CN110607316A

CN110607316A - Adversity stress response related gene in Lycium ruthenicum Murr, and encoding protein and cloning method thereof

Info

Publication number: CN110607316A
Application number: CN201910784372.0A
Authority: CN
Inventors: 袁惠君; 李虎军; 鲍婧婷; 王春梅; 何芳兰; 马倩国; 高泽; 李学勇
Original assignee: Lanzhou University of Technology
Current assignee: Lanzhou University of Technology
Priority date: 2019-08-23
Filing date: 2019-08-23
Publication date: 2019-12-24

Abstract

The invention provides an adversity stress response related gene in Lycium barbarum, wherein the adversity stress response related gene is aldehyde decarbonylase gene LbCER1, and the nucleotide sequence of the adversity stress response related gene is SEQ ID No. 1; or a gene with the similarity of more than 90 percent with the nucleotide sequence shown in SEQ ID NO. 1. The amino acid sequence coded by the LbCER1 gene is SEQ ID NO. 2. The LbCER1 gene transcript abundance is greatly increased under osmotic stress and salt stress by analyzing LbCER1 gene transcript abundance of lycium barbarum leaves through a real-time fluorescent quantitative PCR method, which indicates that LbCER1 plays an important role in plant resistance to abiotic stress such as osmotic stress, salt stress and the like. The invention can be used for genetic transformation of plants, improves the resistance of the plants under drought and high-salt conditions, and has extremely high value in the research and application of plant stress resistance improvement.

Description

Adversity stress response related gene in Lycium ruthenicum Murr, and encoding protein and cloning method thereof

Technical Field

The invention belongs to the technical field of biotechnology, and particularly relates to a gene related to adversity stress response in lycium ruthenicum murr, and a coding protein and a cloning method thereof.

Background

The wax on the surface of the ground is one of the most important barriers for reducing non-porous water loss and resisting plant diseases and insect pests, so that the gene related to wax synthesis is closely related to the drought resistance and disease and insect pest resistance of the plant. Lycium barbarum is a variety of Ningxia Lycium barbarum, and has developed epidermal wax and extremely strong drought and salt resistance characteristics. Lycium barbarum can improve the water retention capacity of the overground part by increasing the accumulation of waxiness on the epidermis so as to improve the drought resistance of the Lycium barbarum, and is one of the main mechanisms for resisting drought and osmotic stress of the Lycium barbarum. The lycium ruthenicum epidermal waxy synthesis related gene has great development potential in the aspect of improving the stress resistance of other mesogenic plants.

Aldehyde decarbonylase (ECERIFERUM1, CER1) is a key enzyme of a decarbonylation pathway which is a main pathway for synthesizing plant epidermal wax, catalyzes long-chain even-numbered aldehydes to be converted into long-chain odd-numbered alkanes, and participates in the synthesis of C29 alkanes and derivatives thereof. The arabidopsis AtCER1 gene is very sensitive to drought stress, and the expression abundance of the arabidopsis AtCER1 reaches the maximum value which is 40 times that of a control group after the arabidopsis AtCER1 gene is processed for 3 hours under a low-humidity condition. The rice Glossyl (GL1) gene is homologous with an Arabidopsis AtCER1 gene, the expression of the gene is induced by osmotic stress, and the aldehyde decarbonylase gene is closely related to plant drought and osmotic stress response, but is only partially verified in a few middle-living plants such as Arabidopsis.

Disclosure of Invention

The invention aims to provide an aldehyde decarbonylase gene LbCER1 which is derived from Lycium ruthenicum Murr and is related to adversity stress response, and a coding protein and a cloning method thereof. The lycium ruthenicum aldehyde decarbonylase disclosed by the invention has homology of less than 80% with arabidopsis thaliana on a nucleotide sequence and an amino acid sequence, shows that the Lb CER1 gene is different from AtCER1 gene, and has great potential in CER1 gene function identification and plant anti-reverse transmission improvement.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides an adversity stress response related gene in Lycium ruthenicum Murr, wherein the adversity stress response related gene is aldehyde decarbonylase gene LbCER 1; preferably, the nucleotide sequence of the LbCER1 gene is SEQ ID NO. 1; or a gene with the similarity of more than 90 percent with the nucleotide sequence shown in SEQ ID NO. 1.

Preferably, the amino acid sequence encoded by the aldehyde decarbonylase gene LbCER1 is shown in SEQ ID NO. 2.

The invention provides a method for cloning adversity stress response related genes in Lycium ruthenicum Murr, which comprises the following steps: seedling culture, extracting total leaf RNA and performing reverse transcription to synthesize first-strand cDNA, designing a primer of a core segment, performing PCR amplification by using the cDNA as a template to obtain a core segment product, recovering the product, cloning the product into a T vector, converting the T vector into competent cells to induce expression, screening positive clones, and sequencing to obtain a core segment sequence; cloning by 5 '-RACE method and 3' -RACE method to obtain 5 'and 3' fragment sequence; obtaining the nucleotide sequence of aldehyde decarbonylase gene LbCER1 gene by sequence splicing;

preferably, the competent cell is E.coli DH5 α.

Preferably, the seedling culture method comprises the following steps: 3 weeks old seedlings of Lycium ruthenicum Murr were osmosized with 1/2Hoagland nutrient solution containing 80mmol/L D-sorbitol for 24 h.

Preferably, the preparation method of the core fragment product comprises the following steps: taking a P1 primer with a sequence of 5 '-CATCAYCAYTCWATTGYWACWGARCC-3' as an upstream primer, taking a primer P2 with a sequence of 5 '-CAACWGCYARDCTRCTTCCRTCYACC-3' as a downstream primer, and performing PCR (polymerase chain reaction) at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 54.5 ℃ for 30s, extension at 72 ℃ for 90s, 30 cycles; extending for 10min at 72 ℃;

in the P1 primer, Y, W and R are degenerate primers, Y is C/T, W is A/T, and R is A/G.

In the P2 primer, W, Y, R and D are degenerate primers, W is A/T, Y is C/T, R is A/G, and D is A/G/T.

Preferably, when cloning the 5' end by the RACE method, the primer GSP1 with the sequence of 5'-GATTACGCCAAGCTTAGCTGCTTCCGTCTACCACCTTCA-3' is used as an upstream primer, and the primer NGSP1 with the sequence of 5'-GATTACGCCAAGCTTAGCATCAGGCACTTCAGCTTCCC-3' is used as a nested downstream primer.

Preferably, when cloning the 3' end by the RACE method, the primer GSP2 with the sequence of 5'-GATTACGCCAAGCTTCCTTGGCCTCGGAGCCTCACACCT-3' is used as an upstream primer, and the primer NGSP2 with the sequence of 5'-GATTACGCCAAGCTTAGCTGAGTGAGGTGGTAGACGG-3' is used as a nested downstream primer.

The invention provides a method for analyzing the transcriptional abundance of genes related to adversity stress response in Lycium barbarum by using a qRT-PCR method, wherein a qRT-PCR reaction system comprises upstream and downstream primers of an internal reference gene LbACT and upstream and downstream primers of an internal reference gene LbCER1 gene; the sequence of an upstream primer QA1 of the internal reference gene LbACT is 5'-CTATGAGTTGCCAGATGGACAG-3', and the sequence of a downstream primer QA2 is 5'-TGGCTGGAAAAGGACTTCTG-3'; the upstream primer QC1 sequence of the LbCER1 gene is 5'-CACTGGGACTGCCTCTATGG-3', and the downstream primer QC2 sequence is 5'-GTAGTGAGTGGTACGAGGGC-3'; the qRT-PCR reaction procedure was 95 ℃ pre-denaturation for 30s, 95 ℃ denaturation for 5s, 60 ℃ annealing for 30s, 72 ℃ for 10s, 40 cycles.

The invention provides the improvement of transcription abundance of adversity stress response related genes in Lycium barbarum under osmotic stress and salt stress.

The invention provides application of the adversity stress response related gene in lycium ruthenicum in preparation of stress-resistant, salt stress-resistant or drought-resistant transgenic plants.

The transcriptional abundance of LbCER1 gene of Lycium barbarum leaf is analyzed by Real-time fluorescent quantitative PCR (qRT-PCR) method, so that the transcriptional abundance of LbCER1 gene is greatly increased under osmotic stress and salt stress (figure 2), and the LbCER1 plays an important role in resisting abiotic adversity stress such as osmotic stress and salt stress of plants. The invention can be used for genetic transformation of plants, improves the resistance of the plants under drought and high-salt conditions, and has extremely high value in the research and application of plant stress resistance improvement.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 shows phylogenetic analyses of amino acid sequences of LbCER1 and CER1 from other plants.

FIG. 2 shows the transcriptional abundance analysis of LbCER1 gene of Lycium barbarum leaf under different conditions.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Example 1

The method comprises the following steps:

(1) preparation of materials: selecting full seeds, sterilizing with 2% sodium hypochlorite solution for 8-10 min, washing with distilled water, uniformly spreading on wet filter paper, culturing in dark for 4-5 days, after the seeds are exposed to white, planting in vermiculite, adding 1/2Hoagland nutrient solution, standing at 26 + -2 deg.C/23 + -2 deg.C, and illuminating for 16 h.d^-1Light intensity of 600. mu. mol. m^-2·s^-1And changing the nutrient solution every 2 to 3 days in a greenhouse with the relative air humidity of 40 to 60 percent. Wait for the childAnd (3) after the seedlings grow to be 3 weeks old, performing osmosis treatment on the seedlings for 24 hours by using 1/2Hoagland nutrient solution containing 80mmol/L D-sorbitol, and placing 80-100 mg of leaves into liquid nitrogen to be frozen at a medium speed for extracting total RNA.

(2) Extraction of total RNA and first Strand cDNA Synthesis: the total RNA extraction and reverse transcription were performed by UNIQ-10 column Trizol total RNA extraction kit from Shanghai and first strand cDNA Synthesis kit from TAKARA PrimeScriptTM II, respectively, according to the respective instructions.

(3) Cloning of core fragment: using cDNA template as template, adding 4 μ L cDNA template, 2 μ L P1 primer with sequence 5 '-CATCAYCAYTCWATTGYWACWGARCC-3' and 2 μ L P2 primer with sequence 5 '-CAACWGCYARDCTRCTTCCRTCYACC-3' into 50 μ LPCR reaction system, circulating 30 times under PCR conditions of pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 54.5 ℃ for 30s, and extension at 72 ℃ for 90s, and then extending at 72 ℃ for 10 min. The core fragment of the LbCER1 gene with the length of 828bp can be obtained. The above target band was recovered by using the DNA gel recovery kit of the SanPrep column type, and the procedure was as described in the kit for gel recovery. According to the specification of a pMD18-T vector PCR product cloning kit, loading a core fragment into pMD18-T, transforming a pMD18-T vector containing the core fragment into competent cell escherichia coli DH5 alpha by a heat shock method, screening positive clones by a blue-white spot screening method, identifying the positive clones by bacterial liquid PCR, and sequencing to obtain a core fragment sequence.

(4) 5' end cloning: according to SMARTer^TMRACE cDNA kit Specification 5' RACE-Ready cDNA first strand was synthesized. Solution 1 was prepared by adding the following solutions to a sterilized 200 μ L PCR tube: 15.5 μ L of PCR-Grade H₂O, 25.0. mu.L of 2 XSeqAmp^TMBuffer, 1.0. mu.L of SeqAmp DNA Polymerase. In another new sterilized 200 μ L PCR tube the following solutions were added for outside PCR amplification: 2.5. mu.L of 5' RACE-Ready cDNA, 5.0. mu.L of 10 × UPM, 1.0. mu.L of GSP1 (10. mu.M), 41.5. mu.L of solution 1. Pre-denaturation of 3mi at 94 ℃n, denaturation at 94 ℃ for 30s, annealing at 68 ℃ for 30s, and extension at 72 ℃ for 3min, and then extension at 72 ℃ for 10min to obtain an outer PCR amplification product.

Pipette 5. mu.L of the outer PCR amplification product into a new sterilized 200. mu.L PCR tube, add 245. mu.L TE buffer for dilution, and use the diluted outer PCR product for nested PCR amplification. Solution 2 was prepared for nested PCR amplification in sterilized 200 μ L PCR tubes by adding: 17.0 μ L of PCR-Grade H₂O, 25.0. mu.L of 2 XSeqAmp^TMBuffer, 1.0. mu.L of SeqAmp DNA Polymerase. In another new sterilized 200. mu.L PCR tube, nested PCR was performed by adding the following solutions: mu.L of diluted outer PCR product, 1.0. mu.L of UPM short, 1.0. mu.L of NGSP1 (10. mu.M), and 43.0. mu.L of solution 2. Circulating for 20 times under the PCR conditions of pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s and extension at 72 ℃ for 3min, and then extending at 72 ℃ for 10min to obtain a nested PCR amplification product. The nested PCR amplification product is purified and recovered by using a NucleoSpin Gel and PCR Clean-Up Gel recovery kit matched with the RACE cDNA PCR amplification kit, and the operation method is referred to the specification. Loading the 5 'end nested PCR amplification product into a pRACE vector by an In-Fusion method, transforming the pRACE vector containing the 5' end nested PCR amplification product into competent cell escherichia coli DH5 alpha by a heat shock method, screening positive clones by a blue-white spot screening method, identifying the positive clones by a bacterial liquid PCR method, and then sequencing.

Wherein, the sequence of GSP1 is: 5'-GATTACGCCAAGCTTAGCTGCTTCCGTCTACCACCTTCA-3' are provided.

The sequence of NGSP1 is: 5'-GATTACGCCAAGCTTAGCATCAGGCACTTCAGCTTCCC-3' are provided.

The following solutions were added to a new sterilized 200 μ L PCR tube for PCR: 8.5. mu.L of sterile water, 1.0. mu.L of UPM short, 1.0. mu.L of NGSP1 (10. mu.M), 12.5. mu.L of Premix Taq, 2.0. mu.L of 5' RACE bacterial suspension. Circulating 30 times under the PCR condition of pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s and extension at 72 ℃ for 3min, then extending at 72 ℃ for 10min, detecting the PCR amplification product of the bacterial liquid by using 1.2% agarose gel electrophoresis, and obtaining the 5'RACE positive clone if the same band as the 5' RACE nested PCR amplification product appears. And then sending the bacterial suspension with the corresponding number to the Beijing sequencing part of the Huada gene for sequencing, wherein the sequencing primer is the universal primer M13F/R. And analyzing the sequencing result according to the forward and reverse sequences in the sequencing result and the inner primers UPM short and NGSP1 to determine the 5' nucleotide sequence.

(5) Cloning of the 3' end: according to SMARTer^TMRACE cDNA kit Specification 3' RACE-Ready cDNA first strand was synthesized. Solution 1 was prepared by adding the following solutions to a sterilized 200 μ L PCR tube: 15.5 μ L of PCR-Grade H₂O, 25.0. mu.L of 2 XSeqAmp^TMBuffer, 1.0. mu.L of SeqAmp DNA Polymerase. In another new sterilized 200 μ L PCR tube the following solutions were added for outside PCR amplification: 2.5. mu.L of 3' RACE-Ready cDNA, 5.0. mu.L of 10 × UPM, 1.0. mu.L of GSP2 (10. mu.M), 41.5. mu.L of solution 1. Circulating for 25 times under the PCR conditions of pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 68 ℃ for 30s and extension at 72 ℃ for 3min, and then extending at 72 ℃ for 10min to obtain an outer PCR amplification product.

Pipette 5. mu.L of the outer PCR amplification product into a new sterilized 200. mu.L PCR tube, add 245. mu.L TE buffer for dilution, and use the diluted outer PCR product for nested PCR amplification. Solution 2 was prepared for nested PCR amplification in sterilized 200 μ LPCR tubes by adding: 17.0 μ L of PCR-Grade H₂O, 25.0. mu.L of 2 XSeqAmp^TMBuffer, 1.0. mu.L of SeqAmp DNA Polymerase. In another new sterilized 200. mu.L PCR tube, nested PCR was performed by adding the following solutions: mu.L of diluted outer PCR product, 1.0. mu.L of UPM short, 1.0. mu.L of NGSP2 (10. mu.M), and 43.0. mu.L of solution 2. Circulating for 20 times under the PCR conditions of pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s, extension at 72 ℃ for 3min and extension at 72 ℃ for 10min to obtain nested PCR amplification products. Purifying and recovering the nested PCR amplification product according to the instruction of a NucleoSpin Gel and PCR Clean-Up glue recovery kit matched with the RACE cDNAPCR amplification kit. Loading the 3 'end nested PCR amplification product into a pRACE vector by an In-Fusion method, transforming the pRACE vector containing the 3' end nested PCR amplification product into a competent cell DH5 alpha by a heat shock method, screening positive clones by a blue-white spot screening method, identifying the positive clones by a bacterial liquid PCR method, and sequencing to obtain the recombinant plasmid.

Wherein, the sequence of GSP2 is: 5'-GATTACGCCAAGCTTCCTTGGCCTCGGAGCCTCACACCT-3' are provided.

The sequence of NGSP2 is: 5'-GATTACGCCAAGCTTAGCTGAGTGAGGTGGTAGACGG-3' are provided.

The following solutions were added to a new sterilized 200 μ L PCR tube for PCR: 8.5. mu.L of sterile water, 1.0. mu.L of UPM short, 1.0. mu.L of NGSP2 (10. mu.M), 12.5. mu.L of Premix Taq, 2.0. mu.L of 5' RACE bacterial suspension. Circulating 30 times under the PCR condition of pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s and extension at 72 ℃ for 3min, then extending at 72 ℃ for 10min, detecting the PCR amplification product of the bacterial liquid by using 1.2% agarose gel electrophoresis, and obtaining the positive clone of 3'RACE if the same band as the nested PCR amplification product of 3' RACE appears. The bacterial suspensions corresponding to the numbers are sequenced, and the sequencing primer is the universal primer M13F/R. And analyzing the sequencing result according to the forward and reverse sequences in the sequencing result and the inner primers UPM short and NGSP2 to determine the 3' nucleotide sequence.

(6) Using DNAman 6.0 software to splice the 5 'end sequence, the 3' end sequence and the core sequence to obtain a DNA sequence shown as SEQ ID NO: 1 and the cDNA sequence of the LbCER1 gene shown in SEQ ID NO: 2 in sequence shown in the specification, and 2 is an amino acid sequence of LbCER.

(7) And (3) taking 3-week-old seedlings, respectively treating the seedlings for 0h, 3h, 6h, 12h, 24h, 36h, 48h, 60h and 72h by using 1/2Hoagland nutrient solution containing 80mmol/L and 160mmol/L sorbitol, then placing 80-100 mg leaves in liquid nitrogen for quick freezing, and analyzing the change of transcription abundance of LbCER1 under osmotic stress by using a qRT-PCR method. Seedlings grown in 1/2Hoagland nutrient solution without sorbitol were used as controls.

(8) And (3) treating 3-week-old seedlings with 1/2Hoagland nutrient solution containing 50, 100, 150, 200 and 300mmol/L NaCl for 0h, 6h, 12h, 24h, 48h and 72h respectively, then placing 80-100 mg leaves in liquid nitrogen for quick freezing, and analyzing the change of transcription abundance of LbCER1 under salt penetration stress by using a qRT-PCR method. Seedlings grown in 1/2Hoagland nutrient solution without NaCl were used as controls.

(9) The method for analyzing the transcriptional abundance of the LbCER1 gene in the Lycium ruthenicum leaves by using the qRT-PCR method comprises the following steps: using first strand cDNA reverse-transcribed from total RNA extracted from leaves as a templatePremix Ex Taq^TMII (Tli RNaseH Plus) kit instructions, adding 0.8 μ L of each of the upstream primer QA1 of the internal Reference gene LbACT with the sequence of 5'-CTATGAGTTGCCAGATGGACAG-3' and the downstream primer QA2 of the internal Reference gene LbACT with the sequence of 5'-TGGCTGGAAAAGGACTTCTG-3' at a concentration of 10 μ M, or adding 0.8 μ L of each of the upstream primer QC1 of the LbCER1 gene with the sequence of 5'-CACTGGGACTGCCTCTATGG-3' and the downstream primer QC2 with the sequence of 5'-GTAGTGAGTGGTACGAGGGC-3', 10.0 μ L of SYBR Premix Ex Taq II (TliRNaseH Plus) (2X), 0.4 μ L of ROX Reference Dye (50X), 2.0 μ L of cDNA template, 6.0 μ L of pure water, and carrying out qRT-PCR in an ABI Onestrep real-time quantitative PCR instrument. The qRT-PCR program was pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s, annealing at 72 ℃ for 10s, 40 cycles.

The implementation result is as follows:

(1) the cDNA sequence of the LbCER1 gene shown in SEQ ID NO.1 is obtained, the length is 2168bp, the open reading frame is 1881bp, the 5' untranslated region (UTR) is 107bp, the 3' untranslated region (3 ' UTR) is 180bp, and the LbCER1 gene comprises a 26bp poly (A) tail. This cDNA encodes 626 amino acids and is predicted to have 4 transmembrane domains, a fatty acid hydroxylase domain and a C-terminal Wax2 domain. The amino acid sequence is shown as SEQ ID NO. 2. Phylogenetic tree analysis is constructed by MEGA 5.0 software based on an ortho-junction method, and shows that the Lycium barbarum LbCER1 has close relationship with solanaceae plants such as tobacco (Nicotiana tabacum), tomato (Solanum lycopersicum), pepper (Capsicum annuum) and the like, the homology reaches 100%, the homology with dicotyledonous plants such as cocoa (Theobroma cacao), rape (Brassica campestris), Arabidopsis (Arabidopsis thaliana) and Camelina sativa (Camelina sativa) is above 72%, but the homology with monocotyledonous plants such as wheat (Triticum aestivum) and corn (Zea mays) is far away (FIG. 1).

Nucleotide sequence of Lycium ruthenicum (Lycium barbarum ssp. bianguo) aldehyde decarbonylase gene LbCER 1:

sequence characteristics: the total length is 2168bp, including an Open Reading Frame (ORF) of 1881bp, a 5 'untranslated region (5' UTR) of 107bp, and a 3 'untranslated region (3' UTR) of 180bp, including a poly (A) tail of 26 bp.

Amino acid sequence of Lycium ruthenicum (Lycium barbarum ssp. bianguo) aldehyde decarbonylase LbCER 1:

sequence characteristics: the full length of 626 amino acids, predicted to have 4 transmembrane regions, a fatty acid hydroxylase domain and a C-terminal Wax2 domain. LbCER1 has close relationship with solanaceae plants, and the homology reaches 100%.

(2) The qRT-PCR method verifies that the leaf transcriptional abundance of the LbCER1 gene is remarkably improved under osmotic stress and salt stress (figure 2).

From the results, the cloning and transcription abundance analysis method of the lycium ruthenicum LbCER1 gene is feasible and has good repeatability. The LbCER1 gene has wide application prospect in the aspect of cultivating drought-resistant and salt-resistant crops.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

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195 200 205

Ala Tyr Val Thr Tyr Ile Asp Phe Met Asn Asn Met Gly His Cys Asn

210 215 220

Phe Glu Leu Ile Pro Lys Trp Met Phe Ser Ile Phe Pro Pro Leu Lys

225 230 235 240

Tyr Leu Met Tyr Thr Pro Ser Tyr His Ser Leu His His Thr Gln Ser

245 250 255

Arg Thr Asn Phe Ser Leu Phe Met Pro Met Tyr Asp Tyr Ile Tyr Asp

260 265 270

Thr Leu Asp Lys Ser Ser Asp Thr Leu Tyr Glu Met Ser Leu Glu Arg

275 280 285

Glu Ala Glu Val Pro Asp Ala Val His Leu Thr His Leu Thr Thr Pro

290 295 300

Glu Ser Ile Tyr His Leu Arg Leu Gly Phe Ala Ser Leu Ala Ser Glu

305 310 315 320

Pro His Thr Ser Lys Trp Tyr Phe Trp Val Met Trp Pro Val Thr Leu

325 330 335

Cys Ser Ile Met Ile Thr Trp Ile Tyr Gly His Thr Phe Val Val Glu

340 345 350

Arg Asn Leu Phe Lys Asn Leu Lys Leu Gln Thr Trp Ala Ile Pro Lys

355 360 365

Tyr Arg Val Gln Tyr Phe Met Lys Trp Gln Arg Glu Thr Ile Asn Asn

370 375 380

Leu Ile Glu Glu Ala Ile Ala Glu Ala Asp Lys Lys Gly Ile Lys Val

385 390 395 400

Leu Ser Leu Gly Leu Leu Asn Gln Glu Glu Gln Leu Asn Ser Asn Gly

405 410 415

Glu Leu Tyr Ile Arg Lys Asn Pro Gln Leu Lys Val Lys Val Val Asp

420 425 430

Gly Ser Ser Leu Ala Val Ala Val Val Ile Asn Ser Ile Pro Lys Gly

435 440 445

Thr Cys Gln Val Val Leu Arg Gly Arg Leu Ser Lys Val Ala Tyr Ser

450 455 460

Ile Ala Leu Ala Leu Cys Gln Gly Gly Ile Gln Val Val Thr Leu Gly

465 470 475 480

Glu Glu Glu Tyr Lys Arg Leu Lys Ala Lys Leu Thr Pro Glu Ala Ala

485 490 495

Ala His Leu Val Leu Ser Lys Ser Tyr Ala Ser Arg Thr Trp Leu Val

500 505 510

Gly Asp Gly Leu Ser Glu Asp Glu Gln Leu Lys Ala Pro Lys Gly Thr

515 520 525

Leu Phe Ile Pro Tyr Ser Gln Phe Pro Pro Arg Lys Ala Arg Lys Asp

530 535 540

Cys Phe Tyr Phe Ser Thr Pro Ala Met Ile Thr Pro Lys Asn Leu Glu

545 550 555 560

Asn Val Asp Ser Cys Glu Asn Trp Leu Pro Arg Arg Val Met Ser Ala

565 570 575

Trp Arg Ile Ala Gly Ile Leu His Ala Leu Glu Gly Trp Asn Glu His

580 585 590

Glu Cys Gly Asn Met Met Phe Asp Ile Asp Lys Val Trp Lys Ala Ser

595 600 605

Leu Asp His Gly Phe Cys Pro Leu Thr Met Ser Ser Ala Thr Glu Ser

610 615 620

Lys Asn

625

Claims

1. The adversity stress response related gene in the lycium ruthenicum is characterized in that: the adversity stress response related gene is aldehyde decarbonylase gene LbCER 1; preferably, the nucleotide sequence of the LbCER1 gene is SEQ ID NO. 1; or a gene with the similarity of more than 90 percent with the nucleotide sequence shown in SEQ ID NO. 1.

2. The adversity stress response-related gene in Lycium ruthenicum according to claim 1, wherein: the amino acid sequence coded by the aldehyde decarbonylase gene LbCER1 is shown in SEQ ID NO. 2.

3. The method for cloning adversity stress response related genes in Lycium ruthenicum Murr of claim 1 or 2, wherein the method comprises the following steps: the method comprises the following steps: seedling culture, extracting total leaf RNA and performing reverse transcription to synthesize first-strand cDNA, designing a primer of a core segment, performing PCR amplification by using the cDNA as a template to obtain a core segment product, recovering the product, cloning the product into a T vector, converting the T vector into competent cells to induce expression, screening positive clones, and sequencing to obtain a core segment sequence; cloning by 5 '-RACE method and 3' -RACE method to obtain 5 'and 3' fragment sequence; obtaining the nucleotide sequence of aldehyde decarbonylase gene LbCER1 gene by sequence splicing;

preferably, the competent cell is E.coli DH5 α.

4. The method for cloning adversity stress response related genes in Lycium ruthenicum Murr of claim 3, wherein the method comprises the following steps: the seedling culture method comprises the following steps: 3 weeks old seedlings of Lycium ruthenicum Murr were osmosized with 1/2Hoagland nutrient solution containing 80mmol/L D-sorbitol for 24 h.

5. The method for cloning adversity stress response related genes in Lycium ruthenicum Murr of claim 3, wherein the method comprises the following steps: the preparation method of the core fragment product comprises the following steps: taking a P1 primer with a sequence of 5 '-CATCAYCAYTCWATTGYWACWGARCC-3' as an upstream primer, taking a primer P2 with a sequence of 5 '-CAACWGCYARDCTRCTTCCRTCYACC-3' as a downstream primer, and performing PCR (polymerase chain reaction) at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 54.5 ℃ for 30s, extension at 72 ℃ for 90s, 30 cycles; extending for 10min at 72 ℃;

6. The method for cloning adversity stress response related genes in Lycium ruthenicum Murr of claim 3, wherein the method comprises the following steps: when cloning the 5' end by the RACE method, a primer GSP1 with the sequence of 5'-GATTACGCCAAGCTTAGCTGCTTCCGTCTACCACCTTCA-3' is used as an upstream primer, and a primer NGSP1 with the sequence of 5'-GATTACGCCAAGCTTAGCATCAGGCACTTCAGCTTCCC-3' is used as a nested downstream primer.

7. The method for cloning adversity stress response related genes in Lycium ruthenicum Murr of claim 3, wherein the method comprises the following steps: when cloning the 3' end by the RACE method, a primer GSP2 with the sequence of 5'-GATTACGCCAAGCTTCCTTGGCCTCGGAGCCTCACACCT-3' is used as an upstream primer, and a primer NGSP2 with the sequence of 5'-GATTACGCCAAGCTTAGCTGAGTGAGGTGGTAGACGG-3' is used as a nested downstream primer.

8. A method for analyzing transcriptional abundance of adversity stress response-related genes in Lycium barbarum of claim 1 or 2 by using qRT-PCR method, wherein the method comprises the following steps: the qRT-PCR reaction system comprises an upstream primer and a downstream primer of an internal reference gene LbACT and an upstream primer and a downstream primer of an internal reference gene LbCER 1; the sequence of an upstream primer QA1 of the internal reference gene LbACT is 5'-CTATGAGTTGCCAGATGGACAG-3', and the sequence of a downstream primer QA2 is 5'-TGGCTGGAAAAGGACTTCTG-3'; the upstream primer QC1 sequence of the LbCER1 gene is 5'-CACTGGGACTGCCTCTATGG-3', and the downstream primer QC2 sequence is 5'-GTAGTGAGTGGTACGAGGGC-3'; the qRT-PCR reaction procedure was 95 ℃ pre-denaturation for 30s, 95 ℃ denaturation for 5s, 60 ℃ annealing for 30s, 72 ℃ for 10s, 40 cycles.

9. The lycium ruthenicum murray of claim 1 or 2, wherein the transcriptional abundance of the gene related to the adversity stress response is increased under osmotic stress and salt stress.

10. The application of the adversity stress response related gene in Lycium ruthenicum Murr of claim 1 or 2 in preparing stress-resistant, salt stress-resistant or drought-resistant transgenic plants.