Leymus chinensis epidermal wax synthetic gene LcMAH1 and application thereof, protein, recombinant vector and method

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to a Leymus chinensis epidermal wax synthetic gene LcMAH1 and application thereof, protein, a recombinant vector and a method. The nucleotide sequence of the Leymus chinensis epidermal wax synthetic gene LcMAH1 is as shown in SEQ ID No. 1. Agrobacterium is utilized to infect tobacco mesophyll cells, an agrobacterium inflorescence dipping method is utilized to infect arabidopsis thaliana, so that the gene is introduced into tobacco for transient expression and is over-expressed in an arabidopsis thaliana model plant, phenotypic analysis is carried out on a transgenic arabidopsis thaliana plant, the gene function of the LcMAH1 is known, and the LcMAH1 can be used for improving the drought-enduring character of the plant.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to a Leymus chinensis epidermal wax synthesis gene LcMAH1 and its application, protein, recombinant vector and method. Background Art

[0002] Drought stress is one of the environmental stressors that affects plant growth and development, severely limiting the production of both food and cash crops. Drought stress causes water loss in plant cells, leading to osmotic stress and cell death. Therefore, cloning key regulatory genes for drought tolerance, studying the molecular mechanisms of plant drought tolerance, and cultivating drought-tolerant crops are of great practical and strategic significance.

[0003] Plant cuticular waxes are a mixture of very long chain (VLC) lipids produced and secreted by the aerial epidermis of terrestrial plants. These lipids aggregate to form a waterproof barrier called the cuticle. Plant cuticular waxes play a key role in helping plants cope with drought stress by limiting non-stomatal water loss from leaves. They also play an important role in maintaining plant surface cleanliness, combating UV damage, protecting against biological invasions such as viruses, bacteria, and insects, and protecting fruits from disease.

[0004] The composition of plant cuticular waxes is highly complex and varies across plant species, tissues, and developmental stages. Numerous studies have found that cuticular waxes are primarily composed of a mixture of C20-C34 very-long-chain fatty acids (VLCFAs) and their derivatives. These derivatives primarily include aldehydes, alkanes, ketones, primary alcohols, secondary alcohols, and esters. In recent years, the cuticular wax biosynthesis pathway in the model plant Arabidopsis thaliana has been extensively studied. This biosynthesis occurs primarily in epidermal cells, primarily involving transport through the plasmids and endoplasmic reticulum (ER), and via both the Golgi and non-Golgi pathways. Pathways involved in plant cuticular wax production primarily include the alcohol biosynthesis pathway and the alkane biosynthesis pathway. In the alkane biosynthesis pathway, MAH1 (MAH1 protein stands for midchain alkane hydroxylase 1) is a key regulatory gene in the alkane biosynthesis pathway. It further catalyzes the conversion of alkanes to secondary alcohols and ketones. However, the role of MAH1 in drought has not been reported. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a sheep fescue epidermal wax synthesis gene LcMAH1 and its application, protein, recombinant vector and method. Overexpression of the sheep fescue epidermal wax synthesis gene LcMAH1 can improve the drought resistance of plants, and the gene can be better applied to the drought resistance genetic improvement of plants and crops.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a Leymus chinensis epidermal wax synthesis gene LcMAH1. The nucleotide sequence of the Leymus chinensis epidermal wax synthesis gene LcMAH1 is shown as SEQ ID No.1.

[0008] The present invention also provides the use of the Leymus chinensis epidermal wax synthesis gene LcMAH1 described in the above technical solution in regulating plant drought resistance.

[0009] Preferably, overexpression of the epidermal wax synthesis gene LcMAH1 in Leymus chinensis positively regulates plant drought tolerance.

[0010] Preferably, the plant comprises Arabidopsis thaliana.

[0011] The present invention also provides a protein encoded by the Leymus chinensis epidermal wax synthesis gene LcMAH1 described in the above technical solution, and the amino acid sequence of the protein is shown in SEQ ID No.2.

[0012] The present invention also provides a recombinant vector, and the Leymus chinensis epidermal wax synthesis gene LcMAH1 described in the above technical solution is connected to a vector to obtain a recombinant vector.

[0013] Preferably, the vector comprises pCAMBIA1300-GFP vector.

[0014] The present invention also provides a method for obtaining drought-tolerant plants, comprising the following steps:

[0015] 1) Transforming the recombinant vector described in the above technical solution into Agrobacterium to obtain transformed bacteria;

[0016] 2) The transformed bacteria obtained in step 1) are used to infect plants using the inflorescence dipping method and cultured conventionally.

[0017] Preferably, the Agrobacterium in step 1) includes Agrobacterium EHA105.

[0018] Preferably, in step 2), the transformed bacteria infects the plant in the form of a bacterial solution, and the OD of the bacterial solution is 600 The value is 0.8;

[0019] The infection time is 10 to 15 minutes;

[0020] The plants include Arabidopsis thaliana.

[0021] Beneficial effects of the present invention:

[0022] The present invention uses Leymus chinensis as experimental material, adopts a transcriptome high-throughput sequencing method to obtain the nucleotide sequence of the Leymus chinensis resistance-related gene LcMAH1, uses molecular cloning technology to obtain the gene, constructs a pCAMBIA1300 plant expression vector, uses Agrobacterium to infect tobacco mesophyll cells, and uses an Agrobacterium inflorescence dip method to infect Arabidopsis thaliana, so that the gene is introduced into tobacco for transient expression and overexpressed in Arabidopsis thaliana model plants. Then, by performing phenotypic analysis on transgenic Arabidopsis thaliana plants, the gene function of LcMAH1 is understood, and LcMAH1 can be used to improve the drought resistance trait of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0024] Figure 1 This is the electrophoresis diagram of LcMAH1 gene cloning;

[0025] Figure 2 This is the subcellular localization map of LcMAH1 protein in tobacco mesophyll cells;

[0026] Figure 3 Figure 2 shows the phenotypes of wild-type Arabidopsis and transgenic plants under drought stress. DETAILED DESCRIPTION

[0027] The present invention provides a Leymus chinensis epidermal wax synthesis gene LcMAH1, the nucleotide sequence of the Leymus chinensis epidermal wax synthesis gene LcMAH1 is shown in SEQ ID No. 1, and is specifically as follows:

[0028]

[0029] The present invention also provides the use of the Leymus chinensis epidermal wax synthesis gene LcMAH1 described in the above technical solution in regulating plant drought tolerance. In the present invention, overexpression of the Leymus chinensis epidermal wax synthesis gene LcMAH1 positively regulates plant drought tolerance. In the present invention, the plant preferably includes Arabidopsis thaliana.

[0030] The present invention also provides a protein encoded by the Leymus chinensis epidermal wax synthesis gene LcMAH1 described in the above technical solution, the amino acid sequence of the protein is shown in SEQ ID No. 2, and is as follows:

[0031] MASVSFLELSLSLLCFVVFYYFHVRSKRKNPVIPLEWPLVGMLPALLANLPRLHDWVTSLLTASPLNFRFVGPPRSGMELFLTSDPANVRHVFTSNFPNYPKGPDFAEIMDILGGGIFNADGDSWRR QRAKAQMLMSGPRFRAFVTRCSRRKVERDLLPLLAHVAAGAGVCDLQDVFLRLTFDTTTTLVFGVDPGCLTIGFPEVPFARAMDDAMDVLLVRNVLPPSWWKLVRWLGVGYERKMAVAWRDIDRFIG DTIAKRREAVKARGGIEDSADLLSSYIDDDDDDSTVVDAFLRDTTTMNLMLAGRDTTGSGLSWFFYLLLTRNPRVVSKILAELDTVNSTTTPDGMVTYDPDELGRLVYLHAALCESLRLYPPVPMEHKG VVAAEALPSGHEVRPGDKVMVSLYAMGRMEAVWGKDCREFRPERWIGEDGKPRYVPSYKFVSFNSGPRTCLGKDMAFVQLKAVAAAVVRNFEVEAVPGHVVEPKISIILHIKNGFKARIKRRQVLD-.

[0032] The present invention also provides a recombinant vector, wherein the Leymus chinensis epidermal wax synthesis gene LcMAH1 described in the above technical solution is connected to a vector to obtain a recombinant vector. In the present invention, the vector preferably includes a pCAMBIA1300-GFP vector. The present invention does not particularly limit the method for connecting the Leymus chinensis epidermal wax synthesis gene LcMAH1 to the vector, and those skilled in the art can use conventional methods.

[0033] The present invention also provides a method for obtaining drought-tolerant plants, comprising the following steps:

[0034] 1) Transforming the recombinant vector described in the above technical solution into Agrobacterium to obtain transformed bacteria;

[0035] 2) The transformed bacteria obtained in step 1) are used to infect plants using the inflorescence dipping method and cultured conventionally.

[0036] The present invention does not specifically limit the method for transforming the recombinant vector into Agrobacterium, and those skilled in the art can use conventional methods. In the present invention, the Agrobacterium preferably includes Agrobacterium EHA105.

[0037] In the present invention, the transformant is preferably used to infect plants in the form of a bacterial solution, and the OD of the bacterial solution is 600 The value is 0.8. In the present invention, the infection time is preferably 10 to 15 minutes. In the present invention, the plant preferably includes Arabidopsis thaliana.

[0038] In order to further illustrate the present invention, the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] Obtaining the gene encoding LcMAH1 protein from Leymus chinensis and constructing its expression vector

[0041] 1.1 Extraction of total RNA from Leymus chinensis seedlings

[0042] RNA was extracted using Trizol method

[0043] (1) Transfer the harvested young leaves of Leymus chinensis to a liquid nitrogen pre-cooled mortar (sterilized by high temperature and high pressure), add a small amount of liquid nitrogen and grind rapidly several times until fully ground. Transfer to an RNase-free 2 ml centrifuge tube, then add 1 ml of Trizol and shake to mix.

[0044] (2) To fully lyse the above mixture, let it stand at room temperature for 5 minutes;

[0045] (3) Add 200 μl of chloroform solution to the above mixture, gently shake and mix for 15 seconds, and then let it stand at room temperature for 3 minutes;

[0046] (4) After low-temperature centrifugation (4°C, 12,000 rpm, 15 min), the mixture was separated into phases, and the colorless supernatant solution was carefully pipetted into a new RNase-free centrifuge tube;

[0047] (5) Add an equal volume of isopropanol solution to the supernatant, mix the mixture thoroughly, and let it stand on ice for 10 minutes;

[0048] (6) After low-temperature centrifugation (4°C, 12,000 rpm, 10 min), a white precipitate appeared at the bottom or wall of the tube. Discard the supernatant, add 1 ml of 75% ethanol prepared with DEPC water, flick the precipitate to wash it, and centrifuge at low temperature (4°C, 12,000 rpm, 5 min).

[0049] (7) Repeat step 6 to thoroughly wash the precipitate;

[0050] (8) Discard the supernatant and place on ice until the precipitate is fully dry;

[0051] (9) Dissolve the precipitate in 50-100 μl of DEPC water, test its concentration and purity, and store the RNA sample at -80°C.

[0052] 1.2 Synthesis of the first strand of cDNA ( Ⅲ1st Strand cDNA Synthesis SuperMixfor qPCR)

[0053] (1) Removal of residual genomic DNA (prepared on ice):

[0054] Table 1 Reaction system

[0055]

[0056]

[0057] Prepare the above mixture in an RNase-free centrifuge tube, gently pipette to mix, and incubate at 42°C for 2 minutes.

[0058] (2) Preparation of reverse transcription reaction system (20 μl system, prepared on ice):

[0059] Directly add the following into the reaction tube in step (1): IIISuperMix plus, mix gently using a pipette.

[0060] Table 2 Reaction system

[0061]

[0062] (3) Mix slowly and place in a 25°C water bath for 5 min;

[0063] (4) 55°C water bath for 15 min;

[0064] (5) Store in a water bath at 85°C for 5 minutes, place on ice and then store at -20°C, or aliquot and store at -80°C for long-term storage.

[0065] 1.3 Cloning primer design

[0066] Primers required for gene cloning were designed using Primer Premier 5.0 and synthesized by Sangon Biotechnology Co., Ltd. (see Table 3 for details).

[0067] Table 3 Gene cloning primers

[0068] Primer name Sequence (5'-3') LcMAH1-F (SEQ ID No. 3) ATGGCGTCCGTCTCCTTCCTC LcMAH1-R (SEQ ID No. 4) TTAGTCAAGCACCTGCCTCCTCTTG

[0069] 1.4 Target gene amplification

[0070] PCR was performed on the cDNA using Leymus chinensis cDNA as a template and gene-specific primers (see Table 3). The amplification procedure was as follows: 94°C pre-denaturation for 2 minutes, one cycle; 94°C denaturation for 30 seconds, 57°C annealing for 45 seconds, 72°C extension for 2 minutes 15 seconds, 30 cycles; and 72°C extension for 10 minutes, one cycle. The amplified gene products were examined by 1.0% agarose gel electrophoresis to determine if the target gene band size was correct. Figure 1 ).

[0071] Table 4 PCR reaction system

[0072]

[0073]

[0074] 1.5 Rubber recycling

[0075] The target band was recovered using a conventional agarose gel DNA recovery kit from Kangwei Century Biotechnology Co., Ltd. For specific experimental methods, refer to the instructions.

[0076] 1.6 Ligation of PCR products with T-vector and transformation of E. coli

[0077] (1) Ligation of PCR products with T vectors

[0078] The recovered PCR product was ligated to the pMD19-T vector using TaKaRa's pMD19-T vector Cloning Kit. The reaction system was as follows:

[0079] Table 5 Ligation reaction system of LcMAH1 gene and pMD19-T vector

[0080] Reaction solution Volume (μl) Recovered PCR products 4 pMD19-T vector 1 solution I 5 Total 10

[0081] Connect overnight in a 16°C constant temperature water bath (12-16 hours)

[0082] (2) Transformation of Escherichia coli

[0083] 1) Thaw 100 μl of prepared E. coli (DH5α) competent cells on ice;

[0084] 2) Add 10 μl of the ligation product to the thawed competent cells, mix gently, and place on ice for 30 minutes;

[0085] 3) Heat shock the cells in a 42°C water bath for 1 minute, then quickly place them in an ice bath for 2 minutes to cool them down. Be gentle and avoid shaking to prevent the competent cells from rupturing.

[0086] 4) Add 400 μl of LB liquid medium (preheated at 37°C for 2 min) to the centrifuge tube, and then shake in a constant temperature shaker at 37°C, 200 rpm for 1 h;

[0087] 5) The transformed E. coli culture was evenly spread on LB screening solid medium (50 mg / ml Amp);

[0088] 6) After the bacterial solution is completely absorbed on the LB screening solid medium, incubate the culture inverted in a 37°C constant temperature incubator overnight (12-16 hours).

[0089] (3) Shake the bacteria and identify the bacterial solution

[0090] A single strain of moderate size was selected and placed in 5 ml of LB liquid medium (50 mg / ml Amp) and cultured in a constant temperature shaker at 37°C, 200 rpm for 12 hours. A 1 μl aliquot of the bacterial culture was then analyzed by PCR. The correct target gene band was then detected by 1.0% agarose gel electrophoresis.

[0091] Table 6 PCR reaction system for bacterial liquid identification

[0092] Reaction solution Volume (μl) EX-Taq 10 Forward primer (10M) 1 Reverse primer(10M) 1 bacterial liquid 1 <![CDATA[ddH2O]]> 7 Total 20

[0093] (4) Strain preservation and sequence determination

[0094] 200 μl of bacterial solution was mixed with 200 μl of 50% glycerol (autoclaved) and stored at -80°C. The E. coli solution containing LcMAH1 that had been correctly identified was sent to Sangon Biotech for sequencing. The sequencing results were assembled and analyzed to obtain the correct sequence of LcMAH1.

[0095] 1.7 Plasmid extraction using kit method

[0096] Use the TIANGEN plasmid extraction kit to extract the plasmid from the cultured bacterial solution. Refer to the instruction manual for the experimental method.

[0097] 1.8 Primer design for adding restriction enzyme cutting sites

[0098] Primers with restriction enzyme sites were designed using primer design software Primer Premier 5.0 and synthesized by Sangon Biotechnology Co., Ltd. The primers are listed in Table 7.

[0099] Table 7 Primers for adding restriction enzyme sites

[0100] Primer name Sequence (5'-3') LcMAH1-F(K) (SEQ ID No. 5) GGTACCTTATGGCGTCCGTCTCCTTC LcMAH1-R(K) (SEQ ID No.6) TCTAGAGTCAAGCACCTGCCTCCTCTTG

[0101] Use primers containing restriction sites to add restriction sites to the target gene through PCR. Use the same PCR reaction system and parameters as in 1.4. Recover the fragment of the correct size using the same gel extraction method as in 1.5. Ligate the recovered fragment to the pMD19-T vector and transform competent E. coli. Culture and bacterial culture identification are the same as in 1.6. Plasmid extraction is the same as in 1.7.

[0102] 1.9 Double enzyme digestion products and gel recovery

[0103] After preparing the double enzyme digestion reaction system of the recombinant plasmid, incubate it in a constant temperature water bath at 37°C for 1 hour. Use 1.0% agarose gel electrophoresis to detect the double enzyme digestion products. The reaction system is as follows (the double enzyme digestion identification system uses a 20 μl system, and the LcMAH1 double enzyme digestion system with sticky ends of the enzyme digestion site is recovered using a 50 μl system). Refer to 1.5 for gel recovery.

[0104] Table 8 KpnI, XbaI double enzyme digestion reaction system

[0105] Reaction solution Volume (μl) Volume (μl) 10×Green Buffer I 2 5 KpN 0.5 1.2 XB 0.5 1.2 1300-GFP / pMD19-T-LcMAH1 5 20 <![CDATA[ddH2O]]> 12 23 Total 20 50

[0106] 1.10 Ligation of recombinant plasmid

[0107] After preparing the ligation reaction system, perform the ligation in a constant temperature water bath at 16°C overnight (12-16 hours), transform the competent E. coli, and select the positive strain for identification as in 1.6.

[0108] Table 9 Ligation reaction system

[0109] Reaction solution Volume (μl) Buffet T4 1 T4 ligase 1 Linearized 1300-GFP vector fragment 5 Linearized LcMAH1 gene fragment 3 Total 10

[0110] 1.11 Sequencing and Analysis of Recombinant Plasmids

[0111] Store the identified positive clones at -80°C. Send the identified E. coli strains containing LcMAH1 to Sangon Biotech for sequencing. After sequencing, assemble the results and analyze the correct LcMAH1 sequence. Determine the amino acid sequence of the LcMAH1 gene using relevant biological software and websites (Bioedit, MEGA, NCBI, and transmembrane domain prediction websites). Plasmid extraction is the same as in 1.7.

[0112] Example 2

[0113] Functional analysis of LcMAH1 protein in Leymus chinensis

[0114] 2.1 Transformation of Agrobacterium competent cells

[0115] (1) Thaw the competent cells of Agrobacterium tumefaciens EHA105 stored at -80°C on ice;

[0116] (2) Add 10 μl of the recombinant plasmid of LcMAH1-1300 to every 100 μl of Agrobacterium EHA105 competent cells;

[0117] (3) After adding the recombinant plasmid to the Agrobacterium competent cells and mixing gently, the cells were placed on ice for 5 min, placed in liquid nitrogen for 5 min, placed in a 42°C metal bath for 5 min, and placed on ice for 5 min;

[0118] (4) Add 0.4 ml of YEP liquid medium to the transformed competent cells, and culture them in a constant temperature shaker at 180 rpm at 28°C for 3-4 h. Then, spread them on YEP solid medium (50 μg / ml Kana and 50 μg / ml Rif) and culture them upside down in a 28°C incubator for two days. During this period, observe the growth of the colonies until single colonies are formed. Perform PCR identification of positive colonies. The identified bacterial solution is cultured in 5 ml of YEP liquid culture (50 μg / ml Kana and 50 μg / ml Rif) and cultured in a constant temperature incubator at 28°C for one day. Take 200 μl of the bacterial solution and store it in 200 μl of glycerol for storage.

[0119] 2.2 Transient expression of LcMAH1 in tobacco mesophyll cells using Agrobacterium injection

[0120] (1) Take the competent Agrobacterium EHA105 out of the -80℃ freezer, wait for it to partially thaw, and then place it in ice to continue thawing. After thawing, add 5μl of the plasmid of the LcMAH1-1300 recombinant vector. Then, place it on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37℃ water bath for 5 minutes, and in an ice bath for 5 minutes. Add 400ml of YEP liquid medium and culture it with shaking at 28℃ for 3 hours. Take 200μl and spread it on YEP solid medium containing 50mg / L Kana and Rif antibiotics. Culture it upside down at 28℃ for 2 days. After identification of the bacterial solution, select the positive bacterial solution and multiply it in 5ml of YEP liquid medium containing 50mg / L Kana and Rif antibiotics. Store the bacterial solution after 24 hours.

[0121] (2) Injection of tobacco

[0122] The previously preserved LcMAH1 bacterial liquid that has been transformed into Agrobacterium is streaked on a YEP solid containing Kana and Rif. After the bacterial plate is placed at room temperature for 2 days, a single colony of appropriate size is picked and placed in a 50ml liquid YEP medium containing the corresponding antibiotics, and cultured at 28°C with shaking for 24 hours. When the bacterial liquid turns orange-yellow, centrifuge it at a speed of 5000r / min for 10 minutes, pour out the supernatant, and use a resuspension solution of 100μM AS, 10mM MES, and 10mM MgCl2 to resuspend the bacterial liquid, adjust the OD value of the bacterial liquid to between 0.8-1.0, let it stand in the dark for 2-3 hours, and then inject it into the back of the tobacco leaves. The tobacco is cultured in the dark for 1 day and under light for two days, and its positioning is observed using a laser confocal microscope. The results are as follows Figure 2 As shown, LcMAH1 protein is localized on the endoplasmic reticulum.

[0123] 2.3 Infection of Arabidopsis thaliana by Agrobacterium inflorescence dip method

[0124] (1) Take 1 ml of the above-mentioned Agrobacterium culture solution with the recombinant vector and place it in 200 ml of YEP liquid culture (50 μg / ml Kana and 50 μg / ml Rif), shake and culture in a constant temperature shaker (28°C, 200 rpm) for two days until the OD 600 is 1-2;

[0125] (2) Centrifuge the Agrobacterium culture at 5000 rpm for 15 min, resuspend the cells in pre-sterilized 1 / 2 MS, transfer to 250 ml of 1 / 2 MS (liquid), and adjust the OD 600 The final value is about 0.8;

[0126] (3) Add 2.5 μl 6-BA (1.0 g / l), 0.25 μl As (100 mM), and 50 μl Silwet-77 to every 250 ml of 1 / 2 MS solution and stir to mix.

[0127] (4) The day before infection, water the Arabidopsis thaliana thoroughly, leaving only the unopened inflorescences. Place the plants upside down and immerse them in the bacterial solution. After infection for 10-15 minutes, place them in the dark for 24 hours and then transfer them to a 12-hour light / 12-hour dark light cycle for cultivation.

[0128] 2.4 Screening of positive transgenic Arabidopsis plants

[0129] After the infected Arabidopsis seeds (T0) mature, they are disinfected with alcohol (75%) for 30 seconds and NaClO for 3 minutes in a sterile operating table. After being washed with sterile water 5 times, the seeds are spread on 1 / 2MS medium containing the antibiotic Hyg (40 μg / ml). After vernalization at 4°C for 48 hours, the seeds are placed in a constant temperature incubator (22°C, 12 hours light / 12 hours dark) and cultured for about 10 days for observation. Normally growing plants with 4 leaves with tender green color and strong root systems are selected and transplanted into vermiculite for 2-3 days of acclimatization. Then, the Arabidopsis thaliana is moved to a planting pot filled with peat soil: vermiculite = 2:1 and continued to be cultured in a plant culture room (22°C, 12 hours light / 12 hours dark) to ensure sufficient water and normal lighting conditions, and pay attention to the prevention and control of diseases and pests.

[0130] 2.5 DNA level detection of transgenic Arabidopsis

[0131] Use the 2×T5 Direct PCR Kit to identify the green plants screened on the Hyg-containing plate. Take 1-2 mm plant leaves and place them in a centrifuge tube. Add 30 μl of lysis buffer Buffer A. Heat at 95°C for 10 minutes and then add Buffer B. After instant centrifugation, take 2 μl as the template for PCR amplification verification and detect whether the target gene band size is correct.

[0132] Table 10 PCR reaction system

[0133] Reaction solution Volume (μl) 2×T5 Direct PCR Mix 12.5 Forward primer (10M) 1 Reverse primer(10M) 1 template 2 <![CDATA[H2O]]> 8.5 Total volume 25

[0134] 2.6 Detection of RNA levels in transgenic Arabidopsis

[0135] Select healthy Arabidopsis leaves, sampling three replicates per treatment. Place the sample into a centrifuge tube with three steel balls, quickly freeze in liquid nitrogen, and grind five times using a vibrating grinder until the plant material is a fine powder. Plant total RNA extraction and reverse transcription follow the same procedures as in 1.1 and 1.2.

[0136] 2.7 Identification of drought tolerance of transgenic plants

[0137] The wild-type Arabidopsis thaliana and the harvested T2 transgenic Arabidopsis thaliana seeds were sterilized and then plated on 1 / 2MS, 1 / 2MS+175mM Mannitol, 1 / 2MS+200mM Mannitol, and 1 / 2MS+225mM Mannitol culture media. Six seeds of each of the four wild-type and transgenic lines were vernalized at 4°C for 2 days and then moved to a constant temperature incubator (22°C, 12 hours of light / 12 hours of darkness) for normal culture. The experiment was repeated three times to observe the phenotype. The results showed that the drought tolerance of the transgenic plants was significantly stronger than that of the wild-type control plants. After drought stress treatment, the wild-type plants showed symptoms such as leaf wilting, while the leaves of the transgenic plants were relatively strong ( Figure 3 ).

[0138] Drought tolerance experiments showed that LcMAH1 can be used to improve plant drought tolerance.

[0139] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A Leymus chinensis epidermal wax synthesis gene LcMAH1, characterized in that: The nucleotide sequence of the Leymus chinensis epidermal wax synthesis gene LcMAH1 is shown in SEQ ID No.

1.

2. Use of the Leymus chinensis epidermal wax synthesis gene LcMAH1 according to claim 1 in regulating plant drought resistance.

3. The use according to claim 2, characterized in that Overexpression of the cuticular wax synthesis gene LcMAH1 in Leymus chinensis positively regulates plant drought tolerance.

4. The use according to claim 2 or 3, characterized in that The plants include Arabidopsis thaliana.

5. A protein encoded by the Leymus chinensis epidermal wax synthesis gene LcMAH1 according to claim 1, characterized in that: The amino acid sequence of the protein is shown in SEQ ID No.

2.

6. A recombinant vector, characterized in that The Leymus chinensis epidermal wax synthesis gene LcMAH1 according to claim 1 is connected to a vector to obtain a recombinant vector.

7. The recombinant vector according to claim 6, characterized in that The vector includes pCAMBIA1300-GFP vector.

8. A method for obtaining drought-tolerant plants, characterized in that: The following steps are involved: 1) transforming the recombinant vector according to claim 6 into Agrobacterium to obtain transformed bacteria; 2) The transformed bacteria obtained in step 1) are used to infect plants using the inflorescence dipping method and cultured conventionally.

9. The method according to claim 8, characterized in that The Agrobacterium in step 1) includes Agrobacterium EHA105.

10. The method according to claim 8, characterized in that In step 2, the transformed bacteria infects the plant in the form of bacterial liquid, and the OD 600 The value is 0.8; The infection time is 10 to 15 minutes; The plants include Arabidopsis thaliana.

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