CN114480416B

CN114480416B - Application of tsaoko AtDRM2 gene in improving cold resistance of plants

Info

Publication number: CN114480416B
Application number: CN202210055245.9A
Authority: CN
Inventors: 潘春柳; 周芸伊; 姚李祥; 李莹; 王春丽; 黄雪彦; 余丽莹
Original assignee: Guangxi Botanical Garden of Medicinal Plants
Current assignee: Guangxi Botanical Garden of Medicinal Plants
Priority date: 2022-01-18
Filing date: 2022-01-18
Publication date: 2023-06-09
Anticipated expiration: 2042-01-18
Also published as: CN114480416A

Abstract

The invention discloses application of a tsaoko AtDRM2 gene in improving cold resistance of plants, wherein the DNA sequence of the tsaoko AtDRM2 gene is shown as SEQ ID NO. 1. The application comprises the following steps: 1) Amplifying the AtDRM2 gene of the tsaoko; 2) Plasmid is linearized by double enzyme digestion of plasmid with Bgl II and BstE II restriction enzyme, homologous recombination reaction is carried out on the linearized plasmid and the tsaoko AtDRM2 gene, the product is transformed into DH5 alpha competence, and is cultured on a culture medium coated with antibiotics, and single colony on the culture medium is positively verified, thus obtaining recombinant plasmid; 3) The recombinant plasmid is transformed into agrobacterium, and agrobacterium is utilized to infect plants, and transgenic plants are screened and identified, thus obtaining plants with improved cold resistance. The invention improves the tolerance of the plant to low temperature stress by constructing the plant transformed with the dormancy related gene DRM, and provides a new idea for cultivating cold tolerance plants.

Description

Application of tsaoko AtDRM2 gene in improving cold resistance of plants

Technical Field

The invention belongs to the field of plant genetic engineering. More specifically, the invention relates to an application of a fructus tsaoko AtDRM2 gene in improving cold resistance of plants.

Background

Fructus Tsaoko (Amomum tsaoko) is a perennial herb of the genus cardamom of the family Zingiberaceae. The fruit is not only a spice, but also an important medicinal material, has the functions of warming the middle-jiao, invigorating stomach, promoting digestion and guiding qi downward, and is mainly used for treating heart and abdomen pain, abdominal distention, nausea and vomiting, cough with excessive phlegm, malaria cold and heat and the like. At present, most of researches on tsaoko fruits at home and abroad are focused on chemical components, pharmacological actions and cultivation and breeding, and few researches on growth adaptability are performed. The tsaoko has special biological characteristics, high temperature requirement and narrow suitable range, and is mainly distributed in southeast and Guangxi West of Yunnan province in China. The produced fructus Tsaoko is mostly cultivated in broad-leaved forest with the altitude of 1100-1800 m, and the annual average temperature in the growing area is required to be 16-22 ℃. Yang Yaowen et al (2016) found that the behavior of the curly lawns is very sensitive to temperature and humidity, and that when the average temperature is greater than 18 ℃, there is hysteresis in the curly lawns, which in turn affects the propagation of plants. Cui Xiaolong et al (1995) found that tsaoko pollen is extremely lost in viability under high temperature and low humidity conditions, and that the optimum temperature for pollen germination and pollen tube growth is between 12 and 24 ℃. The results of these studies indicate that temperature is one of the main factors affecting the growth and propagation of tsaoko fruits. However, no research to improve the temperature adaptability of tsaoko has been reported.

Dormancy-associated genes (DRM) are members of the DRM2/ARP (dormancy-associated genes/auxin repressor) family, involved in regulating dormancy of meristems or organs. Various evidences indicate that DRM is involved in seed dormancy maintenance, bud dormancy and hormone signal regulation, however, no report for improving plant temperature adaptability by applying DRM genes exists at present.

Disclosure of Invention

It is an object of the present invention to solve at least the above problems and to provide at least the advantages to be described later.

It is still another object of the present invention to provide an application of the AtDRM2 gene of the tsaoko fruit in improving cold tolerance of plants, which can reduce and improve the tolerance of the plants to cold stress.

To achieve these objects and other advantages and in accordance with the purpose of the invention, there is provided an application of the AtDRM2 gene of fructus Tsaoko in improving cold tolerance of plants, wherein the DNA sequence of the AtDRM2 gene of fructus Tsaoko is shown as SEQ ID NO. 1.

The application of the tsaoko AtDRM2 gene in improving the cold resistance of plants comprises the following steps:

1) Amplifying the AtDRM2 gene of the tsaoko;

2) Cutting plasmid pCAMBIA1301 by BglII and BstEII restriction enzyme to linearize plasmid pCAMBIA1301, carrying out homologous recombination reaction on the linearized plasmid pCAMBIA1301 and the tsaoko AtDRM2 gene, converting the product into DH5 alpha competence, culturing on a culture medium coated with antibiotics, and carrying out positive verification on single colony on the culture medium to obtain pCAMBIA1301-AtDRM2 recombinant plasmid;

3) The pCAMBIA1301-AtDRM2 recombinant plasmid is transformed into agrobacterium, and agrobacterium is utilized to infect plants, and transgenic plants are screened and identified, thus obtaining plants with improved cold resistance.

Preferably, the extraction method of the tsaoko AtDRM2 gene comprises the following steps:

a) 0.2-1g of tsaoko leaves are taken and put into a centrifuge tube containing grinding beads, quick-frozen by liquid nitrogen and ground into powder to obtain powder;

b) Extracting total RNA of plants in the powder, reversely transcribing the RNA into cDNA, and carrying out PCR amplification and purification by taking the cDNA as a template to obtain the Amomum tsao-ko AtDRM2 gene.

Preferably, the upstream primer used for PCR amplification by using cDNA as a template is SEQ ID NO.2: actcttgaccatggtagatctgatgggtcttctagacaagctctgg; the downstream primer is SEQ ID NO.3: ggggaaattcgagctggtcaccttacacatcgaagggaggataaatg.

Preferably, the reaction conditions for PCR amplification using cDNA as a template are: pre-denaturation at 95℃for 3min, denaturation at 95℃for 15s, annealing at 58℃for 15s, elongation at 72℃for 45s,35 cycles; the reaction system is as follows: 25 mu L of high-fidelity enzyme, 2 mu L of upstream primer, 2 mu L of downstream primer, 2 mu L of cDNA and ddH ₂ O 19μL。

Preferably, the method for infecting plants by using agrobacterium is to disinfect plant seeds, clean the seeds with sterile water, culture the seeds on a culture medium, germinate the seeds and grow 2-4 leaves, then transfer seedlings into peat soil for culture, and when the plants bloom, the agrobacterium containing pCAMBIA1301-AtDRM2 recombinant plasmid is used for impregnating inflorescences, thus realizing the impregnating of the agrobacterium on the plants.

Preferably, the method of impregnating inflorescences with agrobacterium containing the pCAMBIA1301-AtDRM2 recombinant plasmid is to aspirate 2-5mL of agrobacterium containing the pCAMBIA1301-AtDRM2 recombinant plasmid, inoculate in 100-200mL of YEP liquid medium containing 50-100mg/L kanamycin and 50-100mg/L rifampicin, culture at 25-30 ℃ and 200-250rpm until OD value is 1.0-1.2; transferring the bacterial liquid into a sterile centrifuge tube, centrifuging for 10-15min at 5000-6000rpm, discarding the supernatant, re-suspending the bacterial body with the transformation liquid until the OD600 is 0.8-1.0, obtaining an agrobacterium leaching solution containing pCAMBIA1301-AtDRM2 recombinant plasmid, soaking inflorescences of plants in the agrobacterium leaching solution for 30-60s, culturing for 24-36h in a dark place, and repeatedly transforming for 1-2 times.

Preferably, the method for screening and identifying transgenic plants is to utilize agrobacterium to infect plants and then harvest seeds T0 of the plants, disinfect the seeds T0 and clean with sterile water, place the seeds on a resistant culture medium for culture, select plants which grow normally for culture and harvest seeds T1; screening the separation proportion of the seeds T1, selecting plants meeting the separation proportion of 3:1 for culture, and harvesting the seeds of the generation T2; screening the seeds T2 by using a resistance flat plate, selecting seedlings and surviving plants to continue culturing, harvesting the T3 generation seeds, extracting DNA of leaves of the T3 generation plants, and carrying out PCR verification; extracting total RNA of T3 generation plant leaves, reversely transcribing the total RNA into cDNA, and detecting the expression quantity by fluorescent quantitative PCR to obtain the positive plant with high gene expression quantity.

Preferably, the upstream primer of the fluorescent quantitative PCR is SEQ ID NO.4: ttcaggaggaagtcaatg; the downstream primer is SEQ ID NO.5: aatggaagaagaactggaa.

The invention at least comprises the following beneficial effects: the invention improves the tolerance of the plant to low temperature stress by constructing the plant transformed with the dormancy related gene DRM, and provides a new idea for cultivating cold tolerance plants.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a schematic diagram of a pCAMBIA1301-AtDRM2 recombinant plasmid constructed in the present invention;

FIG. 2 is a graph of plant growth variation for low temperature stress; wherein 1 represents before cold stress; 2 represents stress at-10 ℃ for 3 hours; 3 represents resume growth 3d;4 represents day 7 of resume growth;

FIG. 3 is a graph showing plant growth ratio change after low temperature stress;

FIG. 4 is a graph showing changes in SOD activity, POD activity, CAT activity and MDA content after low temperature stress at 4 ℃;

FIG. 5 is a graph showing changes in SOD activity, POD activity, CAT activity and MDA content after low temperature stress at 0deg.C;

FIG. 6 is a graph showing changes in SOD activity, POD activity, CAT activity and MDA content after-10℃low temperature stress;

FIG. 7 is a graph showing changes in expression levels of genes involved in cold stress response after low temperature stress at 4 ℃;

FIG. 8 is a graph showing changes in expression levels of genes involved in cold stress response after low temperature stress at 0 ℃;

FIG. 9 is a graph showing changes in the expression level of a gene involved in cold stress response after low temperature stress at-10 ℃.

Detailed Description

The present invention is described in further detail below with reference to the drawings to enable those skilled in the art to practice the invention by referring to the description.

It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

The experimental methods described in the following embodiments are conventional methods unless otherwise indicated, and the reagents and materials are commercially available.

The invention provides application of a tsaoko AtDRM2 gene in improving cold resistance of plants, wherein the DNA sequence of the tsaoko AtDRM2 gene is shown as SEQ ID NO. 1.

1) Amplifying the AtDRM2 gene of the tsaoko;

In another technical scheme, the amplification method of the tsaoko AtDRM2 gene comprises the following steps:

In another technical scheme, the upstream primer used for PCR amplification by taking cDNA as a template is SEQ ID NO.2: actcttgaccatggtagatctgatgggtcttctagacaagctctgg; the downstream primer is SEQ ID NO.3: ggggaaattcgagctggtcaccttacacatcgaagggaggataaatg.

In another technical scheme, the reaction conditions for PCR amplification using cDNA as a template are as follows: pre-denaturation at 95℃for 3min, denaturation at 95℃for 15s, annealing at 58℃for 15s, elongation at 72℃for 45s,35 cycles; the reaction system is as follows: 25 mu L of high-fidelity enzyme, 2 mu L of upstream primer, 2 mu L of downstream primer, 2 mu L of cDNA and ddH ₂ O 19μL。

In another technical scheme, the method for infecting plants by using agrobacterium is to disinfect plant seeds, clean the seeds by using sterile water, culture the seeds on a culture medium, germinate the seeds and grow 2-4 leaves, transfer seedlings into peat soil for culture, and dip-dye inflorescences by using agrobacterium containing pCAMBIA1301-AtDRM2 recombinant plasmids when the plants bloom, namely, dip-dye the plants by using the agrobacterium.

In another technical scheme, the method for impregnating inflorescences by using agrobacterium containing pCAMBIA1301-AtDRM2 recombinant plasmid comprises the steps of sucking 2-5mL of agrobacterium containing pCAMBIA1301-AtDRM2 recombinant plasmid into 100-200mL of YEP liquid culture medium containing 50-100mg/L kanamycin and 50-100mg/L rifampicin, and culturing at 25-30 ℃ and 200-250rpm until OD value is 1.0-1.2; transferring the bacterial liquid into a sterile centrifuge tube, centrifuging for 10-15min at 5000-6000rpm, discarding the supernatant, re-suspending the bacterial body with the transformation liquid until the OD600 is 0.8-1.0, obtaining an agrobacterium leaching solution containing pCAMBIA1301-AtDRM2 recombinant plasmid, soaking inflorescences of plants in the agrobacterium leaching solution for 30-60s, culturing for 24-36h in a dark place, and repeatedly transforming for 1-2 times.

In another technical scheme, the method for screening and identifying transgenic plants is to utilize agrobacterium to infect plants and then harvest seeds T0 of the plants, disinfect the seeds T0 and clean with sterile water, place the seeds on a resistant culture medium for culture, select plants which grow normally for culture and harvest seeds T1; screening the separation proportion of the seeds T1, selecting plants meeting the separation proportion of 3:1 for culture, and harvesting the seeds of the generation T2; screening the seeds T2 by using a resistance flat plate, selecting seedlings and surviving plants to continue culturing, harvesting the T3 generation seeds, extracting DNA of leaves of the T3 generation plants, and carrying out PCR verification; extracting total RNA of T3 generation plant leaves, reversely transcribing the total RNA into cDNA, and detecting the expression quantity by fluorescent quantitative PCR to obtain the positive plant with high gene expression quantity.

In another technical scheme, the upstream primer of the fluorescent quantitative PCR is SEQ ID NO.4: ttcaggaggaagtcaatg; the downstream primer is SEQ ID NO.5: aatggaagaagaactggaa.

< example >

1. DNA fragment acquisition of Amomum tsao-ko AtDRM2 Gene

0.2g of tsaoko leaves was taken, placed in a 2.0ml centrifuge tube containing grinding beads, quickly frozen with liquid nitrogen, and ground into powder using a tissue grinder (Shanghai Jingxin, JXFSPRP-64L). Total plant RNA was extracted by Takara TaKaRa MiniBEST Plant RNA Extraction Kit using Takara PrimeScript ^TM RT reagent Kit with gDNA Eraser (Perfect Real Time) reverse transcribes RNA into cDNA. According to the AtDRM2 gene DNA sequence SEQ ID NO.1, an upstream primer SEQ ID NO.2: actcttgacc is designed and synthesizedatggtagatctgatgggtcttctagacaagctctgg, wherein actcttgaccatggtagatct is the pCAMBIA1301 partial vector sequence, which is commercially available; the downstream primer SEQ ID NO.3: ggggaaattcgagctggtcaccttacacatcgaagggaggataaatg was designed and synthesized, wherein ggggaaattcgagctggtcacc is the pCAMBIA1301 partial vector sequence. PCR amplification was performed using the Norway Hi-Fi enzyme 2X Phanta Max Master Mix and the Amomum tsao-ko cDNA as a template (reaction conditions: 95℃pre-denatured 3min,95℃denatured 15s,58℃annealed 15s,72℃extended 45s,35 cycles; reaction system: hi-Fi enzyme 25. Mu.L, upstream primer 2. Mu.L, downstream primer 2. Mu.L, cDNA 2. Mu.L, ddH) ₂ O19 μl). Gel recovery was performed using the DNA purification recovery kit from the physcomitrella biochemical technology limited company.

2. Carrier connection

The plasmid pCAMBIA1301 was extracted using the smallcloth plasmid miniprep kit, and double digestion was performed with Bgl II and BstE II to linearize the vector. The purified product was subjected to homologous recombination reaction with linearized vector using ClonExpII One Step Cloning Kit of Norpran (reaction conditions of 37 ℃ C., 30 min). 10. Mu.L of the recombinant reaction product was transformed into DH 5. Alpha. Competence, spread on LB medium containing 50mg/L kanamycin, and single colonies were PCR verified using primers (upstream primer SEQ ID NO.2: actcttgaccatggtagatctgatgggtcttctagacaagctctgg; downstream primer SEQ ID NO.3: ggggaaattcgagctggtcaccttacacatcgaagggaggataaatg) and positive clones were sequenced. The positive clone with correct sequence was named pCAMBIA1301-AtDRM2, and the vector construction schematic is shown in FIG. 1.

3. Genetic transformation

The pCAMBIA1301-AtDRM2 recombinant plasmid was transformed into Agrobacterium GV3101 using heat shock. Arabidopsis thaliana was cultivated and genetically transformed using inflorescence infestation as follows.

1. Soaking wild Col-0 Arabidopsis seeds in 75% ethanol for 30s, soaking in 25% sodium hypochlorite for 10min, and washing with sterile water for 3-5 times. Inoculating the seeds to MS basic culture medium with sterile gun head, placing in refrigerator at 4deg.C for vernalization for 2-3d, and transferring into illumination incubator at 25deg.C for culturing.

2. Long lengthTransferring Arabidopsis seedling into peat soil after 2-4 leaves are removed, and placing at 22deg.C with illumination intensity of 100-150 μmol.m ^-2 ·s ^-1 Is cultured in a culture chamber of (2). When the main inflorescence is pod-bearing and the secondary inflorescence is about 2-10cm long and flowering in small quantity, the arabidopsis inflorescence is infected by agrobacterium GV3101 containing the pCAMBIA1301-AtDRM2 recombinant plasmid.

The preparation method of the agrobacteria GV3101 invasion solution comprises the steps of absorbing 2mL of agrobacteria GV3101 containing pCAMBIA1301-AtDRM2 recombinant plasmid, inoculating the agrobacteria GV3101 into 100mL of YEP liquid culture medium containing 50mg/L kanamycin and 50mg/L rifampicin, and culturing at 28 ℃ and 200rpm until the OD value is 1.0-1.2. The bacterial solution was transferred to a 50mL sterile centrifuge tube, centrifuged at 6000rpm for 10min and the supernatant was discarded. The cells were resuspended to OD with a transformation solution (5% sucrose, 0.02% Silwet, formulated with 1/2 MS) ₆₀₀ The agrobacteria GV3101 invasion solution containing pCAMBIA1301-AtDRM2 recombinant plasmid is obtained at 0.8-1.0.

The method for infecting the arabidopsis inflorescence by using the agrobacterium GV3101 containing the pCAMBIA1301-AtDRM2 recombinant plasmid comprises the steps of immersing the inflorescence of the arabidopsis plant in an agrobacterium GV3101 invasion solution for 30-60s and culturing for 24h in a dark place. The transformation was repeated 1-2 times and the harvested seeds were designated as T0.

4. Homozygous selection and transgenic plant identification

1. Soaking T0 seeds in 75% ethanol for 30s, soaking in 25% sodium hypochlorite for 10min, and washing with sterile water for 3-5 times. Seeds were inoculated onto a resistance plate (MS minimal medium containing 50mg/L hygromycin) with a sterile gun head, placed in a refrigerator at 4℃for vernalization for 2-3d, and transferred to an illumination incubator at 25℃for cultivation.

2. Plants grown normally on the resistance plates were selected for cultivation and T1 generation seed harvested. And screening the T1 generation seeds in a separation proportion, selecting plants meeting the separation proportion of 3:1, further culturing, and harvesting the T2 generation seeds. And screening the T2 generation seeds by using a resistance plate, selecting all the plants which emerge and survive for culture, and harvesting the T3 generation seeds.

3. Identification of transgenic plants: the leaf DNA of the T3 plant was extracted by CTAB method and PCR was performed using primers (upstream primer SEQ ID NO.2: actcttgaccatggtagatctgatgggtcttctagacaagctctgg; downstream primer SEQ ID NO.3: ggggaaattcgagctggtcaccttacacatcgaagggaggataaatg). Extracting total RNA of leaves of the T3 generation plant by using an RNA extraction kit of Takara company, reversely transcribing the total RNA into cDNA, and using an upstream primer SEQ ID NO.4: ttcaggaggaagtcaatg; the downstream primer SEQ ID NO.5: aatggaagaagaactggaA) was subjected to qRT-PCR verification (the kit used for fluorescent quantitative PCR detection was ChamQ Universal SYBR qPCR Master Mix by Novazak, and the instrument was Roche LightCycler 96). Three strains with positive identification result and high gene expression were selected and named OE-2, OE-15 and OE-21.

< verification of Effect >

To study the biological function of the tsaoko AtDRM2 gene under cold stress, wild arabidopsis seedlings and transgenic arabidopsis seedlings were subjected to cold stress treatment at 4 ℃,0 ℃ and-10 ℃. Counting leaf wilting plant proportion, survival plant proportion and normal growth plant proportion after treatment, and taking the leaf to measure the antioxidant enzyme activity and Malondialdehyde (MDA) content. The leaves of wild type Arabidopsis thaliana and the over-expressed plants were taken, total RNA was extracted using the RNA extraction kit from Takara, reverse transcribed into cDNA, and cold stress response gene expression level was detected using specific primers as shown in Table 1. The housekeeping gene Actin is used as an internal reference gene (upstream primer SEQ ID NO.6: gcaccctgttcttcttaccga; downstream primer SEQ ID NO.7: agtaaggtcacgtccagcaagg), 2 is adopted ^-△△CT The relative expression of the gene to be tested is calculated by the method.

Experimental results show that after cold stress treatment for 24 hours at 4 ℃ and 0 ℃, the wild arabidopsis and the transgenic arabidopsis have no leaf wilting and death phenomenon, and the plants can grow normally after the stress treatment. After being subjected to cold stress treatment at the temperature of minus 10 ℃ for 3 hours, the leaf wilting rate (26-35%) of the over-expressed plants is obviously lower than that of wild plants (64%), the survival rate (84-90%) of the plants and the normal growth rate (72-74%) of the plants are obviously higher than that of the wild plants (78%, 46%), as shown in fig. 2 and 3. The overexpression of AtDRM2 can improve the superoxide dismutase (SOD) activity, the Peroxidase (POD) activity and the Catalase (CAT) activity of plant leaves under the cold stress condition, reduce the peroxidation degree of membrane lipid, and is helpful for improving the tolerance of cold stress as shown in figures 4, 5 and 6. Overexpression of AtDRM2 also significantly improved the expression of cold stress responsive genes, including AtCOR15a, atCBF1, atCBF2, atCBF3, atCOR47, atRCI2A, atRD A and AtKINI, indicating that overexpression of AtDRM2 can improve cold tolerance in plants, as shown in FIGS. 7, 8 and 9.

TABLE 1 specific primers

Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.

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<110> Guangxi Zhuang nationality medicinal plant garden

<120>Amomum tsao-koAtDRM2Application of gene in improving cold tolerance of plants

<160> 23

<170> PatentIn version 3.5

<210> 1

<211> 483

<212> DNA

<213> Amomum tsaoko

<400> SEQ ID NO.1

atgggtcttc tagacaagct ctgggacgac accctcgccg gcccccgccc cgactccggc 60

ctaggccgcc tccgcaagcc gccctccttc tcctcctcct cctcttcttc ttcttccccc 120

ggtgtcgttt cacccgtagg ggcggtgacg ccgacggcag ccgcgggggc tgcgggaaga 180

gcgagcagca gcggggaggg cataaacgta attaccggcc cgcagatggg ggaggatcag 240

ccgagggtga cgaggagcat tatgataaag aggcctcccg ggtggtcgtc gcctgggagc 300

ggtactccgc cgtcgtcgcc ggcgagctcc acccctcccc cctctccctt tgcgggaggc 360

ggagtagcgc agcggttcag gaggaagtca atgtcggacg cgcaggagag gaggagttcg 420

ccggtggagt tgccgacggc tgattccagt tcttcttcca tttatcctcc cttcgatgtg 480

taa 483

<210> 2

<211> 46

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.2

actcttgacc atggtagatc tgatgggtct tctagacaag ctctgg 46

<210> 3

<211> 47

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.3

ggggaaattc gagctggtca ccttacacat cgaagggagg ataaatg 47

<210> 4

<211> 18

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.4

ttcaggagga agtcaatg 18

<210> 5

<211> 19

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.5

aatggaagaa gaactggaa 19

<210> 6

<211> 21

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.6

gcaccctgtt cttcttaccg a 21

<210> 7

<211> 22

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.7

agtaaggtca cgtccagcaa gg 22

<210> 8

<211> 25

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.8

ggtaaagcag gagaggctaa ggatg 25

<210> 9

<211> 25

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.9

aagaatgtga cggtgactgt ggata 25

<210> 10

<211> 23

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.10

tgtctcaact tcgctgactc ggc 23

<210> 11

<211> 25

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.11

accttcgctc tgttccggtg tataa 25

<210> 12

<211> 25

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.12

ggtttcctca ggcggtgatt acagt 25

<210> 13

<211> 25

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.13

tcagcggttt ggaaagtccc gagcc 25

<210> 14

<211> 28

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.14

ggaatcaact tgcgctaagg acatccaa 28

<210> 15

<211> 27

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.15

ccaacaaact cggcatctca aacatcg 27

<210> 16

<211> 21

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.16

tatcatgcca agaccactga a 21

<210> 17

<211> 21

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.17

caacgaaagc cacaataaca a 21

<210> 18

<211> 20

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.18

atcgccatcc tcttgcctcc 20

<210> 19

<211> 19

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.19

taggagaaca cgacggaac 19

<210> 20

<211> 23

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.20

gtctgccgtg acgacgaagt tac 23

<210> 21

<211> 25

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.21

tccttcttct cttcttctcc tccaa 25

<210> 22

<211> 25

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.22

ggaccaacaa gaatgccttc caagc 25

<210> 23

<211> 21

<212> DNA

<213> artificial sequence

<400> SEQ ID NO.23

cgctgccgca tccgatacac t 21

Claims

1. The application of the tsaoko AtDRM2 gene in improving the cold resistance of plants is provided, wherein the DNA sequence of the tsaoko AtDRM2 gene is shown as SEQ ID NO. 1.

2. The use according to claim 1, characterized by the steps of:

1) Amplifying the AtDRM2 gene of the tsaoko;

3. The use according to claim 2, characterized in that the method for extracting the AtDRM2 gene of tsaoko comprises the following steps:

4. The method according to claim 3, wherein the upstream primer used in PCR amplification using cDNA as a template is SEQ ID NO.2: actcttgaccatggtagatctgatgggtcttctagacaagctctgg; the downstream primer is SEQ ID NO.3: ggggaaattcgagctggtcaccttacacatcgaagggaggataaatg.

5. The use according to claim 3, wherein the reaction conditions for PCR amplification using cDNA as template are: pre-denaturation at 95℃for 3min, denaturation at 95℃for 15s, annealing at 58℃for 15s, elongation at 72℃for 45s,35 cycles; the reaction system is as follows: 25 mu L of high-fidelity enzyme, 2 mu L of upstream primer, 2 mu L of downstream primer, 2 mu L of cDNA and ddH ₂ O 19μL。

6. The use according to claim 2, wherein the method of infecting plants with agrobacterium is to disinfect the plant seeds, clean them with sterile water, culture them on a culture medium, germinate the seeds and grow 2-4 leaves, transfer the seedlings to peat soil for culture, and when the plants bloom, the agrobacteria containing the pCAMBIA1301-AtDRM2 recombinant plasmid is used to dip the inflorescence, thus realizing the agrobacteria to plant.

7. The use according to claim 6, wherein the inflorescence is transfected with the agrobacterium containing the pCAMBIA1301-AtDRM2 recombinant plasmid by pipetting 2-5mL of agrobacterium containing the pCAMBIA1301-AtDRM2 recombinant plasmid into 100-200mL of YEP liquid medium containing 50-100mg/L kanamycin and 50-100mg/L rifampicin, culturing at 25-30 ℃ and 200-250rpm to an OD value of 1.0-1.2; transferring the bacterial liquid into a sterile centrifuge tube, centrifuging for 10-15min at 5000-6000rpm, discarding the supernatant, re-suspending the bacterial body with the transformation liquid until the OD600 is 0.8-1.0, obtaining an agrobacterium leaching solution containing pCAMBIA1301-AtDRM2 recombinant plasmid, soaking inflorescences of plants in the agrobacterium leaching solution for 30-60s, culturing for 24-36h in a dark place, and repeatedly transforming for 1-2 times.

8. The use according to claim 2, wherein the method of screening and identifying transgenic plants is to harvest the seeds T0 of the plants after infection of the plants with agrobacterium, to disinfect the seeds T0 and to clean them with sterile water, to culture them on a resistant medium, to select plants that grow normally for cultivation, to harvest the seeds T1; screening the separation proportion of the seeds T1, selecting plants meeting the separation proportion of 3:1 for culture, and harvesting the seeds of the generation T2; screening the seeds T2 by using a resistance flat plate, selecting seedlings and surviving plants to continue culturing, harvesting the T3 generation seeds, extracting DNA of leaves of the T3 generation plants, and carrying out PCR verification; extracting total RNA of T3 generation plant leaves, reversely transcribing the total RNA into cDNA, and detecting the expression quantity by fluorescent quantitative PCR to obtain the positive plant with high gene expression quantity.

9. The use according to claim 8, wherein the upstream primer of the fluorescent quantitative PCR is SEQ ID NO.4: ttcaggaggaagtcaatg; the downstream primer is SEQ ID NO.5: aatggaagaagaactggaa.