CN111269298B

CN111269298B - Application of protein GhCCOAOMT7 in regulation and control of plant heat resistance

Info

Publication number: CN111269298B
Application number: CN201811375275.8A
Authority: CN
Inventors: 朱生伟; 黄丹; 隆浩; 罗小敏; 蒙福宁; 曹务强; 陈霞; 毛玉伟
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2018-11-19
Filing date: 2018-11-19
Publication date: 2021-08-31
Anticipated expiration: 2038-11-19
Also published as: CN111269298A

Abstract

The invention discloses an application of protein GhCCOAOMT7 in regulation and control of plant heat resistance, and an amino acid sequence of the protein is shown as a sequence 2 in a sequence table. Experiments prove that the expression quantity and/or activity of the protein GhCCOAOMT7 in wild type arabidopsis is improved to obtain transgenic arabidopsis; compared with wild arabidopsis thaliana, the transgenic arabidopsis thaliana has improved heat resistance, specifically, the survival rate is improved, the germination rate of pollen is improved, and the pollen tube length is increased. Inhibiting the expression quantity and/or activity of protein GhCCOAOMT7 in the upland cotton TM-1 to obtain an upland cotton TM-1 silent strain; compared with the upland cotton TM-1, the heat resistance of the upland cotton TM-1 silent plant is reduced, which is particularly represented by the reduction of plant height and the reduction of half wilting time of the plant stem tip after high-temperature treatment. The protein GhCCOAOMT7 can regulate the heat resistance of plants. The invention has important application value.

Description

Application of protein GhCCOAOMT7 in regulation and control of plant heat resistance

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a protein GhCCOAOMT7 in regulation and control of plant heat resistance.

Background

With the advance of industrialization and the increase of human activities, the global warming trend is increasingly getting worse. In 2018, the book "blue book for climate change in China" issued by the China weather administration indicates that 2017 is the second warm year since complete weather observation and recording all over the world, and China is a sensitive area and an area with significant influence on global climate change. Extreme high temperature weather in China frequently occurs in recent years, the average temperature in the region tends to rise, the temperature increase rate in the northwest region is more obvious than that in the south, and particularly the high temperature (more than or equal to 35 ℃) adversity in the Xinjiang cotton region in the northwest region frequently occurs. Along with the action of agricultural irrigation and climatic factors, the high-temperature situation is increasingly serious, the overall situation is not optimistic, and the high-temperature disaster is treated (Elfatih Eltahir et al, 2018). High temperature not only harms human life health (center for prevention and control of Chinese diseases, 2014), but also stresses the growth and development of plants.

Cotton is an important fiber crop in the world, is also an important oil and biological energy crop, and plays an important role in the development of national economy and society. The frequent high temperature (more than or equal to 35 ℃) adversity causes serious cotton bud and boll shedding and boll forming development deformity, thus causing the cotton to lose yield and fiber quality to be reduced. The longer the high temperature duration, the higher the shedding rate becomes, which becomes one of the important environmental impact factors affecting the yield and quality of cotton. Therefore, the research on the cotton heat-resistance related gene and the enhancement of the heat resistance of the cotton have important significance for improving the yield and the quality of the cotton.

Disclosure of Invention

The object of the present invention is to improve the heat resistance of plants.

The invention firstly protects the application of the protein GhCCOAOMT7 in regulating and controlling the heat resistance of plants.

In the above application, the protein GhCCoAOMT7 may be a1) or a2) or a 3):

a1) the amino acid sequence is protein shown as a sequence 2 in a sequence table;

a2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 2 in the sequence table;

a3) a protein related to plant heat resistance, which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in a1) or a 2).

Wherein, the sequence 2 in the sequence table is composed of 336 amino acid residues.

In order to facilitate the purification of the protein in a1), the amino terminal or the carboxyl terminal of the protein shown in the sequence 2 in the sequence table can be connected with a label shown in the table 1.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
FLAG	8	DYKDDDDK
			Strep-tag II	8	WSHPQFEK
c-myc	10	EQKLISEEDL

The protein according to a3), wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein of a3) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of a3) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence 1 in the sequence table, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 above at the 5 'end and/or 3' end.

The application of the nucleic acid molecule for coding any one of the proteins GhCCOAOMT7 in regulating and controlling the heat resistance of plants also belongs to the protection scope of the invention.

In the above application, the nucleic acid molecule encoding the protein GhCCOAOMT7 can be a DNA molecule shown in b1) or b2) or b3) or b4) as follows:

b1) the coding region is a DNA molecule shown as a sequence 1 in a sequence table;

b2) the nucleotide sequence is a DNA molecule shown as a sequence 1 in a sequence table;

b3) a DNA molecule which has 75 percent or more identity than 75 percent with the nucleotide sequence defined by b1) or (b2) and codes the protein GhCCOAOMT 7;

b4) a DNA molecule which hybridizes with the nucleotide sequence defined in (b1) or (b2) under strict conditions and encodes the protein GhCCOAOMT 7.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, the sequence 1 in the sequence table is composed of 1011 nucleotides, and the nucleotide of the sequence 1 in the sequence table encodes an amino acid sequence shown as a sequence 2 in the sequence table.

The nucleotide sequence of the invention encoding the protein GhCCOAOMT7 can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified and have 75% or more identity to the nucleotide sequence of the protein GhCCOAOMT7 isolated in the present invention, as long as the nucleotide sequence encoding the protein GhCCOAMT 7 is derived from the nucleotide sequence of the present invention and is identical to the sequence of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the protein GhCCOAOMT7 consisting of the amino acid sequence shown in sequence No. 2 of the sequence Listing of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In any of the above applications, the regulating the plant heat tolerance may be improving the plant heat tolerance or reducing the plant heat tolerance.

In the use of any of the above, the plant may be any of the following c1) to c 7): c1) a dicotyledonous plant; c2) a monocot plant; c3) cotton; c4) cotton variety upland cotton TM-1; c5) a cruciferous plant; c6) arabidopsis thaliana; c7) the wild type Arabidopsis thaliana Columbia-0 subtype.

The invention also provides a method for cultivating the transgenic plant A or a method for cultivating the transgenic plant B.

The method for cultivating the transgenic plant A can comprise the following steps: increasing the expression quantity and/or activity of any one of the proteins GhCCOAOMT7 in the starting plant to obtain a transgenic plant A; the transgenic plant A has improved heat resistance compared with the original plant.

In the above method, the "increasing the expression level and/or activity of any one of the above proteins GhCCoAOMT7 in the starting plant" can be achieved by a method known in the art, such as transgene, multicopy, and change of a promoter and a regulatory factor, to increase the expression level and/or activity of any one of the above proteins GhCCoAOMT7 in the starting plant.

In the above method, the "increasing the expression level and/or activity of any of the proteins GhCCoAOMT7 in the starting plant" may be specifically achieved by introducing a nucleic acid molecule encoding the protein GhCCoAOMT7 into the starting plant.

In the above method, the "introducing into the starting plant a nucleic acid molecule encoding the protein GhCCoAOMT 7" may be carried out by introducing into the starting plant a recombinant vector a; the recombinant vector A can be a recombinant plasmid obtained by inserting a nucleic acid molecule encoding the protein GhCCOAOMT7 into an expression vector. The recombinant vector A can be specifically the recombinant plasmid 35S:: GhCoAOMT 7-EYFP mentioned in the examples. The recombinant plasmid 35S is obtained by inserting a double-stranded DNA molecule shown in the 1 st to 744 th positions from the 5' end of a sequence 1 in a sequence table between recognition sequences of restriction enzymes Sal I and BamH I of a vector pEZR-LNY.

The transgenic plants A can be GhCCOAOMT7-OE 6-11 and GhCCOAOMT7-OE 19-3.

In one embodiment of the present invention, the starting plant may be specifically any one of c1), c2), c5), c6) and c 7): c1) a dicotyledonous plant; c2) a monocot plant; c5) a cruciferous plant; c6) arabidopsis thaliana; c7) an ecotype Arabidopsis thaliana Columbia-0 subtype.

The method for cultivating the transgenic plant B can comprise the following steps: inhibiting the expression quantity and/or activity of any one of the proteins GhCCOAOMT7 in the starting plant to obtain a transgenic plant B; the transgenic plant B has reduced heat tolerance compared to the starting plant.

In the above method, the "inhibiting the expression level and/or activity of any one of the proteins GhCCoAOMT7 in the starting plant" can be achieved by a method known in the art, such as gene site-directed editing, RNA interference, homologous recombination, gene knock-out, etc., to inhibit the expression level and/or activity of the protein GhCCoAOMT 7.

In the above method, the "inhibition of the expression level and/or activity of any of the above proteins GhCCoAOMT7 in the starting plant" may be achieved by introducing a substance that inhibits the expression of the protein GhCCoAOMT7 into the starting plant. The substance that inhibits the expression of the protein GhCCoAOMT7 may be a substance that inhibits the expression of a nucleic acid molecule encoding the protein GhCCoAOMT 7. The "substance inhibiting the expression of the nucleic acid molecule encoding the protein GhCCoAOMT 7" can be achieved by introducing a recombinant plasmid PTRV2-GhCCoAOMT7 and a vector PTRV1 into the starting plant. The recombinant plasmid PTRV2-GhCCOAOMT7 can be specifically a DNA molecule shown in 457 th to 656 th positions from the 5' tail end of a sequence 1 in a sequence table by inserting into a recognition sequence of a restriction enzyme Pst I of a vector PTRV 2.

The transgenic plant B can be the upland cotton TM-1 silent strains T2, T3, T5 and T9 mentioned in the examples.

In one embodiment of the present invention, the starting plant may specifically be any one of c1) -c 4): c1) a dicotyledonous plant; c2) a monocot plant; c3) cotton; c4) cotton variety upland cotton TM-1.

The invention also protects a plant breeding method I or a plant breeding method II.

The first plant breeding method can comprise the following steps: increasing the content and/or activity of any one of the proteins GhCCOAOMT7 in the plant, thereby improving the heat resistance.

The second plant breeding method can comprise the following steps: reducing the content and/or activity of any one of the proteins GhCCOAOMT7 in the plant, thereby reducing the heat resistance.

Any of the plants described above may be any of the following c1) to c 7): c1) a dicotyledonous plant; c2) a monocot plant; c3) cotton; c4) cotton variety upland cotton TM-1; c5) a cruciferous plant; c6) arabidopsis thaliana; c7) the wild type Arabidopsis thaliana Columbia-0 subtype.

Experiments prove that the transgenic Arabidopsis thaliana with GhCoAOMT 7 gene is obtained by introducing recombinant plasmid 35S, namely GhCoAOMT 7-EYFP, into wild Arabidopsis thaliana; compared with wild arabidopsis, the GhCCOAOMT7 transgenic arabidopsis has improved heat resistance, and is specifically represented as follows: increased survival rates, increased relative expression levels of the "HSFA 3 gene, DREB2A gene, HSP101 gene, and HSP26 gene", increased germination rates of pollen, and increased pollen tube lengths. Introducing a recombinant plasmid PTRV 2-GhCoAOMT 7 and a vector PTRV1 into the upland cotton TM-1 to obtain an upland cotton TM-1 silent strain; compared with the upland cotton TM-1, the heat resistance of the upland cotton TM-1 silent plant is reduced, which is particularly represented by the reduction of plant height and the reduction of half wilting time of the stem tip of the plant after high-temperature treatment. Therefore, the protein GhCCOAOMT7 can regulate the heat resistance of plants. The invention has important application value.

Drawings

FIG. 1 shows the heat resistance identification and Real-Time PCR detection of GhCCOAOMT7 gene in GhCCOAOMT7 gene of GhCCOAOMT7 gene transferred Arabidopsis thaliana.

FIG. 2 shows the identification of heat resistance of transgenic Arabidopsis pollen with GhCCOAOMT7 gene.

FIG. 3 shows relative expression levels of HSFA3 gene, DREB2A gene, HSP101 gene and HSP26 gene in GhCCOAOMT7 transgenic Arabidopsis thaliana detected by Real-Time PCR.

FIG. 4 is the identification of thermotolerance of cotton Gossypium hirsutum TM-1 silent strain.

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 a conventional biochemical reagent store unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

Upland cotton TM-1 is described in the following documents: tronlinder NL et al, 1989, Plant Cell Rep 8: 133-. The public is available from the plant institute of Chinese academy of sciences (i.e., the applicant) to repeat the experiments of the present application. Hereinafter, upland cotton TM-1 is simply referred to as TM-1.

Agrobacterium tumefaciens GV3101 is described in: zhengyingying, trebaiming, changming, Pengming, the research of transforming Arabidopsis ICE1 gene to enhance the cold resistance of tobacco, the northwest plant declaration, No. 29, No. 1, No. 75-79 in 2009, the public can be obtained from the institute of plants (i.e. the applicant) of Chinese academy of sciences, so as to repeat the experiment of the application.

Both the vector PTRV1 and the vector PTRV2 are described in the following documents: dong Y, Burch-Smith TM, Liu Y, Mamillapalli P, Dinesh-Kumar SP.A. growth-independent cloning of bacteria and virus vector for high-throughput virus-induced genes ingredients rolls for NbMADS4-1 and-2 in flow estimation plant physiology.plant physiology.2007, No. 145 1161 & 1170, publicly available from the plant institute of Chinese academy of sciences (i.e., the Applicant) to repeat the experiments of the present application.

Wild type Arabidopsis thaliana (Arabidopsis thaliana) (Columbia-0 subtype) is described in the following references: kim H, Hyun Y, Park J, Park M, Kim M, Kim H, Lee M, Moon J, Lee I, Kim J.A genetic link between cool responses and flowing time through FVE in Arabidopsis thaliana Nature genetics.2004,36: 167-. Hereinafter, Arabidopsis thaliana (Columbia-0 subtype) is simply referred to as wild type Arabidopsis thaliana.

The nucleotide sequence (circular) of the vector pEZR-LNY is shown as a sequence 3 in a sequence table.

The Phusion enzyme is a product of the Saimerfi company. 5 × Phusionbuffer is a module in Phusion enzyme.

Example 1 cloning of a Gene encoding the protein GhCCOAOMT7 (GhCCOAOMT7 Gene)

1. Extracting total RNA of leaves of 14-day-old upland cotton TM-1 seedlings by a Trizo1 method, and performing reverse transcription by using a reverse transcriptase AMV to obtain first-strand cDNA so as to obtain the upland cotton TM-1 cDNA.

2. Taking the cDNA of the upland cotton TM-1 obtained in the step 1 as a template, adopting a Forward Primer: 5'-ATGATTTGTTTCTCAAGCATATATTTG-3' and Reverse Primer: 5'-TCAACGAATTCGCCTGCA-3', and PCR amplification is carried out to obtain double-stranded DNA molecules of about 750 bp.

The reaction system was 50. mu.L, consisting of 2. mu.L of template (containing 1. mu.g of cDNA of Gossypium hirsutum TM-1), 1. mu.L of Phusion enzyme, 2.5. mu.L of Forward Primer aqueous solution (concentration 10. mu.M), 2.5. mu.L of Reverse Primer aqueous solution (concentration 10. mu.M), 10. mu.L of 5 XPhusionBuffe, 10. mu.L of dNTP mix (concentrations of dATP, dTTP, dGTP and dCTP are all 10mmol), and 22. mu.L of ddH₂0.

The reaction conditions are as follows: 3min at 98 ℃; 30s at 98 ℃, 30s at 60 ℃ and 1min at 72 ℃ for 35 cycles; 10min at 72 ℃; 5min at 20 ℃; storing at 4 ℃.

And (3) sequencing the double-stranded DNA molecules obtained in the step (3). The sequencing result shows that the nucleotide sequence of the double-stranded DNA molecule (GhCCOAOMT7 gene) obtained in the step 3 is shown as the 1 st to 744 th positions from the 5' end of the sequence 1 in the sequence table.

Example 2 acquisition and characterization of transgenic Arabidopsis thaliana with GhCCOAOMT7 Gene

First, the acquisition of recombinant plasmid 35S, GhCoAOMT 7-EYFP and GV3101/35S, GhCoAOMT 7-EYFP

1. Double-stranded DNA molecules shown in 1 st to 744 th positions from the 5' end of a sequence 1 in a sequence table are inserted between recognition sequences of restriction enzymes Sal I and BamH I of a vector pEZR-LNY to obtain a recombinant plasmid 35S, namely GhCCOAOMT 7-EYFP.

The recombinant plasmid 35S is protein GhCCOAOMT7 shown in a sequence 2 in a GhCCOAOMT7-EYFP expression sequence table.

2. The recombinant plasmid 35S is GhCoAOMT 7-EYFP is introduced into Agrobacterium tumefaciens GV3101 to obtain recombinant Agrobacterium, which is named as GhCoAOMT 7-EYFP, GhCoAOMT 3101/35S.

The vector pEZR-LNY is led into the agrobacterium tumefaciens GV3101 to obtain the recombinant agrobacterium tumefaciens which is named as GV 3101/pEZR-LNY.

Second, obtaining of transgenic Arabidopsis thaliana with GhCCOAOMT7 gene

1. GV3101/35S obtained in 2 of the first step (GhCCOAOMT 7-EYFP) was transferred to wild type Arabidopsis thaliana to obtain T.thaliana by the floral dip transformation method (described in Clough, S.J., and Bent, A.F. Floraldip: expressed method for Agrobacterium-mediated transformation of Arabidopsis thaliana. plant J. (1998)16, 735-743.), and T.₁Transferring GhCCOAOMT7 gene Arabidopsis thaliana seeds.

2. The T obtained in the step 1₁Transferring GhCCOAOMT7 gene Arabidopsis seeds to be sown on 1/2MS culture medium containing 30mg/L hygromycin, the Arabidopsis capable of normally growing (resistant seedling) is T₁Transfer GhCCOAOMT7 gene positive seedling, T₁The seeds received by the positive seedlings of the transfer GhCCOAOMT7 gene are T₂Transferring GhCCOAOMT7 gene Arabidopsis thaliana seeds.

3. The T of different strains screened in the step 2₂Transgenic Arabidopsis thaliana with GhCCOAOMT7 geneSeeds were sown on 1/2MS medium containing 30mg/L hygromycin for selection, if the ratio of the number of Arabidopsis thaliana capable of normal growth (resistant shoots) to the number of Arabidopsis thaliana incapable of normal growth (non-resistant shoots) in a certain strain was 3: 1, the strain is a strain with a copy of the GhCCOAOMT7 gene inserted therein, and the seeds received by the resistant seedlings in the strain are T₃Transferring GhCCOAOMT7 gene Arabidopsis thaliana seeds.

4. The T screened out in the step 3₃The transgenic Arabidopsis seeds with the GhCCOAOMT7 gene are sown on 1/2MS culture medium containing 30mg/L hygromycin again for screening, and the transgenic Arabidopsis seeds with the GhCCOAMT 7 gene which are all resistant seedlings are T3 generation homozygous transgenic Arabidopsis with the GhCCOAMT 7 gene. 2T 3-generation homozygous transgenic GhCCOAOMT7 Arabidopsis strains are named as GhCCOAOMT7-OE 6-11 (hereinafter referred to as 6-11 or L6-11) and GhCCOAMT 7-OE19-3 (hereinafter referred to as 19-3 or L19-3), and subsequent experiments are carried out.

According to the method, GhCCOAOMT7-EYFP is replaced by GV3101/pEZR-LNY, and other steps are the same, so that a T3 generation homozygous empty vector transfer Arabidopsis thaliana plant, namely empty vector transfer Arabidopsis thaliana is obtained.

Identification of transgenic Arabidopsis thaliana with GhCCOAOMT7 gene

1. Heat resistance identification of GhCCOAOMT7 gene-transferred Arabidopsis thaliana

The Arabidopsis seeds to be tested are T3 generation seeds of 6-11 and T of 19-3₃T of seed generation and empty vector Arabidopsis thaliana₃Generation seed or wild type arabidopsis seed.

(1) Taking an arabidopsis seed to be detected, soaking the arabidopsis seed in 70% (v/v) ethanol water solution for 30s, and washing the arabidopsis seed with sterile water for 3 times; then, the suspension was spread on 1/2MS solid medium and vernalized at 4 ℃ for 2 days.

(2) And (3) after the step (1) is finished, taking the arabidopsis thaliana seeds to be detected, and carrying out light-dark alternate culture (16h illumination culture/8 h dark culture) at the temperature of 22 ℃ for 7 days to obtain the arabidopsis thaliana seedlings to be detected.

(3) And (3) after the step (2) is finished, taking the arabidopsis thaliana seedlings to be detected, carrying out dark treatment at 42 ℃ for 2.5h, and then carrying out light-dark alternate culture at 22 ℃ (16h light culture/8 h dark culture) for 7 days (for recovery).

(4) After completion of step (3), the phenotype of Arabidopsis thaliana was observed and the survival rate was counted.

The partial survival rate statistics are shown in FIG. 1A (Col is wild type Arabidopsis). The results showed that after high temperature (42 ℃) treatment, 2T were observed compared to wild type Arabidopsis thaliana₃The survival rate of the generation homozygous GhCCOAOMT7 transgenic Arabidopsis strains (6-11 and 19-3) is obviously improved. The survival rates of the empty vector Arabidopsis thaliana and the wild Arabidopsis thaliana have no significant difference.

2. Real-Time PCR detection of expression level of GhCCOAOMT7 gene in GhCCOAOMT7 gene-transferred Arabidopsis thaliana

(1) And (4) placing the arabidopsis seedlings to be detected in the step (3) in the step (1) into liquid nitrogen for preservation to obtain corresponding samples to be detected.

(2) Extracting total RNA of a sample to be detected by using a Trizo1 method, and then carrying out reverse transcription by using a reverse transcriptase AMV to obtain first strand cDNA. The cDNA was used as a template to detect the expression level of the GhCCOAOMT7 gene by real-time quantitative PCR.

Primers for detecting the GhCCOAOMT7 gene are 5'-ACTGGTGAAAGTTGGCGGAT-3' and 5'-ATTCGCCTGCAGATGGTCAA-3', and a target fragment is shown as 540 th to 740 th sites from 5' tail end of a sequence 1 in a sequence table.

The expression level of the GhCCOAOMT7 gene in wild type Arabidopsis seedlings was taken as 1, and the relative expression level of the GhCCOAOMT7 gene in some Arabidopsis seedlings was shown as B in FIG. 1 (Col is wild type Arabidopsis). The results showed 2T compared to wild type Arabidopsis thaliana₃The relative expression quantity of the GhCCOAOMT7 gene in the generation homozygous transgenic GhCCOAOMT7 gene Arabidopsis strains (6-11 and 19-3) is obviously improved; the relative expression quantity of the GhCCOAOMT7 gene in the empty vector transfer arabidopsis seedlings and the wild arabidopsis seedlings has no obvious difference. The higher the heat resistance of the Arabidopsis line in step 1 (as indicated by the higher the survival rate), the higher the relative expression level of the GhCCOAOMT7 gene of the corresponding seedling.

3. Heat resistance identification of GhCCOAOMT7 gene transferred arabidopsis pollen

The solute of the pollen culture medium and the concentration thereof are 20mM KCl and 200mM CaCl₂、20mM MgSO₄30mM boric acid, 5mM MES, 1% (w/v) inositol, 18% (w/v) sucrose and 1% (w/v) agarose, in water and at a pH of 5.8.

Arabidopsis seeds to be testedIs T of 6 to 11₃Seed generation, T of 19-3₃T of seed generation and empty vector Arabidopsis thaliana₃Generation seed or wild type arabidopsis seed.

(2) And (3) after the step (1) is finished, taking the arabidopsis thaliana seeds to be detected, and performing light-dark alternate culture (16h illumination culture/8 h dark culture) at the temperature of 22 ℃ for 4 weeks to obtain an arabidopsis thaliana plant to be detected.

(3) And (3) after the step (2) is finished, collecting the pollen of the arabidopsis thaliana plant to be detected, then paving the pollen on a pollen culture medium, and carrying out dark treatment for 6 hours at 32 ℃ (high temperature) or 28 ℃ (normal temperature).

(4) And (4) after the step (3) is finished, observing the phenotype of the pollen, and measuring the germination rate and the pollen tube length of the pollen.

Part of the phenotypic results are shown in FIG. 2A (Col is wild type Arabidopsis). The result of statistics of germination rates of partial pollen is shown in B in FIG. 2 (Col is wild type Arabidopsis). The statistics of the pollen tube length of part of the pollen are shown in C in FIG. 2 (Col is wild type Arabidopsis). The results show that 2T pollen treated at high temperature (32 ℃) is compared with wild Arabidopsis thaliana₃The germination rate of the pollen of the generation homozygous GhCCOAOMT7 gene transfer Arabidopsis strains (6-11 and 19-3) is obviously improved, and the length of a pollen tube is obviously increased.

4. Real-Time PCR detection of expression levels of HSFA3 gene, DREB2A gene, HSP101 gene and HSP26 gene in GhCCoAOMT7 gene-transferred Arabidopsis thaliana

The seed of Arabidopsis thaliana to be tested is T of 6-11₃Seed generation, T of 19-3₃T of seed generation and empty vector Arabidopsis thaliana₃Generation seed or wild type arabidopsis seed.

(3) And (3) after the step (2) is finished, taking the arabidopsis thaliana seedlings to be detected, carrying out dark treatment for 0h, 0.5h or 1h at 42 ℃, and then carrying out light-dark alternate culture (16h light culture/8 h dark culture) at 22 ℃ for 7 days (for recovery). And (4) placing the arabidopsis seedlings to be detected into liquid nitrogen for preservation to obtain corresponding samples to be detected.

(4) Extracting total RNA of a sample to be detected by using a Trizo1 method, and then carrying out reverse transcription by using a reverse transcriptase AMV to obtain first strand cDNA. The cDNA is used as a template, and the expression level of a gene to be detected (HSFA3 gene, DREB2A gene, HSP101 gene or HSP26 gene) is detected by real-time quantitative PCR.

The expression level of the gene to be tested in the wild type Arabidopsis seedlings is taken as 1, and the relative expression level of the gene to be tested (HSFA3 gene, DREB2A gene, HSP101 gene or HSP26 gene) in part of the Arabidopsis seedlings is shown in FIG. 3. The result shows that after the arabidopsis is subjected to high-temperature treatment for 0.5h or 1h, the relative expression quantity of the gene to be detected in the arabidopsis seedlings is remarkably improved, and the T is₃The relative expression quantity of the generation homozygous transgenic GhCCOAOMT7 gene Arabidopsis strains (6-11 and 19-3) is obviously higher than that of wild Arabidopsis, and the relative expression quantity of genes to be detected in the transgenic empty vector Arabidopsis and the wild Arabidopsis has no obvious difference. The higher the heat resistance of the Arabidopsis line in step 1 (as indicated by the higher the survival rate), the higher the relative expression levels of the HSFA3 gene, DREB2A gene, HSP101 gene and HSP26 gene of the corresponding seedling.

The results show that the overexpression of the GhCCOAOMT7 gene in wild arabidopsis thaliana can improve the heat resistance of arabidopsis thaliana; the improvement in heat resistance is shown as follows: increased survival rates, increased relative expression levels of the "HSFA 3 gene, DREB2A gene, HSP101 gene, and HSP26 gene", increased germination rates of pollen, and increased pollen tube lengths.

Example 3 acquisition and identification of Cotton gossypii TM-1 Silent strains

Infection solution: containing 10mM MES and 10mM MgCl₂And 200mM acetosyringone in water.

Firstly, construction of recombinant plasmid PTRV2-GhCCOAOMT7 and acquisition of recombinant agrobacterium

2. Taking the cDNA of the upland cotton TM-1 obtained in the step 1 as a template, and adopting a primer F: 5'-CGACGACAAGACCCTAAAGATAATGAAGGGAGTTTTGATTTT-3' and primer R: 5'-GAGGAGAAGAGCCCTTCTATGATCGACCTCCTCGATT-3' to obtain PCR amplification product.

The reaction system was 50. mu.L, consisting of 2. mu.L of template (cDNA containing 1. mu.g of Gossypium hirsutum TM-1), 1. mu.L of Phusion enzyme, 2.5. mu.L of aqueous primer F (concentration 10. mu.M), 2.5. mu.L of aqueous primer R (concentration 10. mu.M), 10. mu.L of 5 XPUSionbuffer, 10. mu.L of dNTP mix (concentrations of dATP, dTTP, dGTP and dCTP are all 10mmol), and 22. mu.L of ddH₂0.

The reaction conditions are as follows: 3min at 98 ℃; 30s at 98 ℃, 30s at 60 ℃, 40s at 72 ℃ and 35 cycles; 10min at 72 ℃; 5min at 20 ℃; storing at 4 ℃.

3. After step 2 is completed, taking the PCR amplification product and recovering a DNA fragment of about 200 bp. Placing the DNA fragment in T containing dATP₄Treating in DNA synthetase buffer solution at 22 deg.C for 30min, and standing at 70 deg.C for 20min to obtain fragment A.

4. The vector PTRV2 is digested for 8h by using a restriction enzyme Pst I, and the digestion product is recovered. Placing the enzyme digestion product in T containing dTTP₄Treating in DNA synthetase buffer solution at 22 deg.C for 30min, and standing at 70 deg.C for 20min to obtain carrier skeleton.

5. And mixing the fragment A with a vector skeleton, and connecting for 2min at 65 ℃ to obtain the recombinant plasmid PTRV2-GhCCOAOMT 7.

The recombinant plasmid PTRV2-GhCCOAOMT7 was sequenced. According to the sequencing result, the recombinant plasmid PTRV2-GhCCOAOMT7 is a DNA molecule shown in 457 th to 656 th positions from the 5' end of a sequence 1 in a sequence table inserted into a recognition sequence of a restriction enzyme Pst I of a vector PTRV 2.

The recombinant plasmid PTRV2-GhCCOAOMT7 is introduced into Agrobacterium tumefaciens GV3101 to obtain recombinant Agrobacterium, which is named as GV3101/PTRV2-GhCCOAOMT 7.

The vector PTRV1 is introduced into Agrobacterium tumefaciens GV3101 to obtain recombinant Agrobacterium tumefaciens designated as GV3101/PTRV 1.

II, obtaining of upland cotton TM-1 silent strain

1. A single colony of GV3101/PTRV 2-GhCoAOMT 7 was inoculated into 4mL of LB liquid medium containing 100mg/L rifampicin (Rif) and 50g/L kanamycin (Kan), and cultured at 28 ℃ and 200rpm for 24 hours with shaking to obtain culture solution 1.

2. After the step 1 is finished, taking the culture bacterium solution 1, and mixing the culture bacterium solution 1 according to the volume ratio of 1: 100 into LB liquid medium containing 100mg/L rifampicin (Rif) and 50g/L kanamycin (Kan), and cultured at 28 ℃ for 6 hours with shaking at 200rpm to obtain culture broth 2. 0D of culture broth 2_600nmAbout 0.5.

3. And (3) after the step 1 is finished, centrifuging the culture bacterial liquid 2 at 5000rpm for 5min to obtain a precipitate 1, and then re-suspending the precipitate 1 by using 50mL of infection solution to obtain an infection solution A.

4. Replacing GV3101/PTRV 2-GhCoAOMT 7 in the steps 1 to 3 with GV3101/PTRV1, and obtaining the staining solution B without changing other steps.

5. Mixing the infection liquid A obtained in the step 3 and the infection liquid B obtained in the step 4 (the volume ratio is 1:1) to obtain an infection working solution; the infection working solution is used for infecting the cotyledons of upland cotton TM-1 seedlings which grow for 10 days to obtain 10 quasi-silent upland cotton TM-1 plants which are named as T1-T10 in sequence.

6. After the completion of the step 5 and two weeks, total RNA of 10 gossypium hirsutum TM-1 quasi-silent strains and gossypium hirsutum TM-1 seedlings growing for 24 days are respectively extracted by a Trizo1 method, and then reverse transcription is carried out to obtain first strand cDNA by utilizing reverse transcriptase AMV. The cDNA is used as a template, and the expression level of the GhCCOAOMT7 gene is detected by real-time quantitative PCR.

The experimental result shows that the expression level of the GhCCOAOMT7 gene in T2, T3, T5 and T9 is obviously reduced compared with the expression level of the GhCCOAOMT7 gene in the upland cotton TM-1 seedlings. Thus, T2, T3, T5 and T9 are all cotton upland TM-1 silent strains. Subsequent experiments were carried out with T2, T3, T5 and T9 as research materials, collectively designated VIGS-GhCCOAOMT7 hereinafter.

The empty vector-transferred silent strain was obtained by replacing GV3101/PTRV2-GhCCOAOMT7 with GV3101/PTRV2 (recombinant Agrobacterium obtained by introducing the vector PTRV2 into Agrobacterium tumefaciens GV 3101) according to the above procedure, but not by other procedures.

Identification of Mitsugao TM-1 silent strain

1. Phenotypic identification of upland cotton TM-1 silent strains

And (3) taking a cotton plant to be tested (VIGS-GhCCOAOMT7, a trans-empty vector silent plant or upland cotton TM-1) which grows to 8 weeks, and observing the phenotype of the overground part and leaves of the cotton plant to be tested.

The result shows that the VIGS-GhCCOAOMT7 plant is dwarfed; the plant heights of the empty vector-transferred silent plant and the upland cotton TM-1 are not obviously different and are all obviously higher than those of a VIGS-GhCCCoAOMT 7 plant.

2. Real-Time PCR detection of expression level of GhCCOAOMT7 gene in upland cotton TM-1 silent strain

(1) And (3) taking leaves of the cotton plant to be detected (VIGS-GhCCOAOMT7, the empty vector-transferred silent plant or the upland cotton TM-1) which grows to 8 weeks, and putting the leaves into liquid nitrogen for storage to obtain a corresponding sample to be detected.

The expression level of the GhCCOAOMT7 gene in Gossypium hirsutum TM-1 was defined as 1, and the relative expression level of the GhCCOAMT 7 gene in VIGS-GhCCOAMT 7 is shown in FIG. 4C (CK is an empty vector silencing strain). The result shows that the expression level of the GhCCOAOMT7 gene in VIGS-GhCCOAOMT7 is remarkably reduced compared with that of the empty vector-transferred silent strain. There was no significant difference in the relative expression level of the GhCCOAOMT7 gene between the empty vector-silenced strain and the upland cotton TM-1.

3. Heat resistance identification of upland cotton TM-1 silent strain

The experiment was repeated three times to obtain an average, and the procedure for each repetition was: 20 cotton plants to be tested (VIGS-GhCCOAOMT7, empty vector silent plants or upland cotton TM-1) which grow for 8 weeks are taken and cultured alternately in light and dark at 42 ℃ (high temperature) (14h illumination culture/10 h dark culture), the plant height and stem tip of the cotton plants to be tested are observed, and the number of high-temperature treatment days for half wilting of the stem tip of the plants is recorded.

The stem tip phenotype is shown in A in FIG. 4 (Normal is the stem tip in Normal state, Wilting is the stem tip in Wilting state), and the statistical result of the high temperature treatment days for half Wilting of the tested cotton stem tips is shown in B in FIG. 4 (CK is the empty vector silent strain). The result shows that the stem tip of VIGS-GhCCAOMT 7 reaches half wilting after 6 days of high-temperature treatment; after high temperature treatment for 27 days, the stem tips of the empty carrier silent plants and the upland cotton TM-1 can be half wilted.

Compared with the empty vector-transferred silent strain, the strain height of VIGS-GhCCOAOMT7 is obviously reduced; the plant height of the empty vector-transferred silent plant and the plant height of the upland cotton TM-1 have no obvious difference.

The results show that the heat resistance can be reduced by silencing the GhCCOAOMT7 gene in the upland cotton TM-1; the reduced heat resistance is manifested by reduced plant height and shortened time for half wilting of the plant stem tip after high temperature treatment.

<110> institute of plant of Chinese academy of sciences

Application of <120> protein GhCCOAOMT7 in regulation and control of plant heat resistance

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 747

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 1

atgatttgtt tctcaagcat atatttgaag agtatggaat ttacaacagc cgaaccattt 60

ctccataagg ggttgttgca gagttttgag ttaactaagt atatcttgaa gaccaacgtc 120

taccctaggg aaccatcacc tctaaaggag ctccgagaag ttactgcaaa gcatccaggg 180

aattttatga gcacaacacc agattcaggt caactaatgg ggatgttact taagctaatt 240

aatgcaaaga agacgattga aattggtgtt tacaccggct attctcttct cctcactgct 300

ctttcaatcc ctcacgatgc tatgattata gccatagatc caaacaagga aacatatgag 360

ataggccttc ccattatcca aaaagcaggt gtggaacaca aaatcaactt cattgaatcc 420

caagctttac cagttctgga caagcttctt caaaataaag ataatgaagg gagttttgat 480

tttgcatttg tcgatgcgga taaagaaaat tacctgaatt accacgagag gcttctgaaa 540

ctggtgaaag ttggcggatt gatcgtcttt gacaacacgc tttggggagg cacggtggct 600

caacccgaag aggcggtttc agaagataga aaggaatcga ggaggtcgat catagagttt 660

aacaactcag tttcaatcga tcaacgcatc gaaatcgctc tcactccgtc gggtgatggg 720

ttgaccatct gcaggcgaat tcgttga 747

<210> 2

<211> 248

<212> PRT

<213> Artificial sequence

<220>

<223>

<400> 2

Met Ile Cys Phe Ser Ser Ile Tyr Leu Lys Ser Met Glu Phe Thr Thr

1 5 10 15

Ala Glu Pro Phe Leu His Lys Gly Leu Leu Gln Ser Phe Glu Leu Thr

20 25 30

Lys Tyr Ile Leu Lys Thr Asn Val Tyr Pro Arg Glu Pro Ser Pro Leu

35 40 45

Lys Glu Leu Arg Glu Val Thr Ala Lys His Pro Gly Asn Phe Met Ser

50 55 60

Thr Thr Pro Asp Ser Gly Gln Leu Met Gly Met Leu Leu Lys Leu Ile

65 70 75 80

Asn Ala Lys Lys Thr Ile Glu Ile Gly Val Tyr Thr Gly Tyr Ser Leu

85 90 95

Leu Leu Thr Ala Leu Ser Ile Pro His Asp Ala Met Ile Ile Ala Ile

100 105 110

Asp Pro Asn Lys Glu Thr Tyr Glu Ile Gly Leu Pro Ile Ile Gln Lys

115 120 125

Ala Gly Val Glu His Lys Ile Asn Phe Ile Glu Ser Gln Ala Leu Pro

130 135 140

Val Leu Asp Lys Leu Leu Gln Asn Lys Asp Asn Glu Gly Ser Phe Asp

145 150 155 160

Phe Ala Phe Val Asp Ala Asp Lys Glu Asn Tyr Leu Asn Tyr His Glu

165 170 175

Arg Leu Leu Lys Leu Val Lys Val Gly Gly Leu Ile Val Phe Asp Asn

180 185 190

Thr Leu Trp Gly Gly Thr Val Ala Gln Pro Glu Glu Ala Val Ser Glu

195 200 205

Asp Arg Lys Glu Ser Arg Arg Ser Ile Ile Glu Phe Asn Asn Ser Val

210 215 220

Ser Ile Asp Gln Arg Ile Glu Ile Ala Leu Thr Pro Ser Gly Asp Gly

225 230 235 240

Leu Thr Ile Cys Arg Arg Ile Arg

245

<210> 3

<211> 11726

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 3

catgccaacc acagggttcc cctcgggatc aaagtacttt gatccaaccc ctccgctgct 60

atagtgcagt cggcttctga cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca 120

agtcctaagt tacgcgacag gctgccgccc tgcccttttc ctggcgtttt cttgtcgcgt 180

gttttagtcg cataaagtag aatacttgcg actagaaccg gagacattac gccatgaaca 240

agagcgccgc cgctggcctg ctgggctatg cccgcgtcag caccgacgac caggacttga 300

ccaaccaacg ggccgaactg cacgcggccg gctgcaccaa gctgttttcc gagaagatca 360

ccggcaccag gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta cgccctggcg 420

acgttgtgac agtgaccagg ctagaccgcc tggcccgcag cacccgcgac ctactggaca 480

ttgccgagcg catccaggag gccggcgcgg gcctgcgtag cctggcagag ccgtgggccg 540

acaccaccac gccggccggc cgcatggtgt tgaccgtgtt cgccggcatt gccgagttcg 600

agcgttccct aatcatcgac cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg 660

tgaagtttgg cccccgccct accctcaccc cggcacagat cgcgcacgcc cgcgagctga 720

tcgaccagga aggccgcacc gtgaaagagg cggctgcact gcttggcgtg catcgctcga 780

ccctgtaccg cgcacttgag cgcagcgagg aagtgacgcc caccgaggcc aggcggcgcg 840

gtgccttccg tgaggacgca ttgaccgagg ccgacgccct ggcggccgcc gagaatgaac 900

gccaagagga acaagcatga aaccgcacca ggacggccag gacgaaccgt ttttcattac 960

cgaagagatc gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt 1020

ctcaaccgtg cggctgcatg aaatcctggc cggtttgtct gatgccaagc tggcggcctg 1080

gccggccagc ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt 1140

tgagtaaaac agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag taaataaaca 1200

aatacgcaag gggaacgcat gaaggttatc gctgtactta accagaaagg cgggtcaggc 1260

aagacgacca tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg 1320

ttagtcgatt ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa 1380

ccgctaaccg ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc 1440

cggcgcgact tcgtagtgat cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg 1500

atcaaggcag ccgacttcgt gctgattccg gtgcagccaa gcccttacga catatgggcc 1560

accgccgacc tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa 1620

gcggcctttg tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag 1680

gcgctggccg ggtacgagct gcccattctt gagtcccgta tcacgcagcg cgtgagctac 1740

ccaggcactg ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc 1800

cgcgaggtcc aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt taatgaggta 1860

aagagaaaat gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca 1920

gcaaggctgc aacgttggcc agcctggcag acacgccagc catgaagcgg gtcaactttc 1980

agttgccggc ggaggatcac accaagctga agatgtacgc ggtacgccaa ggcaagacca 2040

ttaccgagct gctatctgaa tacatcgcgc agctaccaga gtaaatgagc aaatgaataa 2100

atgagtagat gaattttagc ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc 2160

accgacgccg tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc 2220

tgggttgtct gccggccctg caatggcact ggaaccccca agcccgagga atcggcgtga 2280

cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg acctggtgga 2340

gaagttgaag gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag cacgccccgg 2400

tgaatcgtgg caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc 2460

cggtgcgccg tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc 2520

gatgctctat gacgtgggca cccgcgatag tcgcagcatc atggacgtgg ccgttttccg 2580

tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc tacgagcttc cagacgggca 2640

cgtagaggtt tccgcagggc cggccggcat ggccagtgtg tgggattacg acctggtact 2700

gatggcggtt tcccatctaa ccgaatccat gaaccgatac cgggaaggga agggagacaa 2760

gcccggccgc gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcgagccga 2820

tggcggaaag cagaaagacg acctggtaga aacctgcatt cggttaaaca ccacgcacgt 2880

tgccatgcag cgtacgaaga aggccaagaa cggccgcctg gtgacggtat ccgagggtga 2940

agccttgatt agccgctaca agatcgtaaa gagcgaaacc gggcggccgg agtacatcga 3000

gatcgagcta gctgattgga tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct 3060

gacggttcac cccgattact ttttgatcga tcccggcatc ggccgttttc tctaccgcct 3120

ggcacgccgc gccgcaggca aggcagaagc cagatggttg ttcaagacga tctacgaacg 3180

cagtggcagc gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc tgatcgggtc 3240

aaatgacctg ccggagtacg atttgaagga ggaggcgggg caggctggcc cgatcctagt 3300

catgcgctac cgcaacctga tcgagggcga agcatccgcc ggttcctaat gtacggagca 3360

gatgctaggg caaattgccc tagcagggga aaaaggtcga aaaggtctct ttcctgtgga 3420

tagcacgtac attgggaacc caaagccgta cattgggaac cggaacccgt acattgggaa 3480

cccaaagccg tacattggga accggtcaca catgtaagtg actgatataa aagagaaaaa 3540

aggcgatttt tccgcctaaa actctttaaa acttattaaa actcttaaaa cccgcctggc 3600

ctgtgcataa ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc ctacccttcg 3660

gtcgctgcgc tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc 3720

aaaaatggct ggcctacggc caggcaatct accagggcgc ggacaagccg cgccgtcgcc 3780

actcgaccgc cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt gatgacggtg 3840

aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg 3900

ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca 3960

tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca 4020

gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 4080

ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4140

gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4200

ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4260

ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4320

acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4380

tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4440

ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 4500

ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4560

ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4620

actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4680

gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4740

tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4800

caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4860

atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4920

acgttaaggg attttggtca tgcattctag gtactaaaac aattcatcca gtaaaatata 4980

atattttatt ttctcccaat caggcttgat ccccagtaag tcaaaaaata gctcgacata 5040

ctgttcttcc ccgatatcct ccctgatcga ccggacgcag aaggcaatgt cataccactt 5100

gtccgccctg ccgcttctcc caagatcaat aaagccactt actttgccat ctttcacaaa 5160

gatgttgctg tctcccaggt cgccgtggga aaagacaagt tcctcttcgg gcttttccgt 5220

ctttaaaaaa tcatacagct cgcgcggatc tttaaatgga gtgtcttctt cccagttttc 5280

gcaatccaca tcggccagat cgttattcag taagtaatcc aattcggcta agcggctgtc 5340

taagctattc gtatagggac aatccgatat gtcgatggag tgaaagagcc tgatgcactc 5400

cgcatacagc tcgataatct tttcagggct ttgttcatct tcatactctt ccgagcaaag 5460

gacgccatcg gcctcactca tgagcagatt gctccagcca tcatgccgtt caaagtgcag 5520

gacctttgga acaggcagct ttccttccag ccatagcatc atgtcctttt cccgttccac 5580

atcataggtg gtccctttat accggctgtc cgtcattttt aaatataggt tttcattttc 5640

tcccaccagc ttatatacct tagcaggaga cattccttcc gtatctttta cgcagcggta 5700

tttttcgatc agttttttca attccggtga tattctcatt ttagccattt attatttcct 5760

tcctcttttc tacagtattt aaagataccc caagaagcta attataacaa gacgaactcc 5820

aattcactgt tccttgcatt ctaaaacctt aaataccaga aaacagcttt ttcaaagttg 5880

ttttcaaagt tggcgtataa catagtatcg acggagccga ttttgaaacc gcggtgatca 5940

caggcagcaa cgctctgtca tcgttacaat caacatgcta ccctccgcga gatcatccgt 6000

gtttcaaacc cggcagctta gttgccgttc ttccgaatag catcggtaac atgagcaaag 6060

tctgccgcct tacaacggct ctcccgctga cgccgtcccg gactgatggg ctgcctgtat 6120

cgagtggtga ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga 6180

tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg cggacgtttt 6240

taatgtactg aattaacgcc gaattaattc gggggatctg gattttagta ctggattttg 6300

gttttaggaa ttagaaattt tattgataga agtattttac aaatacaaat acatactaag 6360

ggtttcttat atgctcaaca catgagcgaa accctatagg aaccctaatt cccttatctg 6420

ggaactactc acacattatt atggagaaac tcgagcttgt cgatcgactc tagctagagg 6480

atcgatccga accccagagt cccgctcaga agaactcgtc aagaaggcga tagaaggcga 6540

tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc 6600

cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca 6660

cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg 6720

gcaagcaggc atcgccatgt gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga 6780

gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat 6840

cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt 6900

cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg 6960

atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc acttcgccca 7020

atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc 7080

ccgtcgtggc cagccacgat agccgcgctg cctcgtcctg gagttcattc agggcaccgg 7140

acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg 7200

catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag 7260

cggccggaga acctgcgtgc aatccatctt gttcaatccc catggtcgat cgacagatct 7320

gcgaaagctc gagagagata gatttgtaga gagagactgg tgatttcagc gtgtcctctc 7380

caaatgaaat gaacttcctt atatagagga aggtcttgcg aaggatagtg ggattgtgcg 7440

tcatccctta cgtcagtgga gatatcacat caatccactt gctttgaaga cgtggttgga 7500

acgtcttctt tttccacgat gctcctcgtg ggtgggggtc catctttggg accactgtcg 7560

gcagaggcat cttgaacgat agcctttcct ttatcgcaat gatggcattt gtaggtgcca 7620

ccttcctttt ctactgtcct tttgatgaag tgacagatag ctgggcaatg gaatccgagg 7680

aggtttcccg atattaccct ttgttgaaaa gtctcaatag ccctttggtc ttctgagact 7740

gtatctttga tattcttgga gtagacgaga gtgtcgtgct ccaccatgtt atcacatcaa 7800

tccacttgct ttgaagacgt ggttggaacg tcttcttttt ccacgatgct cctcgtgggt 7860

gggggtccat ctttgggacc actgtcggca gaggcatctt gaacgatagc ctttccttta 7920

tcgcaatgat ggcatttgta ggtgccacct tccttttcta ctgtcctttt gatgaagtga 7980

cagatagctg ggcaatggaa tccgaggagg tttcccgata ttaccctttg ttgaaaagtc 8040

tcaatagccc tttggtcttc tgagactgta tctttgatat tcttggagta gacgagagtg 8100

tcgtgctcca ccatgttggc aagctgctct agccaatacg caaaccgcct ctccccgcgc 8160

gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg 8220

agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta 8280

tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca 8340

gctatgacca tgattacgaa tttggccaag tcggcctcta atacgactca ctatagggag 8400

ctcgtcgagc ggccgctcga cgaattaatt ccaatcccac aaaaatctga gcttaacagc 8460

acagttgctc ctctcagagc agaatcgggt attcaacacc ctcatatcaa ctactacgtt 8520

gtgtataacg gtccacatgc cggtatatac gatgactggg gttgtacaaa ggcggcaaca 8580

aacggcgttc ccggagttgc acacaagaaa tttgccacta ttacagaggc aagagcagca 8640

gctgacgcgt acacaacaag tcagcaaaca gacaggttga acttcatccc caaaggagaa 8700

gctcaactca agcccaagag ctttgctaag gccctaacaa gcccaccaaa gcaaaaagcc 8760

cactggctca cgctaggaac caaaaggccc agcagtgatc cagccccaaa agagatctcc 8820

tttgccccgg agattacaat ggacgatttc ctctatcttt acgatctagg aaggaagttc 8880

gaaggtgaag gtgacgacac tatgttcacc actgataatg agaaggttag cctcttcaat 8940

ttcagaaaga atgctgaccc acagatggtt agagaggcct acgcagcagg tctcatcaag 9000

acgatctacc cgagtaacaa tctccaggag atcaaatacc ttcccaagaa ggttaaagat 9060

gcagtcaaaa gattcaggac taattgcatc aagaacacag agaaagacat atttctcaag 9120

atcagaagta ctattccagt atggacgatt caaggcttgc ttcataaacc aaggcaagta 9180

atagagattg gagtctctaa aaaggtagtt cctactgaat ctaaggccat gcatggagtc 9240

taagattcaa atcgaggatc taacagaact cgccgtgaag actggcgaac agttcataca 9300

gagtctttta cgactcaatg acaagaagaa aatcttcgtc aacatggtgg agcacgacac 9360

tctggtctac tccaaaaatg tcaaagatac agtctcagaa gaccaaaggg ctattgagac 9420

ttttcaacaa aggataattt cgggaaacct cctcggattc cattgcccag ctatctgtca 9480

cttcatcgaa aggacagtag aaaaggaagg tggctcctac aaatgccatc attgcgataa 9540

aggaaaggct atcattcaag atgcctctgc cgacagtggt cccaaagatg gacccccacc 9600

cacgaggagc atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc aagtggattg 9660

atgtgacatc tccactgacg taagggatga cgcacaatcc cactatcctt cgcaagaccc 9720

ttcctctata taaggaagtt catttcattt ggagaggaca cgctcgagct caagcttcga 9780

attctgcagt cgacggtacc gcgggcccgg gatccggctg ctgccgctgc cgctgcggca 9840

gcggccggac cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg 9900

cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag 9960

ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag 10020

ctgcccgtgc cctggcccac cctcgtgacc accttcggct acggcctgaa gtgcttcgcc 10080

cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac 10140

gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg 10200

aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag 10260

gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc 10320

atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag 10380

gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc 10440

gtgctgctgc ccgacaacca ctacctgagc taccagtccg ccctgagcaa agaccccaac 10500

gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc 10560

atggacgagc tgtacaagta aagcggccgc gactctagag tcctgcttta atgagatatg 10620

cgagacgcct atgatcgcat gatatttgct ttcaattctg ttgtgcacgt tgtaaaaaac 10680

ctgagcatgt gtagctcaga tccttaccgc cggtttcggt tcattctaat gaatatatca 10740

cccgttacta tcgtattttt atgaataata ttctccgttc aatttactga ttgtacccta 10800

ctacttatat gtacaatatt aaaatgaaaa caatatattg tgctgaatag gtttatagcg 10860

acatctatga tagagcgcca caataacaaa caattgcgtt ttattattac aaatccaatt 10920

ttaaaaaaag cggcagaacc ggtcaaacct aaaagactga ttacataaat cttattcaaa 10980

tttcaaaagt gccccagggg ctagtatcta cgacacaccg agcggcgaac taataacgct 11040

cactgaaggg aactccggtt ccccgccggc gcgcatgggt gagattcctt gaagttgagt 11100

attggccgtc cgctctaccg aaagttacgg gcaccattca acccggtcca gcacggcggc 11160

cgggtaaccg acttgctgcc ccgagaatta tgcagcattt ttttggtgta tgtgggcccc 11220

aaatgaagtg caggtcaaac cttgacagtg acgacaaatc gttgggcggg tccagggcga 11280

attttgcgac aacatgtcga ggctcagcag gacctgcagg catgcaagct agcttactag 11340

tgatgcatat tctatagtgt cacctaaatc tgcggccgct gaccaagtca gctagcttgg 11400

cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc 11460

gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc 11520

gcccttccca acagttgcgc agcctgaatg gcgaatgcta gagcagcttg agcttggatc 11580

agattgtcgt ttcccgcctt cagtttaaac tatcagtgtt tgacaggata tattggcggg 11640

taaacctaag agaaaagagc gtttattaga ataacggata tttaaaaggg cgtgaaaagg 11700

tttatccgtt cgtccatttg tatgtg 11726

Claims

1. The application of the protein GhCCOAOMT7 in improving the heat resistance of plants;

the protein GhCCOAOMT7 is a protein with an amino acid sequence shown in SEQ ID NO. 2;

the plant is cotton or arabidopsis thaliana.

2. Use of a nucleic acid molecule encoding a protein GhCCoAOMT7 according to claim 1 for increasing thermotolerance in plants;

the plant is cotton or arabidopsis thaliana.

3. Use according to claim 2, characterized in that: the nucleic acid molecule encoding the protein GhCCOAOMT7 in claim 1 is a DNA molecule shown in b1) or b 2):

b2) the nucleotide sequence is a DNA molecule shown as a sequence 1 in a sequence table.

4. A method of producing a transgenic plant with improved heat tolerance comprising the steps of: introducing a nucleic acid molecule encoding a protein GhCCOAOMT7 according to claim 1 into a starting plant to obtain a transgenic plant; the heat resistance of the transgenic plant is improved compared with that of the original plant;

the plant is cotton or arabidopsis;

the introduction of a nucleic acid molecule encoding the protein GhCCOAOMT7 according to claim 1 into a starting plant is effected by introducing into the starting plant a recombinant plasmid 35S: GhCCOAOMT 7-EYFP;

the recombinant plasmid 35S is obtained by inserting a double-stranded DNA molecule shown in the 1 st to 744 th positions from the 5' end of SEQ ID NO. 1 between recognition sequences of restriction enzymes SalI and BamHI of a vector pEZR-LNY.

5. A method of producing a transgenic plant with reduced heat tolerance comprising the steps of: introducing into a starting plant a substance that inhibits the expression of a nucleic acid molecule encoding a protein GhCCOAOMT7 according to claim 1, to obtain a transgenic plant; the heat tolerance of the transgenic plant is reduced compared to the starting plant;

the plant is cotton or arabidopsis;

the introduction into a starting plant of a substance which inhibits the expression of a nucleic acid molecule encoding the protein GhCCOAOMT7 according to claim 1 is effected by introducing into a starting plant a recombinant plasmid PTRV 2-GhCCOAMT 7 and a vector PTRV 1;

the recombinant plasmid PTRV2-GhCCOAOMT7 is a recombinant plasmid obtained by inserting DNA molecules shown in the 457 th to 656 th positions from the 5' end of SEQ ID NO:1 into the recognition sequence of a restriction enzyme Pst I of a vector PTRV 2.