerant forage varieties have become important ways to solve the problem of forage shortage. As pioneer plants for saline-alkali land utilization, forage grasses exhibit significant differences in salt tolerance among species due to differences in their genetic makeup and cultivation conditions. Discovering salt-tolerant gene resources will provide important guidance for the breeding of salt-tolerant forage grasses and other economic crops.

[0003] Flavonoids are important products of plant secondary metabolism, synthesized via the phenylpropane pathway. They are a class of compounds with a basic C6-C3-C6 structure, consisting of a benzene ring with two phenolic hydroxyl groups linked by three carbon atoms. Chalcone synthase (CHS; EC2.3.1.74) is the first rate-limiting enzyme in the synthesis of flavonoids, catalyzing the condensation of coumaroyl-CoA with malonyl to form chalcones. These chalcones are further derivatized into other flavonoids under the catalysis of other enzymes, playing a crucial role in plant growth, development, and environmental adaptation. Generally, chalcone synthase includes four conserved residues: Cys164, Phe215, His303, and Asn336. These residues are key to the formation of chalcones during the reaction, involving loading, decarboxylation, elongation, and cyclization. The chalcone synthase superfamily plays a crucial role in flavonoid biosynthesis. In some plant flavonoid metabolic pathways, certain enzymes are produced by the chalcone superfamily via a repeat-differentiation pathway, including stilbene synthase (STS), acridinone synthase (ACS), 2-pyranone synthase (2-PS), bibenzyl synthase (BBS), and coumaroyltriacetate synthase (CTAS). Furthermore, they catalyze polyketide condensation using different substrates, resulting in a diverse range of products. Plant flavonoids participate in regulating auxin transport, seed development, and anthocyanin biosynthesis, and also provide protection against abiotic (UV radiation, cold, drought) and biotic (herbivores, bacteria, fungi) stresses. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to improve the stress resistance of plants, especially their salt tolerance.

[0005] To address this problem, the present invention provides the application of a protein, a substance regulating the expression of the gene encoding the protein, or a substance regulating the activity or content of the protein, wherein the protein is an LcSFC6 protein, and is any one of the following:

[0006] A1) The amino acid sequence of this protein is SEQ ID No: 2.

[0007] A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein in A1), which has more than 80% identity with the protein shown in A1) and is associated with plant stress resistance.

[0008] A3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2);

[0009] The application can be any of the following:

[0010] B1) Improve plant stress resistance,

[0011] B2) Enhances plant POD activity.

[0012] B3) Increases plant SOD activity.

[0013] B4) Increase the total flavonoid content of plants.

[0014] The plant's stress resistance may be salt tolerance.

[0015] The protein consists of 389 amino acid residues, including a CHS domain, and belongs to the polyketide synthase (PKSs) type III superfamily. The sequence between amino acids 5-228 is the N-terminal region of Chalcone / stilbene synthase, and the sequence between amino acids 238-382 is the C-terminal region of Chalcone / stilbene synthase. It contains 8 malonyl-CoA binding sites K (55), R (58), I (59), F (215), F (265), G (307), R (308) and A (309), and 3 activation sites C (164), H (304) and N (337).

[0016] In the above applications, the protein may be derived from sheepgrass.

[0017] In the above applications, 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. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0018] The aforementioned 80% or more identity can be 80%, 85%, 90%, or 95% or more identity.

[0019] The 80% or more identity can 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 can 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 can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 95% or more identity can be at least 95%, 96%, 97%, 98%, or 99% identity.

[0020] In the above applications, the regulation can be at least one of the following six types of regulation:

[0021] C1) Regulation at the transcriptional level of the encoded gene.

[0022] C2) Regulation that occurs post-transcriptionally in the encoded gene

[0023] C3) Regulation of RNA transport in the encoded gene.

[0024] C4) regulates the translation of the encoded gene.

[0025] C5) regulates the degradation of the mRNA encoding the gene.

[0026] C6) Post-translational regulation of the gene.

[0027] In the above applications, the substance can be any of the following biomaterials:

[0028] D1) The nucleic acid molecule that encodes the above-mentioned protein;

[0029] D2) An expression cassette containing the nucleic acid molecules described in D1);

[0030] D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0031] D4) Recombinant microorganisms containing the nucleic acid molecules described in D1), or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms containing the recombinant vector described in D3);

[0032] 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);

[0033] D6) Transgenic plant tissue containing the nucleic acid molecules described in D1), or transgenic plant tissue containing the expression cassette described in D2);

[0034] D7) Transgenic plant organs containing the nucleic acid molecules described in D1) or transgenic plant organs containing the expression cassette described in D2).

[0035] Furthermore, in the biological material, the recombinant microorganisms mentioned in D4) can specifically be yeast, bacteria, algae, and fungi.

[0036] Furthermore, the recombinant microorganism may be Agrobacterium. Specifically, Agrobacterium is EHA105.

[0037] Furthermore, in the biological material, D6) the plant tissue may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and / or anthers.

[0038] Furthermore, in the biological material, the transgenic plant organs described in D7) can be the roots, stems, leaves, flowers, fruits, and seeds of the transgenic plant.

[0039] Existing plant expression vectors can be used to construct structures containing... LcSFC6 Recombinant expression vectors for genes. The plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant gene targeting, such as pCAMBIA3301, pCAMBIA1300, pBI121, pCAMBIA1301-Ubi, pS1300, or other derived plant expression vectors. The vector carries the salt stress-specific protein-encoding gene of *Leymus chinensis* of this invention. LcSFC6 Plant expression vectors can be transformed into plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, electroporation, and Agrobacterium-mediated transformation. The transformed plant hosts can be monocotyledons such as sheepgrass, barley, and wheat, or dicotyledons such as Arabidopsis, tomato, soybean, and alfalfa.

[0040] use LcSFC6When constructing recombinant expression vectors, any type of enhancing, constitutive, tissue-specific, or inducible promoter can be added before the transcription initiation nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter or the ubiquitin gene Ubiquitin promoter (pUbi). These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0041] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can express enzymes or luminescent compounds that produce color changes in plants (GUS gene, luciferase gene, etc.), antibiotic resistance markers (gentamicin marker, kanamycin marker, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes).

[0042] Specifically, the recombinant expression vector can be a recombinant expression vector obtained by inserting the coding gene of the above-mentioned *Leymus chinensis* salt stress-specific expression protein into the multiple cloning site of pCAMBIA1300-Ubi, such as... pCAMBIA1300-Ubi-LcSFC6 .

[0043] The present invention also provides a method for improving plant stress resistance, the method comprising improving plant stress resistance by regulating the expression of the gene encoding the protein in the target plant.

[0044] The present invention also provides a method for producing plants with high stress resistance, the method comprising the step of regulating the expression of the encoding 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.

[0045] In the above text, the regulation refers to adjusting, increasing, or raising.

[0046] In the above method, the regulation can be at least one of the following six types of regulation:

[0047] E1) Regulation at the transcriptional level of the encoded gene,

[0048] E2) Regulation that occurs post-transcriptionally in the encoded gene

[0049] E3) regulates the RNA transport of the encoded gene.

[0050] E4) regulates the translation of the encoded gene.

[0051] E5) regulates the degradation of the mRNA encoding the gene.

[0052] E6) Post-translational regulation of the gene.

[0053] Regulating the expression of the gene encoding the protein in the target plant can be achieved by introducing the gene encoding the protein into the target plant.

[0054] In the above method, the protein is derived from sheepgrass.

[0055] The plant mentioned above may be any of the following: F1) monocotyledonous plants, F2) grasses, F3) grasses, F4) Leymus spp., F5) sheepgrass.

[0056] The present invention also provides a product, wherein the product is the aforementioned protein or biological material.

[0057] The expression of Leymus chinensis in different tissues was detected using quantitative PCR. Leymus chinensis seedlings were treated with salt, and the expression before and after stress was assessed using quantitative PCR. LcSFC6 Gene expression status.

[0058] To demonstrate the function of the LcSFC6 protein, the constructed... LcSFC6 Gene overexpression vectors were transferred into Leymus chinensis to construct LcSFC6 overexpression lines.

[0059] This invention provides LcSFC6, a key salt stress-related protein in Leymus chinensis derived from the CHS family, and its encoding gene. Experiments have shown that LcSFC6 is induced by salt stress and participates in the response of Leymus chinensis to salt stress, enhancing the plant's salt tolerance. LcSFC6 and its encoding gene have significant theoretical and practical implications for breeding new varieties of Leymus chinensis and other plants with enhanced stress resistance. They can be used for the breeding and identification of resistant plant varieties needed in agriculture, animal husbandry, and ecological environment management, and have high practical application value. This invention has broad application prospects in the fields of agriculture and economic energy crops. Attached Figure Description

[0060] Figure 1 For PCR amplification LcSFC6 Results of agarose gel electrophoresis detection of full-length cDNA.

[0061] Figure 2 This is a schematic diagram of the domain composition of the LcSFC6 protein.

[0062] Figure 3 for LcSFC6 Results of gene expression levels in different tissues of Leymus chinensis and after salt treatment.

[0063] Figure 4 For PCR testing LcSFC6 Results of gene overexpression vector transformation of Leymus chinensis positive plants.

[0064] Figure 5 for LcSFC6 Phenotypic differences between gene-overexpressing plants and common Leymus chinensis after salt treatment.

[0065] Figure 6 for LcSFC6 The results of physiological index measurements of gene-overexpressing plants compared with ordinary Leymus chinensis after salt treatment. Detailed Implementation

[0066] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0067] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0068] The data in the following examples were processed using SPSS 11.5 statistical software. The experimental results are expressed as mean ± standard deviation and were tested using one-way ANOVA.

[0069] Example 1 LcSFC6 Obtaining Gene Overexpression Plants

[0070] 1. Genes specifically expressed under salt stress in sheepgrass LcSFC6 Sequence cloning and construction of overexpression vectors

[0071] 1.1 Plant material treatment and total RNA extraction

[0072] Total RNA was extracted from Leymus chinensis seedlings and detected 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.

[0073] 1.2 Genes specifically expressed under salt stress in Leymus chinensis LcSFC6 Full-length cDNA cloning

[0074] Using the total RNA extracted from Leymus chinensis seedlings in step 1.1 above as a template, PrimeScript was used... TMFirst-strand cDNA was synthesized using the 1st Strand cDNA Synthesized Kit (Takara) following the kit instructions. The reaction system and conditions were as follows: Oligo-dT (10 pmol / μl) 1 μl, Total RNA (≤ 1 μg) 2 μl, dNTP Mixture (10 mmol / L each) 1.0 μl, 5× Buffer 4.0 μl, RNase Inhibitor (40 U / μl) 0.5 μl, PrimeScript RTase (200 U / μl) 0.5 μl, RNase-free distilled water 11 μl; 65℃ for 5 min, 42℃ for 45 min, 70℃ for 15 min. The synthesized first-strand cDNA was stored at -20℃ for later use.

[0075] Using the obtained first-strand cDNA as a template, and referring to the sequenced genome of Leymus chinensis... LcSFC Homologous genes Lc5Ns04288 Primers were designed based on the CDS sequence. Primer LcSFC-F: 5′-ATGGCAACCGTCCAGCAGATCCG-3′ and primer LcSFC-R: 5′-CTAGTTTTCCTCGAGGCTGCTT-3′ were paired for PCR amplification. The PCR reaction system consisted of: 2 μl each of cDNA template, primer LcSFC-F and primer LcSFC-R, 25 μl of 2*Phanta Max Master Mix (Nanjing Novizan Biotechnology Co., Ltd.), and 19 μl of ddH2O. The reaction conditions were: pre-denaturation at 95℃ for 5 min; followed by 35 cycles of 95℃ for 15 s, 58℃ for 15 s, and 72℃ for 3 min; and finally extension at 72℃ for 5 min.

[0076] After the reaction, the PCR amplification products were detected by 1% agarose gel electrophoresis, and the results are as follows. Figure 1 As shown in the figure, lane M represents the DNA molecular weight standard of TRANS2000 (Beijing TransGen Biotech Co., Ltd.), and lane 1 represents the PCR amplification product. The results showed that a target fragment of approximately 1200 bp was obtained by PCR amplification. The amplification product was recovered and purified, and ligated into the Blunt Zero vector (Beijing TransGen Biotech Co., Ltd., CB501-01). The ligation product was transformed into E. coli DH5α competent cells, and positive clones were screened for identification by bacterial culture PCR. Plasmids of positive clones were extracted and sequenced, and the sequencing results were compared and analyzed. The results showed that the fragment was 1170 bp in length, and its deoxyribonucleotide sequence is shown in sequence 1 of the sequence listing, which is similar to that in Leymus chinensis.Lc5Ns04288 The high homology of the genes indicates that this fragment belongs to Leymus chinensis. LcSFC6 Gene sequence.

[0077] Sequence 1 (SEQ ID NO: 1, 1170bp) is as follows:

[0078]

[0079] The obtained DNAMAN and OMIGA software were used to analyze LcSFC6 Bioinformatics analysis of the full-length cDNA sequence revealed that it encodes a protein consisting of 389 amino acid residues, which is sequence 2 in the sequence listing (SEQ ID NO: 2, 389aa), as follows:

[0080] MATVQQIRRAQRADGPAAVLAIGTANPASSMLQDDYADYYFRVTNSDHHADHKDKLKRICKKSGIERRYAHLDEELLGAHPDFSDRALPTLDARIDMASAAVPALAASAAAKAIAEWGRPAADVTHLVFSTYSGGRAPSADFRLASLLGLRPTVSRTILSLNGCSGGGRALQLAKELAENNRGARVLVACSELTL IAFYGPQEGRLDTILGHGIFGDGAGAVVVGADPVDSVERPLFEMAFATQTTIPETEDEITMRLMKGGLDFHVSIRVPKLLKSNIERCLIDAFESIGVSATWNDLFWAIHPGGRAILDNVEELLGLDAEKLAASRRVLREYGNMSGATVIFVLDELRRRRRAMGEEVAEWGVMMAFGPGITIETMVLHATSSLEEN.

[0081] 2. LcSFC6 Gene sequence analysis and structural-functional prediction of encoded proteins

[0082] Analysis of the LcSFC6 domains using the Pfam server (http: / / pfam.xfam.org / ) revealed that the protein contains a typical CHS domain, belonging to the polyketide synthases (PKSs) type III superfamily. The sequence between amino acids 5-228 is the N-terminal region of the Chalcone / stilbene synthase, and the sequence between amino acids 238-382 is the C-terminal region of the Chalcone / stilbene synthase. It contains eight malonyl-CoA binding sites: K(55), R(58), I(59), F(215), F(265), G(307), R(308), and A(309), and three activation sites: C(164), H(304), and N(337). These results indicate that LcSFC6 belongs to the CHS protein family (…). Figure 2 ).

[0083] 3. LcSFC6 Expression pattern analysis

[0084] 3.1 LcSFC6 Expression pattern analysis in different tissues of Leymus chinensis

[0085] Samples were taken from seven tissues of Leymus chinensis seedlings that had grown normally for 8 weeks: rhizome terminal buds, young leaves, flag leaves, young spikelets, roots, rhizomes, and terminal leaves of vegetative branches. Total RNA was extracted from each tissue and analyzed using quantitative real-time PCR. LcSFC6 Variations in gene expression patterns in different tissues.

[0086] use LcGADPH Genes are used as internal controls for the reaction; primers are designed.

[0087] LcGADPH -F: 5′-CCGTGCTCAATGGGATACTTC-3′;

[0088] LcGADPH -R: 5′-CCCTCGTCTGTGACAATGGTAC-3′.

[0089] According to sheepgrass LcSFC6 Design specific primers for the cDNA sequence:

[0090] QF: 5′-CTACGTGGAACGATCTCTTCT-3′;

[0091] QR: 5′-CCTCGAGGCTGCTTGTGGCGT-3′ was used for Q-PCR amplification. The reaction system consisted of: 10 µL SYBR GreenMix, 0.4 µL QF, 0.4 µL QR, 7.2 µL ddH2O, and 2 µL cDNA template (the reverse transcription product was diluted 10-fold before being used as the template), for a total volume of 20 µL. Real-time quantitative PCR was performed using a two-step method with the following program: 95℃ for 60 s; 95℃ for 15 s, 65℃ for 45 s; 40 cycles. The obtained data and Ct values ​​were analyzed using Mx3000p software.

[0092] The results are as follows Figure 3 As shown in Figure A. The results indicate that, LcSFC6 The gene was expressed at the highest level in the young spikelet, followed by the young leaf and the terminal leaf of the vegetative branch, while the expression level was the lowest in the flag leaf, and the expression level was similar in the root and rhizome.

[0093] 3.2 Sheepgrass LcSFC6 Gene expression pattern analysis after salt treatment

[0094] Eight-week-old Leymus chinensis seedlings were subjected to salt stress (4℃) for different durations (0, 1, 3, 6, 12, 24, and 48 hours). Total RNA was extracted from the seedlings after each treatment and analyzed using quantitative real-time PCR. LcSFC6 Changes in gene expression patterns under salt stress. The primers and reaction systems used followed the same procedure as described above for analyzing expression patterns in different tissues.

[0095] The results are as follows Figure 3 As shown in Figure B. The results indicate that, LcSFC6 After 1 hour of salt treatment, the expression level began to rise slowly, reaching 37 times that of the untreated sample after 3 hours of treatment, and 100 times that of the control sample after 6 hours of treatment. It then began to decline after 12 hours, and by 48 hours of treatment, the relative expression level had decreased to approximately 20-fold. This indicates that... LcSFC6 Gene expression levels increased significantly after salt stress.

[0096] 4. LcSFC6 Construction of overexpression plants

[0097] The amplification obtained in step 1.2 above LcSFC6 Using the gene as a template, the upstream primer 5′- ctggGGTACC ATGGCAACCGTCCAGCAGATC-3′( KpnI (restriction site) and downstream primer 5′- gctGAGCTCCTAGTTTTCCTCGAGGCTGC -3′( SacI Amplification was performed using the enzyme digestion site, with the amplification reaction system and conditions the same as in steps 1 and 2 above. The amplification product was then subjected to double enzyme digestion (…). KpnI and SacI The recombinant expression vector was inserted into the multiple cloning site (MCS) of the plant binary expression vector pCAMBIA1300-Ubi (Changsha Aibiwei Biotechnology Co., Ltd., HG-VZH1683) to obtain the recombinant expression vector named pCAMBIA1300-Ubi - LcSFC6 . pCAMBIA1300-UBi-LcSFC6 Can be expressed in sheepgrass LcSFC6 Gene.

[0098] 1 μg of the above recombinant expression vector was transformed into Agrobacterium tumefaciens strain EHA105. The successfully transformed strain was identified and infected the callus tissue of Leymus chinensis (hereinafter referred to as wild-type Leymus chinensis). After resistance screening and other processes, positive shoots were obtained.

[0099] DNA-level identification was performed on positive buds. Genomic DNA was extracted from 12 T0 generation plants and analyzed using upstream primer F: 5'-CCCTGTTGTTTGGTGTTACTTCTG-3' (corresponding to...). pCAMBIA1300-Ubi - LcSFC6The Ubi promoter (positions 1966-1990) and downstream identification primer R: 5'-GCTGAAGACGAGGTGCGTGACGTC-3' (corresponding to nucleotides 367-390 of SEQ ID NO: 2) were used to amplify the plants by PCR for DNA-level identification of exogenous gene insertion. M is the marker; lanes 1-12 represent 12 T0 generation plants. The identification results indicate that the proposed... LcSFC6 DNA electrophoresis bands of overexpressing plants 1-12 and LcSFC6 The recombinant plasmid electrophoresis bands were of uniform size and brighter than the target bands obtained from amplifying wild-type Leymus chinensis genomic DNA, indicating that the T0 generation plants from generations 1-12 were all... LcSFC6 Sun-loving plants (also known as LcSFC6 (Gene overexpression plants), identification results as follows .

[0100] Harvest T0 generation seeds, self-pollinate T0 generation seeds to obtain T1 generation seeds, plant T1 generation seeds to obtain T2 generation seedlings and perform the following tests.

[0101] extract Figure 4 Total RNA from the T2 generation of positive plants and its recipient material, wild-type Leymus chinensis, was reverse transcribed and then used for real-time quantitative PCR of the transgenic plants using quantitative primers F: 5'-CCCTGTTGTTTGGTGTTACTTCTG-3' and R: 5'-GCTGAAGACGAGGTGCGTGACGTC-3'. LcSFC6 The gene was used as an internal reference gene (upstream internal reference primer: 5'-CCGTGCTCAATGGGATACTTC-3'; downstream internal reference primer: 5'-CCCCTCGTCTGTGACAATGGTAC-3'), using 2 -△△CT Data processing and detection LcGADPH Gene expression in various transgenic lines.

[0102] Compared with the recipient material, wild-type Leymus chinensis, 3 out of 12 transgenic lines showed [a certain characteristic]. LcSFC6 Gene expression was significantly upregulated, and these lines were named L1, L2, and L3, respectively. The transgenic lines L1, L2, and L3 exhibited... LcSFC6 The gene expression levels were 7.5, 4.3, and 6.2 times that of wild-type Leymus chinensis, respectively.

[0103] Example 2 L Phenotypic analysis of plants overexpressing cSFC6 gene

[0104] 1. LcSFC6 Salt phenotype analysis of gene overexpression plants

[0105] The experiment was repeated three times, with the following method used each time:LcSFC6 Seeds of the overexpression lines L1, L2, and L3, and wild-type Leymus chinensis were sown in pots filled with soil. After rooting, the seedlings were cultured together under normal growing conditions (daytime temperature 30±2℃, nighttime temperature 20±2℃) for 3 months in the experimental greenhouse of the Institute of Botany, Chinese Academy of Sciences. Ten seedlings from each line were then treated with 400mM NaCl (500ml of the treatment solution was directly poured into the pots) for 7 days, followed by 20 days of normal culture. Phenotypic data were observed and recorded, and the survival rate was calculated. The results are as follows: LcSFC6 The results showed that under normal growth conditions, the LcSFC6 overexpressing lines L1, L2, and L3 had no significant difference in growth status compared to the common Leymus chinensis lines W1, W2, and W3. However, after salt treatment, the common Leymus chinensis lines died (survival rate was 0%). Figure 5 The survival rates of expression lines L1, L2, and L3 were 86.5%, 79.3%, and 72.6%, respectively. Overexpression of LcSFC6 The expression strains were able to grow normally. This indicates that overexpression... Overexpression of LcSFC6 It can improve the plant's salt tolerance.

[0106] 2. LcSFC6 Physiological parameters of overexpressing plants after salt treatment

[0107] To further verify the salt stress tolerance of LcSFC6 overexpressing plants, a series of physiological and biochemical indicators, including POD and SOD activities, MDA and total flavonoid content, were measured before and after 400 mM NaCl treatment. The detection kits and methods were as follows: 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), and total flavonoid (TF) content determination kit (spectral method, Biosharp, BL867A). Results are as follows: LcSFC6 Figure 6 As shown in the figure. The results showed that before salt treatment, the POD and SOD activities of LcSFC6 overexpressing plants L1, L2, and L3 were slightly higher than those of wild-type Leymus chinensis W1, W2, and W3. The total flavonoid content of the overexpressing lines was also higher than that of the wild-type Leymus chinensis. The MDA content of the overexpressing lines was similar to that of the wild type, with little overall difference. After salt treatment, the POD and SOD activities and the total flavonoid content of LcSFC6 overexpressing plants were significantly higher than those of ordinary Leymus chinensis. In particular, the total flavonoid content reached more than 3 times that of wild-type Leymus chinensis, while the MDA content was less than half that of wild-type Leymus chinensis. This proves that the total flavonoid content of LcSFC6 overexpressing lines is increased and they have a higher tolerance to salt.

[0108] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of substances that upregulate the expression of protein-coding genes or substances that upregulate protein activity or content, characterized in that, The protein is an LcSFC6 protein, and is any one of the following: A1) The amino acid sequence of this protein is SEQ ID No:

2. A2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1); The substance is any of the following biological materials: D1) An expression cassette containing a nucleic acid molecule encoding the protein; D2) A recombinant vector containing a nucleic acid molecule encoding the protein, or a recombinant vector containing the expression cassette described in D1); D2) A recombinant microorganism containing a nucleic acid molecule encoding the protein, or a recombinant microorganism containing the expression cassette of D1), or a recombinant microorganism containing the recombinant vector of D2); D4) A transgenic plant cell line containing a nucleic acid molecule encoding the protein, or a transgenic plant cell line containing the expression cassette described in D1); D5) Transgenic plant tissue containing a nucleic acid molecule encoding the protein, or transgenic plant tissue containing the expression cassette described in D1); D6) A transgenic plant organ containing a nucleic acid molecule encoding the protein, or a transgenic plant organ containing the expression cassette described in D1); The application is any one of the following: B1) Improve plant stress resistance, B2) Under salt stress conditions, increase plant POD activity. B3) Under salt stress conditions, increase plant SOD activity. B4) Increase the total flavonoid content of plants under salt stress conditions; The plant in question is sheepgrass; The stress resistance mentioned refers to salt resistance.

2. The application according to claim 1, characterized in that, The protein is derived from sheepgrass.

3. A method for improving plant stress resistance, characterized in that, The method includes improving plant stress resistance by regulating the expression of the gene encoding the protein described in claim 1 or 2 in the target plant; the regulation is upregulation; the plant is sheepgrass; and the stress resistance is salt tolerance.

4. A method for producing highly stress-resistant plants, characterized in that, The method includes the step of regulating the expression of the gene encoding the protein of claim 1 or 2 in the target plant to obtain a plant with enhanced stress resistance; the enhanced stress resistance plant has higher stress resistance than the target plant; the regulation is upregulation; the plant is sheepgrass; the stress resistance is salt tolerance.

5. The method according to claim 3 or 4, characterized in that, The regulation is at least one of the following four types of regulation: E1) Regulation at the transcriptional level of the encoded gene, E2) Regulation that occurs post-transcriptionally in the encoded gene E3) regulates the RNA transport of the encoded gene. E4) regulates the translation of the encoded gene.

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

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