Application of LcSFC6 protein and related biological materials thereof

Abstract

The invention provides an application of an LcSFC6 protein and a related biological material thereof. The amino acid sequence of the LcSFC6 protein is as shown in SEQ ID No: 2. Wild leymus chinensis is used as a receptor material, and an overexpression plant of the LcSFC6 gene is obtained. Compared with a wild type, the stress resistance of an LcSFC6 overexpressed plant is remarkably improved. The LcSFC6 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 present invention relates to the field of mutation or genetic engineering, and in particular to the application of LcSFC6 protein and related biological materials. Background Art

[0002] In recent years, the annual gap of high-quality forage in my country has reached 50 million tons. At the same time, the red lines of cultivated land protection and ecological protection have restricted the large-scale planting and promotion of forage on cultivated land. Saline-alkali land is an important land reserve resource in my country. By breeding salt-alkali tolerant forage, forage can be planted on saline-alkali land, thus effectively utilizing these land resources that were originally difficult to use. This not only increases the utilization rate of land, but also provides a rich source of feed for animal husbandry, which is of great significance for alleviating the gap of high-quality forage in my country. In addition, breeding salt-alkali tolerant forage is of great significance for promoting sustainable agricultural development. By breeding and promoting salt-alkali tolerant forage, the output capacity of saline-alkali land can be improved, the dependence on chemical fertilizers and pesticides can be reduced, agricultural non-point source pollution can be reduced, and the development of green ecological agriculture can be promoted. Therefore, the research on salt-tolerant plants and the breeding of new varieties of tolerant forage have become an important way to solve the problem of forage shortage. As a pioneer plant for the utilization of saline-alkali land, forage has obvious differences in salt tolerance among species due to its own genetics and cultivation conditions. Exploring salt-alkali tolerant gene resources will provide important guidance for the breeding of salt-alkali tolerant forage grasses and other economic crops.

[0003] Flavonoids are important products of plant secondary metabolism. They are synthesized through the phenylpropanoid pathway and are a class of compounds with a C6-C3-C6 basic structure formed by two phenolic hydroxyl benzene rings connected by three carbon atoms. Chalcone synthase (CHS; EC2.3.1.74) is the first rate-limiting enzyme in the synthesis of flavonoids. It catalyzes the condensation of coumaroyl CoA and malonyl to form chalcone, which is further derived and converted into other flavonoids under the catalysis of other enzymes. It plays an important role in the growth and development of plants and their adaptation to the environment. In general, chalcone synthase includes four conserved residues, Cys164, Phe215, His303 and Asn336, which are the key to the production of chalcone in the reaction process, including loading, decarboxylation, extension and cyclization. The chalcone synthase superfamily plays an important role in flavonoid biosynthesis. In some plant flavonoid metabolic pathways, some enzymes are produced by CHS through duplication-differentiation pathways, including stilbene synthase (STS), acridone synthase (ACS), 2-pyrone synthase (2-PS), bibenzyl synthase (BBS), and coumaroyltriacetic acid synthase (CTAS). In addition, they use different substrates to catalyze polyketide condensation to produce diverse products. Plant flavonoids are involved in regulating auxin transport, seed development, anthocyanin biosynthesis, etc., and also play a protective role against abiotic (ultraviolet radiation, cold, drought) and biotic (herbivores, bacteria, fungi) stresses. Summary of the invention

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

[0005] In order to solve this problem, the present invention provides the 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, wherein the protein is LcSFC6 protein, which is any 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) Increase the total flavonoid content of plants.

[0006] The plant stress resistance may be salt resistance.

[0007] The protein consists of 389 amino acid residues, contains a CHS domain, and belongs to the polyketidesynthases (PKSs) type III superfamily. The sequence between the 5th and 228th amino acids is the N-terminal region of Chalcone / stilbene synthase, and the sequence between the 238th and 382nd amino acids 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). In the above application, the protein may be derived from Leymus chinensis.

[0008] 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.

[0009] The above-mentioned 80% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0010] 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.

[0011] 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.

[0012] 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).

[0013] Furthermore, in the biological material, the recombinant microorganism D4) can specifically be yeast, bacteria, algae and fungi.

[0014] Furthermore, the recombinant microorganism may be Agrobacterium, and the Agrobacterium is EHA105.

[0015] Furthermore, in the biological material, D6) the plant tissue may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and / or anthers.

[0016] Furthermore, in the biological material, D7) the transgenic plant organ can be the root, stem, leaf, flower, fruit and seed of the transgenic plant.

[0017] Existing plant expression vectors can be used to construct LcSFC6 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. LcSFC6 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.

[0018] use LcSFC6 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.

[0019] 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.

[0020] 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 pCAMBIA1300-Ubi, such as pCAMBIA1300-Ubi-LcSFC6 .

[0021] 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.

[0022] 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.

[0023] In the above, the regulation refers to upregulation, improvement or increase.

[0024] 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.

[0025] 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.

[0026] In the above method, the protein is derived from Leymus chinensis.

[0027] In the above, the plant may be any one of the following: F1) monocots, F2) Gramineae, F3) Poaceae, F4) Leymus, F5) Leymus chinensis.

[0028] The present invention also provides a product, which is the above protein or biological material.

[0029] 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. LcSFC6 The expression of genes.

[0030] In order to prove the function of LcSFC6 protein, the constructedLcSFC6 The gene overexpression vector was transferred into Leymus chinensis to construct the LcSFC6 overexpression strain.

[0031] The present invention provides a key protein LcSFC6 related to salt stress of Leymus chinensis from the CHS family and its encoding gene. Experiments have shown that LcSFC6 is induced by salt stress, participates in the response of Leymus chinensis to salt stress, and improves the salt resistance of plants. LcSFC6 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

[0032] Figure 1 For PCR amplification LcSFC6 Agarose gel electrophoresis detection results of full-length cDNA.

[0033] Figure 2 Schematic diagram of the domain structure of LcSFC6 protein.

[0034] Figure 3 for LcSFC6 Gene expression results in different tissues of Leymus chinensis and after salt treatment.

[0035] Figure 4 For PCR testing LcSFC6 The results of transforming positive plants of Leymus chinensis with gene overexpression vector.

[0036] Figure 5 for LcSFC6 Phenotypic differences between gene overexpressing plants and common Leymus chinensis after salt treatment.

[0037] Figure 6 for LcSFC6 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, LcSFC6 Obtaining gene overexpression plants 1. Genes specifically expressed in Leymus chinensis under salt stress LcSFC6 Sequence cloning and overexpression vector construction 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 LcSFC6 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) 1μl, 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 11μl; 65℃ 5 min, 42℃ 45min, 70℃ 15min. The synthesized first strand cDNA was stored at -20℃ for later use.

[0043] The first-strand cDNA was used as a template and the sequenced genome sequence of Leymus chinensis was used as a reference. LcSFC Homologous gene Lc5Ns04288Primers were designed based on the CDS sequence, and primer LcSFC-F: 5′-ATGGCAACCGTCCAGCAGATCCG-3′ was paired with primer LcSFC-R: 5′-CTAGTTTTCCTCGAGGCTGCTT-3′ for PCR amplification; the PCR reaction system was: 2 μl each of cDNA template, primer LcSFC-F and primer LcSFC-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.

[0044] After the reaction, the PCR amplification products were detected by 1% agarose gel electrophoresis. Figure 1 As shown. Among them, 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 1200bp 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 1170bp, and its deoxyribonucleotide sequence was shown in sequence 1 in the sequence list, which was consistent with that in Leymus chinensis. Lc5Ns04288 The gene has a high homology, indicating that the fragment is LcSFC6 Gene sequence.

[0045] Sequence 1 (SEQ ID NO: 1, 1170 bp) is as follows:

[0046] DNAMAN and OMIGA software were used to analyze the LcSFC6 The full-length cDNA sequence was subjected to bioinformatics analysis, and the protein encoded by it, which consists of 389 amino acid residues, is sequence 2 (SEQ ID NO: 2, 389aa) in the sequence list, as follows: MATVQQIRRAQRADGPAAVLAIGTANPASSMLQDDYADYYFRVTNSDHHADHKDKLKRICKKSGIERRYAHLDEELLGAHPDFSDRALPTLDARIDMASAAVPALAASAAAKAIAEWGRPAADVTHLVFSTYSGGRAPSADFRLASLLGLRPTVSRTILSLNGCSGGGRALQLAKELAENNRGARVLVACSELTL IAFYGPQEGRLDTILGHGIFGDGAGAVVVGADPVDSVERPLFEMAFATQTTIPETEDEITMRLMKGGLDFHVSIRVPKLLKSNIERCLIDAFESIGVSATWNDLFWAIHPGGRAILDNVEELLGLDAEKLAASRRVLREYGNMSGATVIFVLDELRRRRRAMGEEVAEWGVMMAFGPGITIETMVLHATSSLEEN.

[0047] 2. LcSFC6 Sequence analysis of genes and prediction of the structure and function of their encoded proteins The domain structure of LcSFC6 was analyzed using the Pfam (http: / / pfam.xfam.org / ) server. The results showed that the protein contained a typical CHS domain and belonged to the polyketide synthases (PKSs) type III superfamily. The sequence between amino acids 5-228 was the N-terminal region of Chalcone / stilbene synthase, and the sequence between amino acids 238-382 was the C-terminal region of Chalcone / stilbene synthase. It contained 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). The above analysis results showed that LcSFC6 belonged to the CHS protein family ( Figure 2 ).

[0048] 3. LcSFC6Expression pattern analysis 3.1 LcSFC6 Analysis of expression patterns in different tissues of Leymus chinensis Total RNA was extracted from seven tissues of the 8-week-old Leymus chinensis seedlings, including the rhizome apical bud, young leaves, flag leaves, young spikes, roots, rhizomes and terminal leaves of vegetative branches. The RNA was analyzed by fluorescence quantitative PCR. LcSFC6 Variations in gene expression patterns in different tissues.

[0049] use LcGADPH The gene is used as the internal reference for the reaction and the primers are designed. LcGADPH -F: 5′-CCGTGCTCAATGGGATACTTC-3′; LcGADPH -R: 5′-CCCTCGTCTGTGACAATGGTAC-3′.

[0050] According to Leymus chinensis LcSFC6 Design specific primers for cDNA sequences: QF: 5′-CTACGTGGAACGATCTCTTCT-3′; QR: 5′-CCTCGAGGCTGCTTGTGGCGT-3′ for Q-PCR amplification. The reaction system is: SYBR GreenMix 10µL, QF 0.4µL, QR 0.4µL, ddH2O 7.2µL, 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.

[0051] The results are as follows Figure 3 As shown in A. The results show that LcSFC6 The gene was expressed at the highest level in young panicles, followed by young leaves and terminal leaves of vegetative branches, and at the lowest level in flag leaves. The expression levels were similar in roots and rhizomes.

[0052] 3.2 Leymus chinensis LcSFC6 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 hours), the total RNA of the Leymus chinensis treated with the above-mentioned different treatments was extracted and analyzed by fluorescence quantitative PCR. LcSFC6 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.

[0053] The results are as follows Figure 3 As shown in B. The results show that LcSFC6 After 1 hour of salt treatment, the expression level began to rise slowly, reaching 37 times of the untreated sample after 3 hours of treatment, and 100 times of the control sample after 6 hours of treatment. Then it began to decline after 12 hours, and after 48 hours of treatment, the relative expression level decreased to about 20 times. LcSFC6 The expression level of the gene increased significantly after salt stress.

[0054] 4. LcSFC6 Construction of gene overexpression plants The amplified product from step 1.2 above LcSFC6 gene as a template, using the upstream primer 5′- ctggGGTACC ATGGCAACCGTCCAGCAGATC-3′ ( KpnI restriction site) and downstream primer 5′- gctGAGCTCCTAGTTTTCCTCGAGGCTGC -3′( SacI The amplification product was amplified by double enzyme digestion ( KpnI and SacI ) 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 Leymus chinensis LcSFC6 Gene.

[0055] 1 μg of the above recombinant expression vector was transferred into Agrobacterium tumefaciens EHA105 strain, and the successfully transformed strain was identified to infect the callus tissue of Leymus chinensis (hereinafter referred to as wild-type Leymus chinensis), and positive buds were obtained after resistance screening and other processes.

[0056] The positive buds were identified at the DNA level. The genomic DNA of 12 T0 plants was extracted and the upstream identification primer F: 5'-CCCTGTTGTTTGGTGTTACTTCTG-3' (corresponding to pCAMBIA1300-Ubi - LcSFC6 PCR amplification was performed on the plants using the primers 5'-GCTGAAGACGAGGTGCGTGACGTC-3' (corresponding to the nucleotides 1966 to 1990 of the Ubi promoter of SEQ ID NO: 2) and downstream identification primer R: 5'-GCTGAAGACGAGGTGCGTGACGTC-3' (corresponding to the nucleotides 367 to 390 of SEQ ID NO: 2) to identify the insertion of foreign genes at the DNA level. M is a marker; lanes 1 to 12 are 12 T0 generation plants. The identification results showed that the plant was LcSFC6 The DNA electrophoresis bands of overexpression plants 1-12 and LcSFC6The recombinant plasmid electrophoresis bands were of the same size and brighter than the target band obtained by amplifying the wild-type Leymus chinensis genomic DNA, indicating that the T0 generation plants of 1-12 were LcSFC6 Positive plants (also called LcSFC6 Gene overexpression plants), identification results Figure 4 .

[0057] 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.

[0058] extract LcSFC6 After reverse transcription, the total RNA of the positive plants T2 generation and the recipient material wild-type Leymus chinensis was used for real-time fluorescence quantitative PCR of transgenic plants using quantitative primers F: 5'-CCCTGTTGTTTGGTGTTACTTCTG-3' and R: 5'-GCTGAAGACGAGGTGCGTGACGTC-3'. LcGADPH The gene was used as an internal reference gene (upstream internal reference primer: 5′-CCGTGCTCAATGGGATACTTC-3′; downstream internal reference primer: 5′- CCCTCGTCTGTGACAATGGTAC-3′), using 2 -△△CT Data processing, detection LcSFC6 Gene expression in each transgenic line.

[0059] Compared with the recipient wild-type Leymus chinensis, 3 of the 12 transgenic lines had LcSFC6 The gene expression was significantly upregulated and they were named L1, L2 and L3. LcSFC6 The expression levels of the genes were 7.5, 4.3 and 6.2 times that of the wild-type Leymus chinensis, respectively.

[0060] Example 2 L Phenotypic analysis of cSFC6 gene overexpressing plants 1. LcSFC6 Analysis of salt phenotypes in gene overexpression plants The experiment was repeated 3 times, and the experimental method was as follows: LcSFC6 Seeds of overexpression lines L1, L2 and L3 and wild-type Leymus chinensis were sown in pots filled with soil. After rooted seedlings were cultivated, they were 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. Ten plants of each line were transferred to 400mMNaCl treatment (500ml of treatment solution was directly poured into the pot) for 7 days, and then normal culture was resumed for 20 days. The phenotypic data were observed and recorded, and the survival rate was calculated. The results are as follows Figure 5The results showed that under normal growth conditions, there was no significant difference in the growth status between LcSFC6 overexpression lines L1, L2 and L3 and common Leymus chinensis W1, W2 and W3. After salt treatment, common Leymus chinensis died (survival rate was 0). Overexpression of LcSFC6 The survival rates of expression lines L1, L2, and L3 were 86.5%, 79.3%, and 72.6%, respectively. Overexpression of LcSFC6 The expression strain can grow normally. This shows that overexpression LcSFC6 It can improve the salt tolerance of plants. 2. LcSFC6 Results of physiological index measurements of gene overexpression plants after salt treatment In order to further verify the salt stress tolerance of LcSFC6 overexpressing plants, a series of physiological and biochemical indicators such as POD and SOD activities and the contents of MDA and total flavonoids of the sheepgrass materials before and after the above-mentioned 400mMNaCl treatment 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) and total flavonoids (TF) content determination kit (spectrophotometry, biosharp, BL867A). The results are as follows: Figure 6 The results showed that before salt treatment, the POD and SOD activities in LcSFC6 overexpression plants L1, L2 and L3 were slightly higher than those in wild-type Leymus chinensis W1, W2 and W3, and the total flavonoids content of the overexpression lines was also higher than that of the wild-type Leymus chinensis lines. The MDA content of the overexpression lines was similar to that of the wild type, with little difference overall. After salt treatment, the POD and SOD activities and the total flavonoids content in LcSFC6 overexpression plants were significantly higher than those in common Leymus chinensis, especially the total flavonoids content reached more than 3 times that of the wild-type Leymus chinensis, while the MDA content was less than half of that of the wild-type Leymus chinensis material, proving that the total flavonoids content of the LcSFC6 overexpression lines was increased and had a higher tolerance to salt.

[0061] 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 LcSFC6 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) Increase the total flavonoid content of plants.

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.

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