Salvia miltiorrhiza SmNCED2 gene and application of encoded protein thereof in drought resistance

Abstract

The invention discloses an application of a salvia miltiorrhiza SmNCED2 gene and an encoding protein thereof in drought resistance. The invention specifically discloses a protein with amino acid shown as SEQ ID No.1, a coding gene, a related biological material and application of the protein in regulation and control of drought resistance of salvia miltiorrhiza. The SmNCED2 gene from salvia miltiorrhiza is introduced into receptor salvia miltiorrhiza to obtain a transgenic salvia miltiorrhiza plant over-expressing the SmNCED2 gene, and drought tolerance agronomic trait identification and ABA index determination results show that compared with control salvia miltiorrhiza, the salvia miltiorrhiza plant over-expressing the SmNCED2 gene can significantly improve the drought resistance of salvia miltiorrhiza under the drought stress condition, so that the drought resistance of salvia miltiorrhiza is improved, and the drought resistance of salvia miltiorrhiza is improved. The SmNCED2 gene and the encoding protein thereof provided by the invention have important application value in regulating the drought resistance of salvia miltiorrhiza.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of a Salvia miltiorrhiza drought-resistant SmNCED2 gene and its encoded protein in drought resistance. Background Art

[0002] Salvia miltiorrhiza (Salvia miltiorrhiza) is a perennial herbaceous plant of the genus Salvia in the family Lamiaceae. It is widely cultivated in my country as a commonly used medicinal herb. With the national ban on the conversion of arable land to non-grain or non-agricultural use, recent years have seen the adaptive cultivation of Salvia miltiorrhiza in forest vacant lots, hilly wastelands, and saline-alkali land, as well as the development of new stress-resistant varieties. 9-cis-epoxycarotenoid dioxygenase (NCED) is a key gene in the plant abscisic acid (ABA) biosynthesis pathway. Changes in NCED levels affect ABA biosynthesis and participate in plant growth and development, as well as in responses to abiotic stresses. Plant growth is inhibited by adverse conditions such as drought and soil salinity. ABA, as one of the key hormones in plants, can enhance plant tolerance to abiotic stresses, thereby improving plant resistance. ABA is a sesquiterpenoid compound primarily synthesized through the mevalonate pathway (MEP) in plants. NCEDs are key rate-limiting enzymes in ABA biosynthesis. NCEDs were first discovered in maize ABA-deficient mutants, which impair ABA biosynthesis and revealed their role in regulating ABA synthesis. Subsequently, NCEDs were cloned and studied in Arabidopsis thaliana, cotton, zucchini, garlic, tea plant, and coconut. Numerous studies have demonstrated that stress can induce NCED expression, thereby regulating the synthesis of the endogenous hormone ABA and improving plant stress tolerance. For example, overexpressing the tomato LeNCED1 gene in drought-stressed petunias significantly increased ABA content and proline concentration in petunias, thereby enhancing their stress tolerance. Seven NCED genes have been identified in Asian cotton, of which overexpression of the GaNCED3 gene can enhance drought tolerance in transgenic Arabidopsis thaliana. Identification of the NCED gene family and drought stress response analysis in zucchini showed that the expression levels of six CpNCED1-6 genes were upregulated under simulated drought and natural drought stress, especially the expression levels of CpNCED2 and CpNCED4 genes were significantly different, suggesting that CpNCEDs play a positive regulatory role in the drought stress response and ABA biosynthesis process in zucchini. Summary of the Invention

[0003] The technical problem to be solved by the present invention is how to improve the stress resistance of plants (such as Salvia miltiorrhiza). The technical problem to be solved is not limited to the technical subject described above, and those skilled in the art can clearly understand other technical subjects not mentioned herein through the following description.

[0004] To solve the above technical problems, the present invention first provides an application of a protein or a substance that regulates the activity and / or content of the protein, and the application can be any of the following:

[0005] D1) Use of a protein or a substance that regulates the activity and / or content of the protein in regulating plant stress resistance;

[0006] D2) Use of a protein or a substance that regulates the activity and / or content of the protein in the preparation of a product for cultivating plant stress resistance;

[0007] D3) Use of proteins or substances that regulate the activity and / or content of said proteins in cultivating stress-resistant plants;

[0008] D4) Use of a protein or a substance that regulates the activity and / or content of the protein in the preparation of a product for cultivating stress-resistant plants;

[0009] D5) Use of proteins or substances that regulate the activity and / or content of said proteins in plant breeding;

[0010] The protein is named SmNCED2 and can be any of the following:

[0011] A1) a protein having an amino acid sequence of SEQ ID No. 1;

[0012] A2) a protein having at least 80% identity with the protein of A1) and having the same function as the protein of A1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence of SEQ ID No. 1;

[0013] A3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

[0014] In order to facilitate purification or detection of the protein in A1), a tag protein may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No. 1 in the sequence listing.

[0015] The tag protein includes but is not limited to: GST (glutathione sulfhydryl transferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0016] Those skilled in the art can readily mutate the nucleotide sequence encoding the SmNCED2 protein of the present invention using known methods, such as directed evolution or point mutagenesis. Artificially modified nucleotide sequences that are 75% or more identical to the nucleotide sequence of the isolated SmNCED2 protein of the present invention are derived from and are equivalent to the nucleotide sequence of the present invention, as long as they encode the SmNCED2 protein and possess the function of the SmNCED2 protein.

[0017] The aforementioned 75% or greater identity may be 80%, 85%, 90% or 95% or greater identity.

[0018] As used herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a search to calculate the identity of the amino acid sequence, the identity value (%) can then be obtained.

[0019] Herein, the greater than 80% 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.

[0020] Herein, the substance that regulates the activity and / or content of the protein may be a substance that regulates the expression of a gene encoding the protein SmNCED2.

[0021] In the above, the substance that regulates gene expression may be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcription level of the gene; 2) regulation after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the cell nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0022] The substance that regulates gene expression can specifically be any of the biological materials described in B1) to B3) herein.

[0023] Furthermore, the substance that regulates gene expression may be a substance (including a nucleic acid molecule or a vector) that increases or upregulates the expression of the gene encoding the protein SmNCED2.

[0024] Furthermore, the substance that regulates gene expression may also be a substance (including a nucleic acid molecule or vector) that inhibits, reduces or downregulates the expression of the gene encoding the protein SmNCED2.

[0025] In the above application, the protein SmNCED2 can be derived from Salvia miltiorrhiza.

[0026] Furthermore, the protein SmNCED2 may be the Salvia miltiorrhiza stress resistance-related protein SmNCED2.

[0027] Furthermore, the protein SmNCED2 may be the Salvia miltiorrhiza drought tolerance-related protein SmNCED2.

[0028] The present invention also provides applications of biomaterials related to the protein SmNCED2, which can be as follows:

[0029] Any of the following:

[0030] E1) Use of biomaterials related to the protein SmNCED2 in regulating plant stress resistance;

[0031] E2) Use of biomaterials related to the protein SmNCED2 in the preparation of products for regulating plant stress resistance;

[0032] E3) Use of biomaterials related to the protein SmNCED2 in cultivating stress-resistant plants;

[0033] E4) Use of biological materials related to the protein SmNCED2 in the preparation of products for cultivating stress-resistant plants;

[0034] E5) Use of biological materials related to the protein SmNCED2 in plant breeding;

[0035] The biological material may be any one of the following B1) to B7):

[0036] B1) a nucleic acid molecule encoding the protein SmNCED2;

[0037] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0038] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0039] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0040] B5) a transgenic plant cell line containing the nucleic acid molecule described in B1) or a transgenic plant cell line containing the expression cassette described in B2);

[0041] B6) transgenic plant tissue containing the nucleic acid molecule described in B1) or transgenic plant tissue containing the expression cassette described in B2);

[0042] B7) A transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2).

[0043] In the above application, the nucleic acid molecule in B1) may be any of the following:

[0044] C1) a DNA molecule whose coding sequence is SEQ ID No. 2;

[0045] C2) The nucleotide sequence of the DNA molecule is SEQ ID No. 2.

[0046] The DNA molecule (SmNCED2 gene) shown in SEQ ID No. 2 encodes the protein SmNCED2 whose amino acid sequence is SEQ ID No. 1.

[0047] The nucleotide sequence shown in SEQ ID No. 2 is the nucleotide sequence of the gene encoding protein SmNCED2 (CDS).

[0048] B1) The nucleic acid molecule may also include a nucleic acid molecule obtained by modifying the codon preference based on the nucleotide sequence shown in SEQ ID No. 2.

[0049] B1) The nucleic acid molecule also includes a nucleic acid molecule having a sequence identity of more than 95% with the nucleotide sequence shown in SEQ ID No. 2 and originating from the same species.

[0050] The gene encoding the protein SmNCED2 (SmNCED2 gene) of the present invention can be any nucleotide sequence capable of encoding the protein SmNCED2. Considering the degeneracy of codons and the codon preferences of different species, those skilled in the art can use codons suitable for expression in a specific species as needed.

[0051] The expression cassette includes a promoter, a nucleic acid molecule encoding the protein SmNCED2, and a terminator. The promoter can be a CaMV35S promoter, a NOS promoter, or an OCS promoter. The terminator can be a NOS terminator or an OCS polyA terminator.

[0052] The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0053] The vectors described herein are well known to those skilled in the art, and include, but are not limited to, plasmids, phages (such as lambda phage or M13 filamentous phage), cosmids (i.e., cosmids), Ti plasmids, or viral vectors. Specifically, they may be pTOPO-Blunt vectors and / or pCAMBIA1300 vectors.

[0054] Existing plant expression vectors can be used to construct a recombinant expression vector containing the SmNCED2 gene. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors suitable for plant microprojectile bombardment. These plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylic acid to the 3' end of the mRNA precursor. Examples include, but are not limited to, Agrobacterium crown gall-inducing (Ti) plasmid genes (e.g., the rouge synthase Nos gene) and the 3' transcribed untranslated region of plant genes (e.g., the soybean storage protein gene), all of which have similar functions.

[0055] When using the SmNCED2 gene to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CaMV) 35S promoter and the maize ubiquitin promoter. These 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, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are diverse and can be natural or synthetic. The translation initiation region can be derived from the transcription initiation region or the structural gene.

[0056] To facilitate identification and screening of transgenic plant cells or plants, the plant expression vectors used can be modified to include, but are not limited to, genes encoding enzymes or luminescent compounds that can be expressed in plants (such as the GUS gene, GFP gene, and luciferase gene), antibiotic resistance markers (such as gentamicin markers and kanamycin markers), or chemical resistance marker genes (such as herbicide resistance genes). For the safety of transgenic plants, it is possible to omit any selectable marker genes and directly screen for transformed plants using stress.

[0057] By introducing the SmNCED2 gene or gene fragment provided by the present invention into plant cells or recipient plants using any vector capable of directing exogenous gene expression in plants, stress-resistant plants with greater stress resistance than the recipient plants can be obtained. Expression vectors carrying the SmNCED2 gene can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultivated into plants.

[0058] The microorganisms described herein may be yeast, bacteria, algae, or fungi. The bacteria may be from the genera Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus, and the like. Specifically, the bacteria may be Agrobacterium tumefaciens EHA105.

[0059] The recombinant vector may specifically be the recombinant vector pCB-SmNCED2.

[0060] The recombinant vector pCB-SmNCED2 is a recombinant expression vector obtained by replacing the fragment (small fragment) between the restriction endonuclease recognition sites of the pCAMBIA1300 vector, BamH I and Kpn I, with the DNA fragment having the nucleotide sequence SEQ ID No. 2 in the sequence listing, while maintaining the other sequences of the pCAMBIA1300 vector unchanged. The recombinant vector pCB-SmNCED2 expresses the SmNCED2 protein shown in SEQ ID No. 1 in the sequence listing.

[0061] The recombinant microorganism can be obtained by introducing the recombinant vector into a microorganism.

[0062] The recombinant microorganism can specifically be recombinant Agrobacterium EHA105 / pCB-SmNCED2.

[0063] The recombinant Agrobacterium EHA105 / pCB-SmNCED2 is a recombinant bacterium obtained by introducing the recombinant vector pCB-SmNCED2 into Agrobacterium tumefaciens EHA105.

[0064] The present invention also provides a method for cultivating stress-resistant plants, which comprises increasing the content and / or activity of the protein SmNCED2 in a target plant to obtain a stress-resistant plant having higher stress resistance than the target plant.

[0065] In the above method, increasing the content and / or activity of the protein SmNCED2 in the target plant can be achieved by increasing the expression level of the gene encoding the protein SmNCED2 in the target plant.

[0066] In the above method, increasing the expression level of the gene encoding the protein SmNCED2 in the target plant can be achieved by introducing the gene encoding the protein SmNCED2 into the target plant.

[0067] In the above method, the stress-resistant plant may be a plant with improved (up-regulated) stress resistance (such as drought resistance).

[0068] In the above method, the gene encoding the protein SmNCED2 may be any one of the following:

[0069] F1) a DNA molecule whose coding sequence is SEQ ID No. 2;

[0070] F2) The nucleotide sequence of the DNA molecule is SEQ ID No. 2.

[0071] Specifically, in one embodiment of the present invention, increasing the expression level of the gene encoding the protein SmNCED2 in the target plant is achieved by introducing the DNA molecule shown in SEQ ID No. 2 into the target plant.

[0072] In one embodiment of the present invention, the method for cultivating stress-resistant plants comprises the following steps:

[0073] (1) constructing a recombinant vector comprising the DNA molecule shown in SEQ ID No. 2;

[0074] (2) introducing the recombinant vector constructed in step (1) into a target plant (such as a crop or Salvia miltiorrhiza);

[0075] (3) Obtain the stress-resistant plant through screening and identification.

[0076] The introduction refers to recombinant means, including but not limited to Agrobacterium-mediated transformation, biolistic methods, electroporation or inplanta technology.

[0077] In the above method, the plant may be any of the following:

[0078] G1) Monocotyledonous or dicotyledonous plants;

[0079] G2) Lamiaceae plants;

[0080] G3) Salvia;

[0081] G4) plants from the Salvia miltiorrhiza group;

[0082] G5) Salvia miltiorrhiza.

[0083] The salvia miltiorrhiza can specifically be the salvia miltiorrhiza variety Jidan No. 4.

[0084] The protein SmNCED2 and / or the biological material also fall within the protection scope of the present invention.

[0085] Herein, the stress resistance may be drought tolerance.

[0086] Herein, the plant may be a crop (eg, an agricultural crop).

[0087] The present invention also provides the use of the method for cultivating stress-resistant plants in creating stress-resistant plants, and / or in plant breeding or plant germplasm resource improvement.

[0088] The plant breeding described herein may be crop stress resistance breeding.

[0089] The stress-resistant plants may be drought-resistant plants, but are not limited thereto.

[0090] The regulation of plant stress resistance described herein may be upregulation (increase) or downregulation (reduction) of plant stress resistance.

[0091] Furthermore, the regulating plant stress resistance may be up-regulating (increasing) or down-regulating (reducing) the drought resistance of Salvia miltiorrhiza.

[0092] As used herein, transgenic plants are understood to include not only first-generation transgenic plants obtained by transforming the target plant with the SmNCED2 gene or knocking out the SmNCED2 gene, but also their progeny. The gene can be propagated within the species or transferred into other varieties of the same species, particularly commercial varieties, using conventional breeding techniques. Transgenic plants include seeds, callus tissue, whole plants, and cells.

[0093] The present invention introduces the SmNCED2 gene, which regulates plant stress resistance, from Salvia miltiorrhiza into a recipient plant, the Salvia miltiorrhiza variety Jidan No. 4, to obtain transgenic Salvia miltiorrhiza plants that overexpress the SmNCED2 gene. The transgenic plants are then subjected to drought resistance identification. Comprehensive measurement results of various physiological and biochemical indices show that, compared with the uncontrolled Salvia miltiorrhiza variety Jidan No. 4 (WT), the transgenic Salvia miltiorrhiza plants that overexpress the SmNCED2 gene have significantly improved stress resistance under drought stress conditions, namely, the drought resistance of the transgenic Salvia miltiorrhiza plants that overexpress the SmNCED2 gene is significantly improved.

[0094] Experiments have demonstrated that the SmNCED2 protein and its encoding gene, SmNCED2, disclosed herein can regulate plant stress tolerance (e.g., drought resistance). The drought-resistance-related protein SmNCED2 and its encoding gene provided herein can enhance plant stress tolerance. Overexpression of the SmNCED2 gene in Salvia miltiorrhiza can improve the plant's drought resistance. Therefore, the drought-resistance-related gene SmNCED2 and its encoded protein have important theoretical and practical value in regulating plant drought resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 PCR amplification results of SmNCED2 gene transgenic Salvia miltiorrhiza

[0096] Figure 2 The expression of SmNCED2 gene in transgenic positive plants of Salvia miltiorrhiza

[0097] Figure 3 The growth status of transgenic Salvia miltiorrhiza plants under drought stress

[0098] Figure 4 ABA content in transgenic Salvia miltiorrhiza plants under drought stress DETAILED DESCRIPTION

[0099] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0100] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. They were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0101] Example 1: Obtaining drought-resistant genes and their encoded proteins

[0102] The cDNA of SmNCED2 protein from Salvia miltiorrhiza was cloned. The Salvia miltiorrhiza variety Jidan 4 was used as the experimental material.

[0103] 1. Extraction of total RNA from Salvia miltiorrhiza

[0104] 0.1 g of young leaves of Salvia miltiorrhiza were ground into powder in liquid nitrogen and added to a 2 mL centrifuge tube. Total RNA of Salvia miltiorrhiza was extracted using TIANGEN's RNApreppure Plant Total RNA Extraction Kit (Catalog Number: DP432). The kit includes: lysis buffer RL, deproteinization buffer RW1, rinse buffer RW, RNase-Free ddH2O, RNase-Free adsorption column CR3, RNase-Free filter column CS, DNase I, buffer RDD, RNase-Free centrifuge tube, and RNase-Free collection tube. Take 10 μL and run it on 1.0% agarose gel electrophoresis to detect its integrity, take another 2 μL and dilute it to 500 μL, and use UV spectrophotometer to detect its quality (OD260) and purity (OD260 / OD280). The total RNA extracted from the Salvia miltiorrhiza variety Jidan No. 4 was detected by non-denaturing gel agarose gel electrophoresis. The 28S and 18S bands were clear, and the brightness ratio of the two was 1.5 to 2:1, indicating that the total RNA was not degraded. The obtained mRNA met the experimental requirements and could be used for cloning the full-length cDNA of the Salvia miltiorrhiza SmNCED2 protein.

[0105] 2. Full-length cloning of SmNCED2 protein cDNA

[0106] Primers for the SmNCED2 gene were designed based on the Salvia miltiorrhiza transcriptome database constructed in our laboratory, and the full-length cDNA of the SmNCED2 protein was cloned. The gene sequence of the SmNCED2 gene was obtained from the Salvia miltiorrhiza transcriptome database, and primers for the 5′- and 3′-ends of the SmNCED2 gene were designed for PCR reactions. The primer sequences are as follows:

[0107] Primer 1: 5′-ATGAGAAATCATTCAACAAG-3′

[0108] Primer 2: 5′-TCACCACACCTGCTTCTCCA-3′

[0109] The full-length ORF of the SmNCED2 gene was obtained by PCR, recovered, connected to the pTOPO-Blunt vector for TA cloning, and sequenced using the M13F / M13R universal primers.

[0110] The total RNA extracted above was reverse transcribed using QuantScript RT Kit (TIANGEN, Beijing) as a template and PCR amplification was performed using the high-fidelity FastPfu enzyme. The PCR amplification product was detected by agarose gel electrophoresis, and an amplified fragment of 1665 bp in length was obtained.

[0111] After sequencing, the PCR product has the nucleotides shown in SEQ ID No. 2 in the sequence listing, the gene shown in the sequence is named SmNCED2, the coding region of the gene is nucleotides 1 to 1665 from the 5' end of SEQ ID No. 2 in the sequence listing; SEQ ID No. 2 in the sequence listing consists of 1665 bases; the protein encoded by the gene is named SmNCED2, and the amino acid sequence of the protein is SEQ ID No. 1 in the sequence listing; SEQ ID No. 1 in the sequence listing consists of 554 amino acids.

[0112] Example 2: Application of Salvia miltiorrhiza SmNCED2 protein in improving plant drought resistance

[0113] 1. Construction of plant expression vector

[0114] Based on the coding sequence of the SmNCED2 protein cDNA of Salvia miltiorrhiza, primer sequences were designed to amplify the complete coding sequence. The forward and reverse primers introduced BamH I and Kpn I restriction sites, respectively. The primer sequences are as follows:

[0115] Primer 3: 5'-CGGGATCCATGAGAAATCATTCAACAAG-3' (the underlined part is the BamHI restriction site),

[0116] Primer 4: 5'- GGGGATCC TCACCACACCTGCTTCTCCA-3' (the underlined part is the Kpn I restriction enzyme cleavage site).

[0117] Using SEQ ID No. 2 in the artificially synthesized sequence list as a template, after PCR amplification, the product was ligated into the pTOPO-Blunt vector (purchased from Beijing Adlai Biotechnology Co., Ltd., product catalog number CV16), named pTOPO-SmNCED2 vector, and M13F / M13R sequencing was performed to ensure the correct reading frame and restriction enzyme cleavage sites of the Salvia miltiorrhiza SmNCED2 protein cDNA.

[0118] The expression vector pCambia1300-GFP was digested with BamHI and KpnI to recover the large vector fragment. Simultaneously, the vector pTOPO-SmNCED2 was digested with BamHI and KpnI to recover the approximately 1.6 kb intermediate fragment. The recovered large vector fragment was ligated with the approximately 1.6 kb intermediate fragment to obtain the target plasmid. The target plasmid was transformed into Escherichia coli DH5a (purchased from Beijing Quanshijin Biotechnology Co., Ltd., catalog number CD201-01) and incubated at 37°C for 20 hours. The recombinant vector was analyzed by PCR and enzyme digestion, and verified by sequencing. Sequencing results revealed that the sequence shown in SEQ ID No. 2 (from positions 1 to 1665 of the 5′ end) was inserted between the BamHI and KpnI restriction sites of the pCambia1300 vector, indicating that the recombinant vector was constructed correctly. The recombinant vector was named pCB-SmNCED2.

[0119] 2. Transformation of plant expression vector into Agrobacterium

[0120] (1) Take out 200 μL of EHA105 competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) from a -80°C freezer, thaw on ice, add 1 μg of the plant expression vector pCB-SmNCED2 obtained in step 1 above, and mix well.

[0121] (2) Freeze in liquid nitrogen for 1 min and incubate at 37°C for 5 min.

[0122] (3) Add 800 μL of LB liquid culture medium and culture at 28°C for 2-6 hours.

[0123] (4) Take 100 μL of bacterial solution and spread it evenly on LB solid medium (containing 100 μg / mL rifampicin (Rif) and 50 μg / mL kanamycin (Kan)). Seal the culture dish and incubate it at 28°C for 2 days.

[0124] (5) A single colony that was positive for PCR was inoculated into LB liquid medium containing 100 μg / mL Rif and 50 μg / mL Kan. The culture was cultured at 28°C for 30 h until the logarithmic growth phase. An appropriate amount of Agrobacterium was diluted 50-fold with liquid MS medium for later use. This yielded an Agrobacterium culture solution containing the pCB-SmNCED2 vector.

[0125] 3. Genetic transformation and regeneration of SmNCED2-transfected Salvia miltiorrhiza

[0126] The cDNA coding sequence of SmNCED2 was introduced into Salvia miltiorrhiza using Agrobacterium-mediated transfection. The specific method is as follows:

[0127] (1) Take the leaves of sterile Salvia miltiorrhiza seedlings that have been subcultured for 4-6 weeks, cut 5×5 Salvia miltiorrhiza leaf discs (remove the main leaf veins) in a clean bench, and suspend them in the EHA105 / pCB-SmNCED2 Agrobacterium solution prepared in step 2 above. After 10 minutes, inoculate the infected Salvia miltiorrhiza leaf discs on solid culture medium (MS with 1.0 mg / L 6-BA and 0.1 mg / L NAA) and culture them in the dark at 28°C for 3 days.

[0128] (2) After 3 days of co-cultivation, the leaf discs of Salvia miltiorrhiza were washed twice with MS liquid medium containing 500 mg / L Car, 1.0 mg / L 6-BA, and 0.1 mg / L NAA. The leaf discs were then transferred to solid MS medium containing 1.0 mg / L 6-BA, 0.1 mg / L NAA, and 100 mg / L Kam for selection culture. The culture conditions were 28°C, 13 hours per day, and 3000 lx light intensity. After 4-6 weeks of culture, the adventitious buds of Salvia miltiorrhiza were transferred to 1 / 2 MS medium containing 1.0 mg / L 6-BA, 0.1 mg / L NAA, and 100 mg / L Kam for adventitious root induction. The culture conditions were 28°C, 13 hours per day, and 3000 lx light intensity. After 4-8 weeks, complete regenerated plants were formed, and the Salvia miltiorrhiza plants intended to be transformed with the SmNCED2 gene were obtained.

[0129] (3) Genomic DNA from the proposed transgenic Salvia miltiorrhiza plants and the control Salvia miltiorrhiza plants was extracted using the CTAB method. PCR detection was performed using conventional methods. The primers used were: Primer 5 (with vector sequence): 5'-AATTCGAGCTCATGAGAAATCATTCAACAAG-3', Primer 6 (with vector sequence): 5'-GTCGACTCTATCACCACACCTGCTTCTCCA-3'. In a 0.2 mL Eppendorf centrifuge tube, 2 μL of 10× PCR buffer, 1 μL of 4 dNTPs (10 mol / L), 1 μL of each primer (10 μmol / L), 2 μL of template DNA (50 ng / μL), and 1 μL of Taq DNA polymerase were added. H2O was added to a total volume of 20 μL. The reaction procedure was denaturation at 94°C for 4 min, annealing at 58°C for 1 min, and extension at 72°C for 2 min, for a total of 35 cycles.

[0130] The experimental results are shown in Figure 1(Marker is DNA molecular weight, water is negative control, plasmid is positive control, CK is wild type plant of Salvia miltiorrhiza variety Jidan No. 4), the results showed that a total of 5 transgenic positive plants were obtained, named The transgenic Salvia miltiorrhiza plants were propagated and their drought resistance was identified and the ABA content was determined.

[0131] 4. Detection of the relative expression of the SmNCED2 gene

[0132] Positive transgenic plants (OE-2 and OE-5) and control plants (CK) were subcultured, ensuring consistent culture conditions for all plants. After a period of time, RNA was extracted from the entire plant or leaves from the same site and reverse transcribed. qRT-PCR was performed to measure SmNCED2 gene expression in each line. The cDNA was diluted to a uniform concentration of 100 ng / μL for each sample. qRT-PCR was performed on an ABI PRISM 7500 (operating software: 7500 and 7500 Fast Real-Time PCR Systems, v2.0.1, USA) according to the instructions in the PerfectStart Green qPCR SuperMix kit. A 20 μL reaction system consisted of the following: 10 μL PerfectStart Green qPCR SuperMix, 0.4 μL Primer-F, 0.4 μL Primer-R, 2.0 μL cDNA (1 ng), and 7.2 μL ddH2O. Amplification conditions: 95℃30s; 95℃5s, 60℃30s, 40 cycles in total; stored at 4℃. Each reaction was repeated 3 times, using 2 -△△CT The expression level of SmNCED2 gene in Salvia miltiorrhiza was analyzed by the method.

[0133] The primers used to detect the SmNCED2 gene are as follows:

[0134] qSmNCED2-F: 5'-GAGCTCCACGGCCACCTCGG-3'

[0135] qSmNCED2-R: 5'-GATCGCCGGAGGGGGTGACG-3'

[0136] The primers used to detect the Actin gene are as follows:

[0137] SmActin-F: 5'-TGCTGTGCTGAGGACGATAC-3'

[0138] SmActin-R: 5'-CCATGAGCCTCCAAACCTAA-3'

[0139] The results of the relative expression of SmNCED2 gene are shown in Figure 2 The results showed that the expression level of the SmNCED2 gene in the transgenic Salvia miltiorrhiza overexpressing the SmNCED2 gene was significantly higher than that in the control variety, Jidan No. 4. The difference was significant. The SmNCED2 gene was not only successfully integrated into the recipient genome but also transcribed and expressed normally in the transgenic Salvia miltiorrhiza.

[0140] 5. Identification of drought resistance of transgenic Salvia miltiorrhiza

[0141] 5.1 Phenotypic identification

[0142] Two SmNCED2 gene-overexpressing Salvia miltiorrhiza plants (OE-2, OE-5) and a control Salvia miltiorrhiza (CK) were inoculated on a culture medium (MS+IBA0.5 mg / L+NAA0.2 mg / L) for propagation and culture. After one month of greenhouse cultivation, drought stress was applied. Figure 3 As shown in the results, under drought stress, the growth and development of SmNCED2-transgenic Salvia miltiorrhiza was better than that of the control Salvia miltiorrhiza. The identification results showed that overexpression of SmNCED2 gene could provide drought resistance to Salvia miltiorrhiza.

[0143] 5.2 ABA content determination

[0144] Abscisic acid (ABA) is a key stress-resistant factor in plants and is involved in the plant's response to various adverse stresses. For example, when plants suffer from stresses such as drought, low temperature, salt damage, and disease, ABA will rapidly accumulate in the plant body, thereby inducing the upregulation of a series of stress-resistant genes that depend on ABA signals, thereby improving plant stress resistance.

[0145] (1) Sample preparation and extraction

[0146] First, weigh approximately 0.5 grams of transgenic Salvia miltiorrhiza plant sample and grind it in liquid nitrogen until finely ground. Then, add 5 milliliters of isopropanol / hydrochloric acid buffer and shake at 4°C for 30 minutes. Next, add 10 milliliters of dichloromethane and shake again at 4°C for 30 minutes. Finally, centrifuge at 13,000 rpm at 4°C for 5 minutes, and remove the organic phase.

[0147] (2) Purification

[0148] The organic phase was dried with nitrogen in the dark, then dissolved with 250-500 μl of methanol (containing 0.1% formic acid), and finally filtered through a 0.45 μm microporous filter membrane for analysis.

[0149] (3) Liquid chromatography analysis

[0150] Liquid chromatography conditions included an Agilent C18 ZORBAX reversed-phase column (150 mm x 4.6 mm, 5 μm), a column temperature of 30°C, a flow rate of 1 mL / min, an injection volume of 20 μL, and a detection wavelength of 265 nm. The flow rate was methanol:acetonitrile:0.6% acetic acid = 40:5:55.

[0151] like Figure 4 The figure shows the changes in ABA content in transgenic Salvia miltiorrhiza plants under drought stress. After being subjected to drought stress, the SmNCED2 gene-transgenic Salvia miltiorrhiza plants accelerated the synthesis of endogenous ABA to cope with the external adverse environment. Therefore, overexpression of the SmNCED2 gene can increase ABA biosynthesis in Salvia miltiorrhiza, thereby improving the drought resistance of Salvia miltiorrhiza.

Claims

1. The use of protein, characterized in that The application is any of the following: D1) Application in increasing plant stress tolerance; D2) Application in cultivating stress-tolerant plants; The protein is any one of the following: A1) Protein whose amino acid sequence is SEQ ID No. 1 A2) a fusion protein having the same function as A1) obtained by connecting a tag to the N-terminus and / or C-terminus; The stress resistance is to improve the drought resistance of plants, and the plant is a dicotyledonous plant Salvia miltiorrhiza.

2. The use according to claim 1, characterized in that The protein is derived from Salvia miltiorrhiza.

3. Application, characterized in that, The application is any of the following: E1) Use of a biological material related to the protein according to any one of claims 1 to 2 for increasing drought tolerance in plants; E2) Use of a biological material related to the protein according to any one of claims 1 to 2 in cultivating drought-tolerant plants; The biological material is any one of the following B1) to B7): B1) a nucleic acid molecule encoding the protein according to any one of claims 1 to 2; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) a transgenic plant cell line containing the nucleic acid molecule described in B1) or a transgenic plant cell line containing the expression cassette described in B2); B6) transgenic plant tissue containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B7) transgenic plant organ containing the nucleic acid molecule described in B1) or the expression cassette described in B2); The plant is a dicotyledonous plant Salvia miltiorrhiza.

4. The use according to claim 3, characterized in that B1) The nucleic acid molecule is any one of the following: C1) a DNA molecule whose coding sequence is SEQ ID No. 2; C2) The nucleotide sequence of the DNA molecule is SEQ ID No.

2.

5. A method for cultivating drought-tolerant plants, characterized in that: The method comprises increasing the content of the protein according to any one of claims 1 to 2 in a target plant, thereby obtaining drought resistance higher than that of the target plant, wherein the plant is a dicotyledonous plant Salvia miltiorrhiza.

6. The method according to claim 5, characterized in that The increasing of the content of the protein according to any one of claims 1 to 2 in the target plant is achieved by increasing the expression level of the gene encoding the protein in the target plant.

7. The method according to claim 6, characterized in that The increasing of the expression level of the gene encoding the protein in the target plant is achieved by introducing the gene encoding the protein according to any one of claims 1 to 2 into the target plant.

8. The method according to claim 7, characterized in that The protein encoding gene is any one of the following: F1) a DNA molecule whose coding sequence is SEQ ID No. 2; F2) The nucleotide sequence of the DNA molecule is SEQ ID No.

2.

9. The protein according to any one of claims 1-2, or the biomaterial according to B1) to B4) in claim 3.

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