CN114381462A

CN114381462A - Application of tsaoko AtDRM1 gene in improving heat resistance of plants

Info

Publication number: CN114381462A
Application number: CN202210056548.2A
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: 2022-04-22
Anticipated expiration: 2042-01-18
Also published as: CN114381462B

Abstract

The invention discloses an application of a tsaoko AtDRM1 gene in improving heat resistance of plants, wherein a DNA sequence of the tsaoko AtDRM1 gene is shown as SEQ ID No. 1. The application of the tsaoko AtDRM1 gene in improving the heat resistance of plants comprises the following steps: 1) amplifying the tsaoko AtDRM1 gene; 2) carrying out double enzyme digestion on plasmids by using restriction enzymes, carrying out homologous recombination reaction on linearized plasmids and the tsaoko AtDRM1 gene, transforming a product into DH5 alpha competence, culturing on a culture medium coated with antibiotics, and carrying out positive verification on a single colony on the culture medium to obtain recombinant plasmids; 3) the recombinant plasmid is transformed into agrobacterium, and the agrobacterium is utilized to infect plants, and the transgenic plants are screened and identified, so that the plants with improved heat resistance are obtained. According to the invention, the plant of the trans-dormancy related gene DRM is constructed, so that the tolerance of the plant to heat stress is improved, and a new thought is provided for cultivating heat-resistant plants.

Description

Application of tsaoko AtDRM1 gene in improving heat resistance of plants

Technical Field

The invention belongs to the field of plant genetic engineering. More particularly, the invention relates to application of a tsaoko AtDRM1 gene in improving heat resistance of plants.

Background

Amomum tsaoko (Amomum tsaoko) is a perennial herb of the genus Amomum of the family Zingiberaceae. The fruit is an spice and an important medicinal material, has the functions of warming spleen and stomach, invigorating stomach, promoting digestion and guiding qi downward, and is mainly used for treating heart and abdomen pain, abdominal fullness and distention, nausea and vomiting, cough with excessive phlegm, malaria, chills and fever and the like. At present, the research on the tsaoko amomum fruits at home and abroad is mostly focused on the aspects of chemical components, pharmacological action and cultivation and breeding, and the research on the growth adaptability is less. The tsaoko has special biological characteristics, higher requirement on temperature and narrow growth range, and is mainly distributed in southeast and western Guangxi provinces of Yunnan province of China. The produced strawberries are mostly cultivated in broad-leaved forests with the altitude of 1100-1800 m, and the annual average temperature of a growing area is required to be 16-22 ℃. Yangling et al (2016) found that the curling character of the flower column of the tsaoko amomum fruit is very susceptible to temperature and humidity, and when the average temperature is higher than 18 ℃, the hysteresis phenomenon of the flower column curling exists, so that the plant breeding is influenced. The Yuanlong et al (1995) find that the viability of the tsaoko pollen is extremely easy to lose under the conditions of high temperature and low humidity, and the optimal temperature for pollen germination and pollen tube growth is between 12 and 24 ℃. These findings indicate that temperature is one of the major factors affecting the growth and reproduction of tsaoko amomum fruits. However, no research report for improving the temperature adaptability of the amomum tsao-ko exists at present.

Dormancy-associated genes (DRM) are members of the DRM1/ARP (dormancy-associated gene/auxin repressor) family, and are involved in regulating the dormancy of meristems or organs. There are several evidences that DRM is involved in seed dormancy maintenance, shoot dormancy and regulation of hormone signaling, however, there is no report on the use of DRM genes to improve plant temperature adaptability.

Disclosure of Invention

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

Still another object of the present invention is to provide a use of the tsaoko AtDRM1 gene for improving heat resistance of plants, which can improve the tolerance of plants to heat stress.

To achieve these objects and other advantages of the present invention, there is provided a use of a tsaoko AtDRM1 gene having a DNA sequence as set forth in SEQ ID No.1 for improving heat resistance of plants.

The application of the tsaoko AtDRM1 gene in improving the heat resistance of plants comprises the following steps:

1) amplifying the tsaoko AtDRM1 gene;

2) carrying out double digestion on a plasmid pCAMBIA1301 by Bgl II and BstEII restriction endonucleases, linearizing the plasmid pCAMBIA1301, carrying out homologous recombination reaction on the linearized plasmid pCAMBIA1301 and a tsaoko AtDRM1 gene, transforming a product into a DH5 alpha competence, culturing on a culture medium coated with antibiotics, and carrying out positive verification on a single colony on the culture medium to obtain a pCAMBIA1301-AtDRM1 recombinant plasmid;

3) the pCAMBIA1301-AtDRM1 recombinant plasmid is transformed into agrobacterium, and the agrobacterium is utilized to infect plants, and transgenic plants are screened and identified, so that the plants with improved heat resistance are obtained.

Preferably, the extraction method of the tsaoko AtDRM1 gene comprises the following steps:

a) taking 0.2-1g of tsaoko leaves, putting into a centrifugal tube containing grinding beads, quickly freezing with liquid nitrogen, and grinding into powder to obtain powder;

b) and extracting the total plant RNA in the powder, carrying out reverse transcription on the RNA to obtain cDNA, carrying out PCR amplification by using the cDNA as a template, and purifying to obtain the amomum tsao-ko AtDRM1 gene.

Preferably, in the step b), the upstream primer used for PCR amplification by taking the cDNA as a template is SEQ ID NO.2: actcttgaccatggtagatctgatggttctccttgataagatgtgg; the downstream primer is SEQ ID NO.3: ggggaaattcgagctggtcacctcaacggtgggtgctcttcg.

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, extension at 72 ℃ for 45s, and 35 cycles; the reaction system is as follows: Hi-Fi enzyme 25. mu.L, forward primer 2. mu.L, reverse primer 2. mu.L, cDNA 2. mu.L, ddH₂O 19μL。

Preferably, the method for infecting plants by using agrobacterium comprises the steps of disinfecting plant seeds, cleaning the plant seeds by using sterile water, culturing the plant seeds on a culture medium, transferring seedlings into peat soil for culturing after the seeds germinate and grow 2 to 4 leaves, and carrying out dip dyeing on inflorescences by using agrobacterium containing pCAMBIA1301-AtDRM1 recombinant plasmids when the plants bloom, namely, the dip dyeing of the agrobacterium on the plants is realized.

Preferably, the inflorescence is impregnated with Agrobacterium containing the pCAMBIA1301-AtDRM1 recombinant plasmid by pipetting 2-5mL of Agrobacterium containing the pCAMBIA1301-AtDRM1 recombinant plasmid into 100-200mL of YEP liquid medium containing 50-100mg/L kanamycin and 50-100mg/L rifampicin, and culturing at 25-30 ℃ and 200-250rpm until the OD value is 1.0-1.2. Transferring the bacterial liquid into a sterile centrifuge tube, centrifuging for 10-15min at 5000-.

Preferably, the method for screening and identifying transgenic plants comprises the steps of harvesting seeds T0 of the plants after the plants are infected by agrobacterium, disinfecting the seeds T0, cleaning the seeds with sterile water, culturing the seeds on a resistance culture medium, selecting the plants with normal growth for culturing, and harvesting the seeds T1; screening the separation ratio of the seeds T1, selecting plants according with the separation ratio of 3:1, culturing, and harvesting seeds of T2 generations; screening seeds T2 with a resistance plate, selecting plants which emerge and survive to continue culturing, harvesting seeds of T3 generations, extracting DNA of leaves of plants of T3 generations, and carrying out PCR verification; extracting total RNA of T3 plant leaves, reverse transcribing into cDNA, and performing fluorescent quantitative PCR detection on the expression quantity to obtain positive plants with high gene expression quantity.

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

The invention at least comprises the following beneficial effects: according to the invention, through constructing the plant with the trans-dormancy related gene DRM, the tolerance of the plant to heat stress is improved, and a new thought is provided for cultivating heat-resistant 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 pCAMBIA1301-AtDRM1 recombinant plasmids constructed by the invention;

FIG. 2 is a graph of plant growth variation under temperature stress; wherein, 1 represents before heat stress; 2 represents stress at 40 ℃ for 24 h; 3 denotes the recovery growth 7 d;

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

FIG. 4 is a graph showing the variation of antioxidant enzyme activity and Malondialdehyde (MDA) content of plants after temperature stress;

FIG. 5 is a graph showing the change in expression of genes associated with stress response after temperature stress.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

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.

It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.

The invention provides application of a tsaoko AtDRM1 gene in improving heat resistance of plants, wherein a DNA sequence of the tsaoko AtDRM1 gene is shown as SEQ ID No. 1.

1) amplifying the tsaoko AtDRM1 gene;

In another technical scheme, the method for amplifying the amomum tsao-ko AtDRM1 gene comprises the following steps:

In another technical scheme, the upstream primer when the cDNA is taken as a template for PCR amplification is SEQ ID NO.2: actcttgaccatggtagatctgatggttctccttgataagatgtgg; the downstream primer is SEQ ID NO.3: ggggaaattcgagctggtcacctcaacggtgggtgctcttcg.

In another technical scheme, the reaction conditions for PCR amplification by using cDNA as a template are as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 15s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 45s, and 35 cycles; the reaction system is as follows: Hi-Fi enzyme 25. mu.L, forward primer 2. mu.L, reverse primer 2. mu.L, cDNA 2. mu.L, ddH₂O 19μL。

In another technical scheme, the method for infecting the plant by using the agrobacterium comprises the steps of disinfecting plant seeds, cleaning the plant seeds by using sterile water, culturing the plant seeds on a culture medium, transferring seedlings into peat soil for culturing after the seeds germinate and grow 2 to 4 leaves, and carrying out dip dyeing on inflorescences by using the agrobacterium containing pCAMBIA1301-AtDRM1 recombinant plasmids when the plant blooms, namely, the dip dyeing of the plant by using the agrobacterium is realized.

In another embodiment, the method for impregnating the inflorescence with Agrobacterium containing the pCAMBIA1301-AtDRM1 recombinant plasmid is to aspirate 2-5mL of Agrobacterium containing the pCAMBIA1301-AtDRM1 recombinant plasmid into 100-200mL of YEP liquid medium containing 50-100mg/L kanamycin and 50-100mg/L rifampicin, and culture the Agrobacterium at 25-30 ℃ and 200-250rpm until the OD value is 1.0-1.2. Transferring the bacterial liquid into a sterile centrifuge tube, centrifuging for 10-15min at 5000-; soaking the inflorescence of the plant in the agrobacterium tumefaciens dip-dyeing solution for 30-60s, culturing for 24-36h in the dark, and repeatedly transforming for 1-2 times.

In another technical scheme, the method for screening and identifying transgenic plants comprises the steps of utilizing agrobacterium to infect plants, then harvesting seeds T0 of the plants, disinfecting the seeds T0, cleaning the seeds with sterile water, placing the seeds on a resistance culture medium for culture, selecting plants with normal growth for culture, and then harvesting seeds T1; screening the separation ratio of the seeds T1, selecting plants according with the separation ratio of 3:1, culturing, and harvesting seeds of T2 generations; screening seeds T2 with a resistance plate, selecting plants which emerge and survive to continue culturing, harvesting seeds of T3 generations, extracting DNA of leaves of plants of T3 generations, and carrying out PCR verification; extracting total RNA of T3 plant leaves, reverse transcribing into cDNA, and performing fluorescent quantitative PCR detection on the expression quantity to obtain positive plants with high gene expression quantity.

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

< example >

An application method of a tsaoko AtDRM1 gene in improving heat resistance of plants comprises the following steps:

first, DNA fragment acquisition of Amomum tsao-ko AtDRM1 gene

Taking 0.2g of tsaoko leaf, putting into a 2.0mL centrifuge tube containing grinding beads, quickly freezing with liquid nitrogen, and grinding into powder with a tissue grinder (JXFSTPRP-64L, Shanghai Jingxin). By using TakaTotal Plant RNA was extracted using TaKaRa MiniBEST Plant RNA Extraction Kit from Takara, PrimeScript^TMThe RT reagent Kit with gDNA Eraser (Perfect Real Time) Kit reverse transcribes RNA to cDNA. Designing and synthesizing an upstream primer SEQ ID NO.2: actcttgaccatggtagatctgatggttctccttgataagatgtgg according to the tsaoko AtDRM1 gene sequence SEQ ID NO.1, wherein actcttgaccatggtagatct is a PCAMBIA1301 partial vector sequence, and the vector can be obtained through a commercial channel; designing and synthesizing a downstream primer SEQ ID NO.3: ggggaaattcgagctggtcacctcaacggtgggtgctcttcg, wherein ggggaaattcgagctggtcacc is a pCAMBIA1301 partial vector sequence. PCR amplification was performed using Novozan Hi-Fi enzyme 2 × Phanta Max Master Mix using Amomum tsao-ko cDNA as template, under the following reaction conditions: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 15s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 45s, and 35 cycles; the reaction system is as follows: Hi-Fi enzyme 25. mu.L, forward primer 2. mu.L, reverse primer 2. mu.L, cDNA 2. mu.L, ddH₂O19. mu.L. The gel was recovered using DNA purification recovery kit from Tiangen Biochemical technology Ltd.

II, carrier ligation

The pCAMBIA1301 plasmid was extracted using the Tiangen plasmid miniprep kit, and the vector was linearized by double digestion with Bgl II and BstE II. The purified product was subjected to homologous recombination with linearized vector using Novozan Clonexpress II One Step Cloning Kit at 37 ℃ for 30 min. mu.L of the recombinant reaction product was transformed into DH 5. alpha. competence, spread on LB medium containing 50mg/L kanamycin, single colonies were verified by PCR using primers (upstream primer SEQ ID NO.2: actcttgaccatggtagatctgatggttctccttgataagatgtgg; downstream primer SEQ ID NO.3: ggggaaattcgagctggtcacctcaacggtgggtgctcttcg), and positive clones were sequenced. The positive clone with correct sequencing was named pCAMBIA1301-AtDRM1, and the vector construction scheme is shown in FIG. 1.

Third, genetic transformation

The pCAMBIA1301-AtDRM1 recombinant plasmid was transformed into Agrobacterium GV3101 using a heat shock method. The method comprises the following steps of culturing Arabidopsis and carrying out genetic transformation by using an inflorescence infection method:

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 an MS basic culture medium by using a sterile gun head, performing vernalization in a refrigerator at 4 ℃ for 2-3 days, and transferring to a light incubator at 25 ℃ for culture.

2. After 2-4 leaves are grown, the Arabidopsis seedlings are transferred to peat soil and are placed at 22 ℃ with the illumination intensity of 100-^-2·s^-1Culturing in the culture chamber of (1). When the main inflorescence is pod-bearing, the minor inflorescence is about 2-10cm long and flowers a little, the arabidopsis inflorescence is infected by using the dip dyeing solution.

The invaded solution was prepared by pipetting 2mL of Agrobacterium GV3101 containing the recombinant plasmid pCAMBIA1301-AtDRM1 and inoculating it into 100mL of YEP liquid medium containing 50mg/L kanamycin and 50mg/L rifampicin, and culturing at 28 ℃ and 200rpm until the OD value was 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 in a transformation solution (containing 5% sucrose, 0.02% Silwet, formulated with 1/2 MS) until OD600 was 0.8-1.0, to obtain a stock solution of Agrobacterium GV3101 containing the recombinant plasmid pCAMBIA1301-AtDRM 1.

The method for infecting arabidopsis inflorescence by using agrobacterium GV3101 staining solution comprises soaking the inflorescence of arabidopsis plant in the agrobacterium GV3101 staining solution for 30-60s, and culturing for 24h in dark. Transformation was repeated 1-2 times and the harvested seeds were scored as T0.

Screening of homozygote and identification of transgenic plant

1. Soaking T0 seed 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 a resistant plate (MS minimal medium containing 50mg/L hygromycin) by using a sterile gun head, performing vernalization for 2-3 days in a refrigerator at 4 ℃, and then transferring to a light incubator at 25 ℃ for culture.

2. Plants grown normally on resistant plates were selected for culture and seeds from T1 generations were harvested. And screening the separation ratio of the T1 generation seeds, selecting plants meeting the separation ratio of 3:1, further culturing, and harvesting T2 generation seeds. Seeds of T2 generations were screened with a resistant plate, plants that all emerged and survived were selected for culture, and seeds of T3 generations were harvested.

3. Identification of transgenic plants: DNA of T3 plant leaves is extracted by a CTAB method, and PCR verification is carried out by using primers (an upstream primer SEQ ID NO.2: actcttgaccatggtagatctgatggttctccttgataagatgtgg; a downstream primer SEQ ID NO.3: ggggaaattcgagctggtcacctcaacggtgggtgctcttcg). Total RNA of T3 plant leaves is extracted by an RNA extraction kit of Takara company and is reversely transcribed into cDNA, qRT-PCR verification is carried out by using primers (an upstream primer SEQ ID NO.4: ggtctctccaaagccttt; a downstream primer SEQ ID NO.5: agatattgaacggtggaacat) (a kit used for fluorescent quantitative PCR detection is a ChamQ Universal SYBR qPCR Master Mix of Novowed company, and an instrument is LightCycler 96 of Roche). Three strains with positive identification results and high gene expression amount are selected and named as OE-3, OE-27 and OE-34.

< verification of Effect >

In order to research the biological function of the tsaoko AtDRM2 gene under heat stress, heat stress treatment at 40 ℃ is carried out on wild type Arabidopsis seedlings and transgenic Arabidopsis seedlings. And (4) counting the leaf wilting plant ratio, the survival plant ratio and the normal growth plant ratio after treatment, and taking leaves to perform antioxidase activity and MDA content determination. Taking wild arabidopsis thaliana and over-expressed plant leaves, extracting total RNA by using an RNA extraction kit of Takara company, carrying out reverse transcription to obtain cDNA, and carrying out heat stress response gene expression quantity detection by using specific primers which are shown in table 1. The housekeeping gene Actin is taken as an internal reference gene (an upstream primer SEQ ID NO.6: gcaccctgttcttcttaccga and a downstream primer SEQ ID NO.7: agtaaggtcacgtccagcaagg), 2 is adopted^-△△CTThe relative expression quantity of the gene to be detected is calculated by the method.

The experimental result shows that after 24 hours of heat stress treatment, the ratio of the plants with withered leaves of wild arabidopsis thaliana is more than 90%, and the ratio of the plants with wilted leaves of transgenic arabidopsis thaliana is less than 60%. The heat stress obviously reduces the proportion of the wild arabidopsis surviving plants, only 54.1 percent, and the proportion of the plants which can normally grow after the plants are recovered to grow for 7 days is only 15.8 percent; the ratio of the transgenic arabidopsis surviving plants after the heat stress treatment reaches more than 90%, and more than 80% of the plants can normally grow after the growth is recovered for 7 days, as shown in fig. 2 and fig. 3. The over-expression of AtDRM1 can improve the activity of plant leaf superoxide dismutase (SOD), the activity of Peroxidase (POD) and the activity of Catalase (CAT) under the condition of heat stress and reduce the degree of membrane lipid peroxidation, and is helpful for improving the tolerance of the heat stress as shown in figure 4. Overexpression of AtDRM1 also significantly improved the expression of heat stress response genes including AtHsp25.3-P, AtHsp18.2-CI, AtHsp70B, AtHsp101, AtHsfA2, AtHsfB1, AtHsfB2a, AtGolS and AtMBF1c, indicating that overexpression of AtDRM1 can improve the heat resistance of plants, as shown in FIG. 5.

TABLE 1

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

<110> Guangxi Zhuang autonomous region medicinal plant garden

<120>Amomum tsaokoAtDRM1Application of gene in improving heat resistance of plant

<160> 25

<170> PatentIn version 3.5

<210> 1

<211> 378

<212> DNA

<213> Amomum tsaoko

<400> SEQ ID NO.1

atggttctcc ttgataagat gtgggacgac gtcttcggcg gtcctcagcc tgagaaaggc 60

ctaggcaagc tgaggaaggt ctctccaaag cctttgtcca tcaaagaagg ggaaggcagc 120

agtgaaggtg gcggcatgtt ccaccgttca atatctctac cccagacgcc gacgacgccg 180

accactccag tgacgccgtc gtcgtccagc tactccccga ctcagcggca gggcaacgtg 240

tggaggagcg tgttcaaccc cggcagcaac ctcgccacca agactgtcgg agctcactat 300

ttcgacaagc cccaacccaa ctcccccact gtctacgact ggttgtacag cggtgagacg 360

aagagcaccc accgttga 378

<210> 2

<211> 46

<212> DNA

<213> Artificial sequence

<400> SEQ ID NO.2

actcttgacc atggtagatc tgatggttct ccttgataag atgtgg 46

<210> 3

<211> 42

<212> DNA

<213> Artificial sequence

<400> SEQ ID NO.3

ggggaaattc gagctggtca cctcaacggt gggtgctctt cg 42

<210> 4

<211> 18

<212> DNA

<213> Artificial sequence

<400> SEQ ID NO.4

ggtctctcca aagccttt 18

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence

<400> SEQ ID NO.5

agatattgaa cggtggaaca t 21

<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

acggagtcct ctttatcact atccc 25

<210> 9

<211> 22

<212> DNA

<213> Artificial sequence

<400> SEQ ID NO.9

gatcgagtcc tactgaatct gg 22

<210> 10

<211> 22

<212> DNA

<213> Artificial sequence

<400> SEQ ID NO.10

ttcacgccat cttctgcgtt gg 22

<210> 11

<211> 23

<212> DNA

<213> Artificial sequence

<400> SEQ ID NO.11

tgtaaacgct gccacatcac gag 23

<210> 12

<211> 27

<212> DNA

<213> Artificial sequence

<400> SEQ ID NO.12

agctattggt atcgatctcg gcactac 27

<210> 13

<211> 28

<212> DNA

<213> Artificial sequence

<400> SEQ ID NO.13

aagcctcagc gacttccttc atcttcac 28

<210> 14

<211> 21

<212> DNA

<213> Artificial sequence

<400> SEQ ID NO.14

accgagaaga agtcctctgg c 21

<210> 15

<211> 21

<212> DNA

<213> Artificial sequence

<400> SEQ ID NO.15

tttcccacga agcttctcaa c 21

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence

<400> SEQ ID NO.16

tggtgtgctt gtagctgagg 20

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence

<400> SEQ ID NO.17

cataaccgca aactgctgaa 20

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence

<400> SEQ ID NO.18

tcgtgtggaa aacagcagag 20

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence

<400> SEQ ID NO.19

atttacgccg tcgtatgtcc 20

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence

<400> SEQ ID NO.20

aacgaagacga ctgggaatg 20

<210> 21

<211> 20

<212> DNA

<213> Artificial sequence

<400> SEQ ID NO.21

cgggagaata actccgatca 20

<210> 22

<211> 22

<212> DNA

<213> Artificial sequence

<400> SEQ ID NO.22

agccgttcat caccgctctt ac 22

<210> 23

<211> 22

<212> DNA

<213> Artificial sequence

<400> SEQ ID NO.23

actcctggca acattcaagc ag 22

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence

<400> SEQ ID NO.24

agaggtgagg ttgatgatac 20

<210> 25

<211> 17

<212> DNA

<213> Artificial sequence

<400> SEQ ID NO.25

gcacagcctg attagga 17

Claims

1. The application of the tsaoko AtDRM1 gene in improving the heat resistance of plants is disclosed, wherein the DNA sequence of the tsaoko AtDRM1 gene is shown in SEQ ID NO. 1.

2. Use according to claim 1, characterized in that it comprises the following steps:

1) amplifying the tsaoko AtDRM1 gene;

3. The use of claim 2, wherein the method for amplifying the Amomum tsao-ko AtDRM1 gene comprises the following steps:

4. The use of claim 3, wherein the upstream primer for PCR amplification using cDNA as a template is SEQ ID NO.2: actcttgaccatggtagatctgatggttctccttgataagatgtgg; the downstream primer is SEQ ID NO.3: ggggaaattcgagctggtcacctcaacggtgggtgctcttcg.

5. The use of claim 3, wherein the reaction conditions for PCR amplification using cDNA as template are as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 15s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 45s, and 35 cycles; the reaction system is as follows: Hi-Fi enzyme 25. mu.L, forward primer 2. mu.L, reverse primer 2. mu.L, cDNA 2. mu.L, ddH₂O 19μL。

6. The application of claim 2, wherein the plant is infected by the agrobacterium, the plant seeds are disinfected, washed by sterile water, cultured on a culture medium, seedlings are transferred to peat soil for culture after the seeds germinate and grow 2 to 4 leaves, and when the plant blooms, inflorescences are impregnated by the agrobacterium containing the pCAMBIA1301-AtDRM1 recombinant plasmid, namely the plant is impregnated by the agrobacterium.

7. The use as claimed in claim 6, wherein the method for impregnating inflorescences with Agrobacterium containing the pCAMBIA1301-AtDRM1 recombinant plasmid comprises the steps of sucking 2-5mL of Agrobacterium containing the pCAMBIA1301-AtDRM1 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 until the OD value is 1.0-1.2; transferring the bacterial liquid into a sterile centrifuge tube, centrifuging for 10-15min at 5000-.

8. The use of claim 2, wherein the method of screening and identifying transgenic plants comprises harvesting plant seed T0 after infecting the plant with agrobacterium, sterilizing and washing seed T0 with sterile water, culturing on a resistant medium, selecting a normally growing plant for culturing, harvesting seed T1; screening the separation ratio of the seeds T1, selecting plants according with the separation ratio of 3:1, culturing, and harvesting seeds of T2 generations; screening seeds T2 with a resistance plate, selecting plants which emerge and survive to continue culturing, harvesting seeds of T3 generations, extracting DNA of leaves of plants of T3 generations, and carrying out PCR verification; extracting total RNA of T3 plant leaves, reverse transcribing into cDNA, and performing fluorescent quantitative PCR detection on the expression quantity to obtain positive plants with high gene expression quantity.

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