CN111303262A

CN111303262A - Plant heat resistance and root development related protein and coding gene and application thereof

Info

Publication number: CN111303262A
Application number: CN202010293716.0A
Authority: CN
Inventors: 朱生伟; 曹务强; 李彬; 毛玉伟; 罗小敏
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2020-04-15
Filing date: 2020-04-15
Publication date: 2020-06-19
Anticipated expiration: 2040-04-15
Also published as: CN111303262B

Abstract

The invention discloses a plant heat resistance and root development related protein, a coding gene and an application thereof, wherein the protein is protein GhBRK7, and the amino acid sequence of the protein GhBRK7 is shown as SEQ ID NO: 2, respectively. Experiments prove that the expression quantity and/or activity of the protein GhBRK7 in wild arabidopsis is improved to obtain transgenic arabidopsis; compared with wild arabidopsis thaliana, the transgenic arabidopsis thaliana has improved heat resistance and enhanced root development. Expression of protein GhBRK7 in inhibition of upland cotton TM-1Obtaining a cotton upland TM-1 silent strain by the amount and/or activity; compared with upland cotton TM-1, the heat resistance of the upland cotton TM-1 silent strain is reduced; the decrease in heat resistance is manifested by decrease in plant height, yellowing of leaves, H in leaves₂O₂Increased content of (a) and an increased number of dead cells. Therefore, the protein GhBRK7 can regulate the heat resistance and root development of plants. The invention has important application value.

Description

Plant heat resistance and root development related protein and coding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a plant heat resistance and root development related protein, and a coding gene and application thereof.

Background

With the advance of industrialization and the increase of human activities, the global warming trend is increasingly getting worse. 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. 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. In addition, pollen abortion and vitality reduction can be caused by high temperature, so that the cross pollination is difficult, the boll forming rate is low, and the production of hybrid cotton seeds is seriously influenced. 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 invention aims to improve the heat resistance of plants and promote root development.

The invention firstly protects the application of the protein GhBRK7, which can be D1) or D2):

D1) regulating and controlling heat resistance and/or root development of the plant;

D2) transgenic plants with altered heat tolerance and/or altered root development are grown.

The invention also protects the application of the nucleic acid molecule for coding the protein GhBRK7, which can be D1) or D2):

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 any of the above applications, the regulating the development of plant roots may be promoting the development of plant roots or inhibiting the development of plant roots.

In any of the above applications, the cultivation of the transgenic plant with altered heat tolerance may be cultivation of a transgenic plant with increased heat tolerance or cultivation of a transgenic plant with decreased heat tolerance.

In any of the above applications, the cultivation of the transgenic plant with altered root development may be the cultivation of a transgenic plant with enhanced root development or the cultivation of a transgenic plant with reduced root development.

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, which comprises the following steps: improving the expression quantity and/or activity of the protein GhBRK7 in the original plant to obtain a transgenic plant A; the transgenic plant A has improved heat tolerance and/or enhanced root development compared to the starting plant.

In the above method, the "improving the expression level and/or activity of the protein GhBRK7 in the starting plant" can be achieved by a method known in the art, such as transgenosis, multicopy, promoter change, regulatory factor change, etc., to improve the expression level and/or activity of the protein GhBRK7 in the starting plant.

In the above method, the "improvement in the expression level and/or activity of the protein GhBRK7 in the starting plant" may be achieved by introducing a nucleic acid molecule encoding the protein GhBRK7 into the starting plant.

In the above method, the "introducing into the starting plant a nucleic acid molecule encoding the protein GhBRK 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 GhBRK7 into an expression vector. The recombinant vector A can be specifically the recombinant plasmid 35S:: GhBRK7-GFP mentioned in the examples.

The preparation method of the recombinant plasmid 35S comprises the following steps:

(1) converting SEQ ID NO: 1 connecting the double-stranded DNA molecule shown in the 1 st to 1464 th positions from the 5' tail end with a pENTR/SD/D-TOPO carrier to obtain an intermediate carrier;

(2) the intermediate vector and the vector pMDC83 are subjected to LR recombination reaction to obtain a recombinant plasmid 35S, namely GhBRK 7-GFP.

The transgenic plant A can be at least one of GhBRK7-OE 1-8, GhBRK7-OE 5-6 and GhBRK7-OE 5-8 mentioned in the examples.

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 invention also provides a method for cultivating the transgenic plant B, which comprises the following steps: inhibiting the expression quantity and/or activity of protein GhBRK7 in the original plant to obtain transgenic plant B; the transgenic plant B has a reduced heat tolerance and/or a reduced root development compared to the starting plant.

In the above method, the "inhibiting the expression level and/or activity of the protein GhBRK7 in the starting plant" can be achieved by methods known in the art, such as gene site-directed editing, RNA interference, homologous recombination, gene knockout, and the like, to achieve the purpose of inhibiting the expression level and/or activity of the protein GhBRK 7.

In the above method, the expression level and/or activity of the protein GhBRK7 in the starting plant can be inhibited by introducing a substance that inhibits the expression of the protein GhBRK7 into the starting plant. The substance inhibiting the expression of the protein GhBRK7 may be a substance inhibiting the expression of a nucleic acid molecule encoding the protein GhBRK 7. The "substance inhibiting the expression of the nucleic acid molecule encoding the protein GhBRK 7" can be obtained by introducing a recombinant plasmid PTRV2-GhBRK7 and a vector PTRV1 into the starting plant. The recombinant plasmid PTVR2-GhBRK7 is specifically a recombinant plasmid with SEQID NO inserted between recognition sequences of restriction enzyme Pst I of a vector PTRV 2: 1 from 159 to 492 from the 5' -end.

The transgenic plant B can be at least one of the silent strains T2, T3, T5 and T6 of Gossypium hirsutum TM-1 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 A or a plant breeding method B.

The plant breeding method A can comprise the following steps: increasing the content and/or activity of the protein GhBRK7 in the plant, thereby improving heat resistance and/or enhancing root development.

The plant breeding method b may comprise the steps of: reduce the content and/or activity of protein GhBRK7 in the plant, thereby reducing heat resistance and/or reducing root development.

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.

Any of the aforementioned promoting root development or enhancing root development is specifically manifested by an increase in at least one of main root length, lateral root length, number of lateral roots, root hair density, and root hair length.

Any of the above-mentioned heat resistance reductions is specifically expressed by a reduction in plant height, H₂O₂And an increase in the number of dead cells. Said H₂O₂The content of (A) can be H in plant leaves₂O₂The content of (a). The dead cells may be dead cells in the leaves of the plant.

Any of the above heat resistances may be a resistance to temperatures of 37 ℃ to 44 ℃ (e.g., 37 ℃ to 40 ℃, 40 ℃ to 42 ℃, 42 ℃ to 44 ℃, 37 ℃, 40 ℃, 42 ℃, or 44 ℃).

Any one of the proteins GhBRK7 described above may be a1) or a2) or a3) or a 4):

a1) the amino acid sequence is SEQ ID NO: 2;

a2) in SEQ ID NO: 2, the N end or/and the C end of the protein shown in the figure is connected with a label to obtain a fusion protein;

a3) a protein which is derived from cotton and is related to plant heat resistance and/or root development and 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);

a4) protein which has 80% or more of identity with the protein shown in a1) or a2), is derived from cotton and is related to heat resistance and/or root development of plants.

Wherein, SEQ ID NO: 2 consists of 488 amino acid residues.

To facilitate purification of the protein in a1), the protein of SEQ ID NO: 2 to the amino terminus or carboxy terminus of the protein shown in 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 converting the amino acid sequence of SEQ ID NO: 1, and/or by missense mutation of one or more base pairs, and/or by attaching to its 5 'and/or 3' end a coding sequence for the tag shown in table 1 above.

The nucleic acid molecule encoding the GhBRK7 can be a DNA molecule shown in the following b1) or b2) or b3) or b 4):

b1) the coding region is SEQ ID NO: 1;

b2) the nucleotide sequence is SEQ ID NO: 1;

b3) a DNA molecule which has 80 percent or more than 80 percent of identity with the nucleotide sequence defined by b1) or b2) and codes the protein GhBRK 7;

b4) a DNA molecule which is hybridized with the nucleotide sequence limited by b1) or b2) under strict conditions and encodes the protein GhBRK 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, SEQ ID NO: 1 consists of 1467 nucleotides, SEQ ID NO: 1 encodes the nucleotide sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

The nucleotide sequence of the GhBRK7 encoding protein of the invention 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 to have 80% or more identity to the nucleotide sequence of the protein GhBRK7 isolated in accordance with the present invention, provided that they encode the protein GhBRK7, are derived from and identical to the nucleotide sequence of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes the identity to the nucleotide sequence of the present invention encoding SEQ ID NO: 2, or 85% or more, or 90% or more, or 95% or more, of the sequence of nucleotides of the protein GhBRK 7. 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.

Experiments prove that the transgenic arabidopsis is obtained by introducing the recombinant plasmid 35S, namely GhBRK7-GFP, into the wild arabidopsis; compared with wild arabidopsis, the transgenic arabidopsis has improved heat resistance and enhanced root development; the increased heat tolerance is manifested by increased survival rates and increased relative expression levels of heat shock-associated genes (e.g., HSFA2 gene, HSFA7a gene, DREB2A gene, HSP70 gene, HSP90 gene, HSP101 gene). Introducing a recombinant plasmid PTVR2-GhBRK7 and a vector PTRV1 into upland cotton TM-1 to obtain a silent upland cotton TM-1 strain; compared with upland cotton TM-1, the heat resistance of the upland cotton TM-1 silent strain is reduced; the decrease in heat resistance is manifested by decrease in plant height, yellowing of leaves, H in leaves₂O₂Increased content of (a) and an increased number of dead cells. Therefore, the protein GhBRK7 can regulate the heat resistance and root development of plants. The invention has important application value.

Drawings

FIG. 1 shows the heat resistance identification and real-time quantitative PCR detection of GhBRK7 gene of GhBRK7 transgenic Arabidopsis.

FIG. 2 shows the relative expression levels of HSFA2 gene, HSFA7a gene, DREB2A gene, HSP70 gene, HSP90 gene and HSP101 gene in GhBRK7 transgenic Arabidopsis thaliana detected by real-time quantitative PCR.

FIG. 3 is T₃And (3) statistical results of the main root length, the lateral root number, the root hair density and the root hair length of the generation homozygous transgenic GhBRK7 gene Arabidopsis line.

FIG. 4 shows phenotypic and thermotolerant identification of cotton upland TM-1 silent strains.

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.

In the following examples, primers 5'-ACCTATCTGCATGTTGTTGGAG-3' and 5'-CATCTGGAGTGTTTCAATTGAG-3' for detecting the GhBRK7 gene were used in real-time quantitative PCR.

Upland cotton TM-1 is described in the following documents: tronlinder NL et al, 1989, Plant Cell Rep8:133-136, publicly 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. 2009, No. 29, No. 1, No. 75-79, the public can be obtained from the plant research institute (i.e. the applicant) of Chinese academy of sciences, so as to repeat the application experiment.

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 geneticin between colour responses and marketing 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.

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 ratio vector for high-throughput video-induced gene cloning experiments for NbMADS4-1 and 2in flow degradation. plant physiology.2007, No. 145 1161 and 1170, publicly available from the plant institute of Chinese academy of sciences (i.e., the Applicant) to repeat the experiments of the present application.

The pENTR/SD/D-TOPO vector is a product of Invitrogen corporation.

The vector pMDC83 was a product of Invitrogen, publicly available from the plant institute of Chinese academy of sciences (i.e., the Applicant) to replicate the experiments of the present application.

The Phusion enzyme is a product of the Saimerfi company. 5 XPPhusion Buffer is a module in Phusion enzymes.

Example 1 cloning of the Gene encoding the protein GhBRK7 (i.e., the GhBRK7 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'-ATGGGGTGTGGGTGTTCAA-3' and Reverse Primer: 5'-AGAAGTTGCATTCTTTTTATTTTCGA-3', and obtaining a double-stranded DNA molecule of about 1464bp by PCR amplification.

The reaction system was 50. mu.L, consisting of 2. mu.L of template (containing 1. mu.g of cDNA from Gossypium hirsutum TM-1), 1. mu.L of Phusion enzyme, 2.5. mu.L of aqueous solution of LForward Primer (concentration 10. mu.M), 2.5. mu.L of aqueous solution of Reverse Primer (concentration 10. mu.M), 10. mu.L of 5 XPusion Buffer, 10. mu.L of dNTP mix (concentrations of dATP, dTTP, dGTP and dCTP are 10mmol), and 22. mu.L of ddH ₂0.

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 (2). The sequencing result shows that the nucleotide sequence of the double-stranded DNA molecule (namely GhBRK7 gene) obtained in the step 2 is shown as SEQ ID NO: 1 is shown at positions 1 to 1464 from the 5' end.

Example 2 obtaining and identification of GhBRK7 Gene-transferred Arabidopsis thaliana

First, the recombinant plasmid 35S, GhBRK7-GFP and GV3101/35S, the acquisition of GhBRK7-GFP

1. The double-stranded DNA molecule obtained in step 2 of example 1 was ligated with pENTR/SD/D-TOPO vector to obtain an intermediate vector.

2. After the step 1 is completed, the intermediate vector and the vector pMDC83 are subjected to LR recombination reaction to obtain a recombinant plasmid 35S, GhBRK 7-GFP.

The recombinant plasmid 35S comprises GhBRK7-GFP expression SEQ ID NO: 2, and a protein GhBRK 7.

3. The recombinant plasmid 35S, GhBRK7-GFP, is introduced into Agrobacterium tumefaciens GV3101 to obtain recombinant Agrobacterium, which is named as GhBRK7-GFP, and GhBRK 3101/35S.

Second, obtaining of transgenic Arabidopsis thaliana with GhBRK7 gene

1. GV3101/35S obtained in 3 of step one, GhBRK7-GFP, was transferred to wild type Arabidopsis thaliana to obtain T by floral dip transformation (described in Clough, S.J., and Bent, A.F. Floraldip: amplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana plant J. (1998)16, 735-743), of Arabidopsis thaliana inflorescence₁Transferring GhBRK7 gene Arabidopsis thaliana seed.

2. The T obtained in the step 1₁The transgenic Arabidopsis seeds with the GhBRK7 gene are sown on 1/2MS culture medium containing 30mg/L hygromycin, and the Arabidopsis seeds capable of growing normally (resistant seedlings) are T₁Transgenic positive seedling of GhBRK7 gene, T₁The seeds received by the positive seedlings of the transfer GhBRK7 gene are T₂Transferring GhBRK7 gene Arabidopsis thaliana seed.

3. The T of different strains screened in the step 2₂Transgenic Arabidopsis thaliana seeds with GhBRK7 gene are sown in 1/2MS culture medium containing hygromycin at 30mg/L for screeningIf the ratio of the number of Arabidopsis thaliana capable of normal growth (resistant shoot) to the number of Arabidopsis thaliana incapable of normal growth (non-resistant shoot) in a certain line is 3: 1, the strain is a strain in which a GhBRK7 gene is inserted into one copy, and seeds received by resistant seedlings in the strain are T₃Transferring GhBRK7 gene Arabidopsis thaliana seed.

4. The T screened out in the step 3₃The transgenic Arabidopsis seeds with the GhBRK7 gene are sown again on 1/2MS culture medium containing 30mg/L hygromycin for screening, and the seeds which are all resistant seedlings are T₃The transgenic Arabidopsis with GhBRK7 gene is homozygous.

3 of them are T₃The generation homozygous transgenic Arabidopsis thaliana strains with the GhBRK7 gene are named GhBRK7-OE 1-8 (1-8 for short), GhBRK7-OE 5-6 (5-6 for short) and GhBRK7-OE 5-8 (5-8 for short), and subsequent experiments are carried out.

Third, real-time quantitative PCR detection T₃Relative expression level of GhBRK7 gene in transgenic Arabidopsis thaliana with homozygous GhBRK7 gene (namely transgenic Arabidopsis thaliana with GhBRK7 gene)

The seeds of Arabidopsis thaliana to be tested are 1-8T₃Seed generation, T of 5-6₃Seed generation, T of 5-8₃Generation 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, putting the arabidopsis seedlings to be detected into liquid nitrogen for preservation, and obtaining corresponding samples to be detected.

4. And 3, after the step 3 is finished, extracting the total RNA of the sample to be detected by adopting a Trizo1 method, and then reversely transcribing the first strand cDNA by utilizing a reverse transcriptase AMV to obtain the cDNA of the sample to be detected.

5. And (4) after the step 4 is completed, using cDNA of a sample to be detected as a template, and detecting the relative expression level of the GhBRK7 gene by real-time quantitative PCR.

The GhBRK7 radical in wild type arabidopsis seedlingsThe expression level of the gene was regarded as 1, and 3T were obtained₃Relative expression level of GhBRK7 gene in generation-homozygous transgenic Arabidopsis lines (1-8, 5-6 and 5-8) with GhBRK7 gene. The results show that T is comparable to wild type Arabidopsis thaliana₃The relative expression level of GhBRK7 genes in generation-homozygous transgenic Arabidopsis strains (1-8, 5-6 and 5-8) is obviously improved.

Fourth, heat resistance identification of GhBRK7 transgenic arabidopsis

The experiment was repeated three times to obtain an average, and the procedure for each repetition was as follows:

3. And (3) after the step (2) is finished, taking 30 arabidopsis 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 Arabidopsis phenotype was observed and the survival rate was counted.

The statistical results of partial survival rates are shown in the left panel of FIG. 1 (Col is wild type Arabidopsis). The results showed that 3T's after high temperature (42 ℃) treatment were comparable to wild type Arabidopsis thaliana₃The survival rate of the generation homozygous GhBRK7 transgenic Arabidopsis strains (1-8, 5-6 and 5-8) is obviously improved.

5. And 3, placing the to-be-detected arabidopsis seedlings before being processed at 42 ℃ and the to-be-detected arabidopsis seedlings processed at 42 ℃ for 30min into liquid nitrogen for preservation to obtain corresponding to-be-detected samples.

6. After the step 5 is completed, extracting the total RNA of the sample to be detected by adopting a Trizo1 method, and then reversely transcribing the first strand cDNA by utilizing a reverse transcriptase AMV to obtain the cDNA of the sample to be detected.

7. And (5) after the step 6 is finished, using cDNA of a sample to be detected as a template, and detecting the relative expression level of the GhBRK7 gene by real-time quantitative PCR.

The expression level of the GhBRK7 gene in the wild type Arabidopsis seedlings is taken as 1 to obtain 3T₃Relative expression level of GhBRK7 gene in generation-homozygous transgenic Arabidopsis lines (1-8, 5-6 and 5-8) with GhBRK7 gene. Part of the results are shown in the right panel of FIG. 1 (Col is wild type Arabidopsis). The results show that T is comparable to wild type Arabidopsis thaliana₃The relative expression level of GhBRK7 genes in generation-homozygous transgenic Arabidopsis strains (1-8, 5-6 and 5-8) is obviously improved.

8. And (3) detecting the relative expression level of heat shock related genes (HSFA2 gene, HSFA7a gene, DREB2A gene, HSP70 gene, HSP90 gene or HSP101 gene) by using cDNA of a sample to be detected as a template through real-time quantitative PCR. The HSFA2 gene, the HSFA7a gene and the DREB2A gene encode different heat shock transcription factors, and the HSP70 gene, the HSP90 gene and the HSP101 gene encode different heat shock proteins.

Primers for detecting the HSFA2 gene were 5'-GGAAGCAGCGTTGGATGTGA-3' and 5'-TAGATCTTGGCTGTCCCAATCCA-3'.

Primers for detecting the HSFA7a gene were 5'-TGCATTCTTTCTCCACGATTCTCC-3' and 5'-CAAATTCCCATCTCTCTGCTTCT-3'.

Primers for detecting the DREB2A gene are 5'-GCGTCTGAGGTTACGAGTAC-3' and 5'-CATGAACACAACCAGGAGTCTCA-3'.

Primers for detecting the HSP70 gene are 5'-CCTACCAACACCGTCTTCGA-3' and 5'-CTTCTCACCTGGACCGGAAA-3'.

Primers for detecting the HSP90 gene are 5'-GGTATTGGCATGACCAAAGCAGAT-3' and 5'-ATGCTTACATCAGCTCCAGCTTGA-3'.

Primers for detecting the HSP101 gene are 5'-CAGAGAGTTATGACCCGGTGTA-3' and 5'-ATCGATTTCCTCACGCACAACCA-3'.

The expression level of the GhBRK7 gene in the wild type Arabidopsis seedlings is taken as 1 to obtain 3T₃Relative expression level of heat shock related genes in transgenic Arabidopsis lines (1-8, 5-6 and 5-8) with generation-homozygous GhBRK7 gene. Part of the results are shown in FIG. 2(Col is wild type Arabidopsis). The results show that the south of PaeoniaAfter 2.5h of high temperature treatment of Arabidopsis thaliana, T is compared with that of wild Arabidopsis thaliana₃The relative expression level of heat shock related genes in the generation homozygous GhBRK7 transgenic arabidopsis strain is obviously improved.

The results show that the overexpression of the GhBRK7 gene in wild arabidopsis thaliana can improve the heat resistance of arabidopsis thaliana; increased thermotolerance is manifested by increased survival and increased relative expression levels of heat shock-associated genes.

Fifth, the root development identification of GhBRK7 transgenic Arabidopsis

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 3 days to obtain the arabidopsis thaliana seedlings to be detected.

3. After the step 2 is completed, the arabidopsis seedlings to be tested with basically consistent growth are transferred to 1/2MS solid culture medium (5 seedlings are transferred to each culture dish), vertical culture is carried out for 4 days, and the length of the main root, the number of lateral roots, the length of the lateral roots, the density of root hairs and the length of the root hairs are counted.

The method for counting the main root length and the lateral root length comprises the following steps: marking the position of the root tip of the arabidopsis seedling to be detected by using a marker pen before vertical culture; after vertical culture, marking the position of the root tip of the arabidopsis seedling to be detected by using a marker pen again; the distance between the two positions is measured, i.e. the length of the respective root is obtained.

The method for counting the root hair density and the root hair length comprises the following steps: after step 3, the seedlings are placed on a stereoscope stage, the number of root hairs on the roots at the second and third millimeters from the root tip is counted (the root hair density is obtained), a picture is taken, and then the root hair length is measured by using Image J software.

The statistical results are shown in FIG. 3(Col is wild)A generative type arabidopsis). The results showed 3T's as compared to wild type Arabidopsis thaliana₃The main root length, the lateral root number, the root hair density and the root hair length of the generation homozygous GhBRK7 transgenic Arabidopsis strains (1-8, 5-6 and 5-8) are obviously increased.

The results show that the overexpression of the GhBRK7 gene in wild Arabidopsis can promote root development, and the promotion of root development is represented by the increase of main root length, lateral root number, root hair density and root hair length.

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

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

Construction of recombinant plasmid PTVR2-GhBRK7 and acquisition of recombinant agrobacterium tumefaciens

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

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 aqueous solution of primer F (concentration 10. mu.M), 2.5. mu.L of aqueous solution of primer R (concentration 10. mu.M), 10. mu.L of 5 XPusion Buffer, 10. mu.L of dNTP mix (concentrations of dATP, dTTP, dGTP and dCTP are all 10mmol), and 22. mu.L of ddH ₂0.

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 completing step 2, taking the PCR amplification product and recovering a DNA fragment of about 362 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. Restriction of the vector PTRV2And (3) carrying out enzyme digestion for 8h by using the endonuclease Pst I, and recovering an enzyme digestion product. 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 PTVR2-GhBRK 7.

The recombinant plasmid PTVR2-GhBRK7 was sequenced. According to the sequencing result, the recombinant plasmid PTVR2-GhBRK7 is a plasmid which is obtained by inserting SEQ ID NO: 1 from 159 to 492 from the 5' -end.

The recombinant plasmid PTVR2-GhBRK7 is introduced into Agrobacterium tumefaciens GV3101 to obtain recombinant Agrobacterium tumefaciens which is named as GV3101/PTVR2-GhBRK 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/PTVR2-GhBRK7 was inoculated into 4mL of YEP liquid medium containing 100mg/L rifampicin (Rif) and 50g/L kanamycin (Kan), and shake-cultured at 28 ℃ and 200rpm for 24 hours 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 YEP liquid medium containing 100mg/L rifampicin (Rif) and 50g/L kanamycin (Kan), and culturing at 28 deg.C and 200rpm for 6 hr with shaking to obtain 0D_600nmAbout 0.5 of the culture broth 2.

3. After the step 2 is finished, taking the culture bacterial liquid 2, centrifuging at 5000rpm for 5min, and collecting precipitates; then, 50mL of infection solution is used for resuspending the precipitate, and an infection solution A is obtained.

4. Replacing GV3101/PTVR2-GhBRK7 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. And respectively using cDNA as templates to detect the expression level of the GhBRK7 gene by real-time quantitative PCR.

The experimental result shows that the expression level of the GhBRK7 gene in T2, T3, T5 and T6 is obviously reduced compared with the cotton upland TM-1 seedlings. Thus, T2, T3, T5 and T6 are all cotton upland TM-1 silent strains. Subsequent experiments were carried out with T2, T3, T5 and T6 as research materials, collectively designated VIGS-GhBRK7 hereinafter.

The empty vector-transferred silent strain was obtained by replacing GV3101/PTVR2-GhBRK7 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. There was no significant difference in the relative expression level of GhBRK7 gene between the empty vector-silenced strain and upland cotton TM-1.

Third, real-time quantitative PCR detection of the relative expression level of GhBRK7 gene and homologous gene thereof in upland cotton TM-1 silent strain

1. And (3) taking leaves of the cotton plant to be detected (VIGS-GhBRK7, 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.

2. After the step 1 is completed, extracting the total RNA of the sample to be detected by adopting a Trizo1 method, and then reversely transcribing the first strand cDNA by utilizing a reverse transcriptase AMV to obtain the cDNA of the sample to be detected.

3. And (3) after the step 2 is completed, using cDNA of a sample to be detected as a template, and carrying out real-time quantitative PCR detection on the relative expression level of the target gene (GhBRK7 gene, GhBRK2 gene, GhBRK3 gene, GhBRK12 gene or GhBRK15 gene).

Primers for detecting the GhBRK2 gene are 5'-AACCTCAACGACTCCTCCGA-3' and 5'-GAGCTTTTTCTCCATGCTCCG-3'.

Primers for detecting the GhBRK3 gene are 5'-GACGAAACAATCCAGCTTGATG-3' and 5'-GCTAGTGATGACAGCACATTGACC-3'.

Primers for detecting the GhBRK12 gene are 5'-CGGATAACGAAAGTGTTAAGATCAC-3' and 5'-TTCTTCACTGAAATTGTTGGTCACA-3'.

Primers for detecting the GhBRK15 gene are 5'-TTACGCTGTGTGGACCCAA-3' and 5'-CTTGTGGGACCGCATGTTT-3'.

The expression level of the target gene in the upland cotton TM-1 is taken as 1, and the relative expression levels of the target gene in the VIGS-GhBRK7 and the transgenic empty vector silent strain are obtained. Part of the results are shown in A of FIG. 4 (CK is cotton. RTM. -1). The results show that compared with the Gossypium hirsutum TM-1, the expression level of the GhBRK7 gene in VIGS-GhBRK7 is remarkably reduced, and the expression levels of 4 homologous genes (GhBRK 2 gene, GhBRK3 gene, GhBRK12 gene and GhBRK15 gene) of the GhBRK7 gene in VIGS-GhBRK7 are reduced and increased. The expression level of the target gene in the empty vector-transferred silent strain and the upland cotton TM-1 has no obvious difference.

Phenotypic identification and thermotolerance identification of upland cotton TM-1 silent strains

1. 20 cotton plants to be tested (VIGS-GhBRK7, empty vector transfer silent plants or upland cotton TM-1) which grow to 8 weeks are taken and cultured alternately in light and dark at 42 ℃ (14h illumination culture/10 h dark culture) for 16 days.

2. After completion of step 1, phenotypes were observed and survival was counted.

The phenotype of some of the cotton plants tested is shown in B of FIG. 4 (CK is cotton. hirsutum TM-1). The survival rate statistics of some cotton plants to be tested are shown in C in FIG. 4 (CK is upland cotton TM-1).

The results show that after the treatment for 16 days at high temperature (42 ℃), the plant height of VIGS-GhBRK7 is obviously reduced, the leaves are yellowed and the survival rate is obviously reduced compared with the upland cotton TM-1. There is no significant difference between the phenotype (such as plant height) and survival rate of upland cotton TM-1 and the empty vector-transferred silent strain.

3. During the step 1, detecting H in the leaves of the cotton plant to be detected before 42 ℃ treatment and 24H after 42 ℃ treatment by adopting a DAB dyeing method₂O₂The content of (a).

The test results of some cotton plants to be tested are shown in D in FIG. 4 (CK is upland cotton TM-1). The results showed that the leaf blades of VIGS-GhBRK7 had H content comparable to that of Gossypium hirsutum TM-1 when treated at 42 ℃ for 24H₂O₂The content of (A) is remarkably increased; upland cotton TM-1 and in leaves of transgenic empty vector silent plants H₂O₂There was no significant difference in the content of (c).

4. During the step 1, the death condition of the cells in the leaves of the cotton plants to be detected before the treatment at 42 ℃ and 48 hours after the treatment at 42 ℃ is detected by adopting a trypan blue staining method.

The test results of some cotton plants to be tested are shown in E in FIG. 4 (CK is upland cotton TM-1). The results show that the number of dead cells in the leaf blade of VIGS-GhBRK7 is remarkably increased compared with the upland cotton TM-1 when the leaf blade is treated at 42 ℃ for 48 hours; there was no significant difference in the number of dead cells in the leaves of upland cotton TM-1 and the empty vector-silenced strains.

5. After the step 1 is completed, the cotton plant to be tested is taken and is subjected to light-dark alternate culture (14h light culture/10 h dark culture) at the temperature of 25 ℃ for 20 days (for recovery).

6. After completion of step 5, phenotypes were observed and survival was counted.

The survival rate statistics of some cotton plants to be tested are shown in F in FIG. 4 (CK is upland cotton TM-1).

The result shows that after 20 days of recovery culture at 25 ℃, the plant height of VIGS-GhBRK7 is obviously reduced and the survival rate is obviously reduced compared with the terrestrial cotton TM-1. There is no significant difference between the phenotype (such as plant height) and survival rate of upland cotton TM-1 and the empty vector-transferred silent strain.

<110> institute of plant of Chinese academy of sciences

<120> plant heat resistance and root development related protein, coding gene and application thereof

<160>2

<170>PatentIn version 3.5

<210>1

<211>1467

<212>DNA

<213> Gossypium hirsutum of upland cotton

<400>1

atggggtgtg ggtgttcaaa cctatctgca tgttgttgga gtttggatca gaatggatca 60

attcctgaag ctgataatgt tgaaaatgaa gacaagggtg aagttgatga tttgcctgca 120

ttccgtgaat actcaattga aacactccag atggctacat ctggatttgc catggagaat 180

atagtttcag aacatggtga gaaggctcct aatgtagttt acaagggcaa gttggaaaat 240

caaaggcgta ttgctgttaa acgttttaat aggtctgcat ggccagatgc ccgacaattc 300

ttggaagaag caaaagctgt tggtcagctc cgaaatcaca ggttggcaaa cttacttggt 360

tgttgctgtg aaggtgatga gaggttactt gtcgcagaat ttatgactaa tgatacactg 420

gcaaaacact tattccattg ggaagcacaa ccaatgaaat gggcaatgcg attaagggtt 480

gctttgcatc ttgcagaagc tctagagtac tgcaccagca aaggacgtgc cctatatcat 540

gacctaaatg catatagaat tgttttcgat gatgagggca accctagact ttcatgcttt 600

ggactaatga agaacagtag agatggaaaa agttacagca caaacttggc atttactcct 660

ccagagtatt tgaggacagg aagagtaaca ccagaaagtg taatttatag ttttggcacc 720

cttctacttg atcttctcag tggaaaacat attcctccaa gtcacgctct ggaccttata 780

cgtgacagga acattcagat gctgacagat tcttgtttgg aagggcaatt ttcaaatgat 840

gatggcactg agttagtgcg tctagcatca cgatgtttac aatatgagcc ccgtgagcgt 900

ccaaatccaa agtcattggt tactgcatta attcctcttc agagggacac tgaggttcct 960

tctcatgaat taatgggcat acttcatgga actgatgctg ttcctttgtc accacttggg 1020

gaagcctgct taagaatgga tttgactgcc attcatgatg tcctagaaaa gcttggatat 1080

aaagatgatg agggtgctgc tactgagctt tccttccaga tgtggacaaa ccagatgcag 1140

gaaacactga catcaaagaa aaagggtgat gttgctttta ggcataagga ttttagagct 1200

gctcttgaat gctataccca gttcattgat gttggaaccg tggtttcccc aacggtttat 1260

gctcggcgta gtttgtctta cctcatgagc aacatgcctc aagaagccct caatgatgca 1320

gtgcaagcac aagtaatatc tcctatttgg catattgcat cctacttgca agcgtctgct 1380

ctacttgctc taggaaagga ggatgaggca caagctgctc ttagggaggc tactgagctc 1440

gaaaataaaa agaatgcaac ttcttga 1467

<210>2

<211>488

<212>PRT

<213> Gossypium hirsutum of upland cotton

<400>2

Met Gly Cys Gly Cys Ser Asn Leu Ser Ala Cys Cys Trp Ser Leu Asp

1 5 10 15

Gln Asn Gly Ser Ile Pro Glu Ala Asp Asn Val Glu Asn Glu Asp Lys

20 25 30

Gly Glu Val Asp Asp Leu Pro Ala Phe Arg Glu Tyr Ser Ile Glu Thr

35 40 45

Leu Gln Met Ala Thr Ser Gly Phe Ala Met Glu Asn Ile Val Ser Glu

50 55 60

His Gly Glu Lys Ala Pro Asn Val Val Tyr Lys Gly Lys Leu Glu Asn

65 70 75 80

Gln Arg Arg Ile Ala Val Lys Arg Phe Asn Arg Ser Ala Trp Pro Asp

85 90 95

Ala Arg Gln Phe Leu Glu Glu Ala Lys Ala Val Gly Gln Leu Arg Asn

100 105 110

His Arg Leu Ala Asn Leu Leu Gly Cys Cys Cys Glu Gly Asp Glu Arg

115 120 125

Leu Leu Val Ala Glu Phe Met Thr Asn Asp Thr Leu Ala Lys His Leu

130 135 140

Phe His Trp Glu Ala Gln Pro Met Lys Trp Ala Met Arg Leu Arg Val

145 150 155 160

Ala Leu His Leu Ala Glu Ala Leu Glu Tyr Cys Thr Ser Lys Gly Arg

165 170 175

Ala Leu Tyr His Asp Leu Asn Ala Tyr Arg Ile Val Phe Asp Asp Glu

180 185 190

Gly Asn Pro Arg Leu Ser Cys Phe Gly Leu Met Lys Asn Ser Arg Asp

195 200 205

Gly Lys Ser Tyr Ser Thr Asn Leu Ala Phe Thr Pro Pro Glu Tyr Leu

210 215 220

Arg Thr Gly Arg Val Thr Pro Glu Ser Val Ile Tyr Ser Phe Gly Thr

225 230 235 240

Leu Leu Leu Asp Leu Leu Ser Gly Lys His Ile Pro Pro Ser His Ala

245 250 255

Leu Asp Leu Ile Arg Asp Arg Asn Ile Gln Met Leu Thr Asp Ser Cys

260 265 270

Leu Glu Gly Gln Phe Ser Asn Asp Asp Gly Thr Glu Leu Val Arg Leu

275 280 285

Ala Ser Arg Cys Leu Gln Tyr Glu Pro Arg Glu Arg Pro Asn Pro Lys

290 295 300

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

305 310 315 320

Ser His Glu Leu Met Gly Ile Leu His Gly Thr Asp Ala Val Pro Leu

325 330 335

Ser Pro Leu Gly Glu Ala Cys Leu Arg Met Asp Leu Thr Ala Ile His

340 345 350

Asp Val Leu Glu Lys Leu Gly Tyr Lys Asp Asp Glu Gly Ala Ala Thr

355 360 365

Glu Leu Ser Phe Gln Met Trp Thr Asn Gln Met Gln Glu Thr Leu Thr

370 375 380

Ser Lys Lys Lys Gly Asp Val Ala Phe Arg His Lys Asp Phe Arg Ala

385 390 395 400

Ala Leu Glu Cys Tyr Thr Gln Phe Ile Asp Val Gly Thr Val Val Ser

405 410 415

Pro Thr Val Tyr Ala Arg Arg Ser Leu Ser Tyr Leu Met Ser Asn Met

420 425 430

Pro Gln Glu Ala Leu Asn Asp Ala Val Gln Ala Gln Val Ile Ser Pro

435 440 445

Ile Trp His Ile Ala Ser Tyr Leu Gln Ala Ser Ala Leu Leu Ala Leu

450 455 460

Gly Lys Glu Asp Glu Ala Gln Ala Ala Leu Arg Glu Ala Thr Glu Leu

465 470 475 480

Glu Asn Lys Lys Asn Ala Thr Ser

485

Claims

1. The application of the protein GhBRK7 is D1) or D2):

D2) cultivating a transgenic plant with altered heat tolerance and/or altered root development;

the protein GhBRK7 is a1) or a2) or a3) or a 4):

a1) the amino acid sequence is SEQ ID NO: 2;

2. The application of the nucleic acid molecule for coding the protein GhBRK7 in the claim 1, which is D1) or D2):

3. Use according to claim 1 or 2, characterized in that:

the regulation and control of the plant heat resistance is to improve the plant heat resistance or reduce the plant heat resistance;

regulating the development of the plant root into promoting the development of the plant root or inhibiting the development of the plant root;

the cultivation of the transgenic plant with changed heat resistance is to cultivate a transgenic plant with improved heat resistance or cultivate a transgenic plant with reduced heat resistance;

the transgenic plant with the changed root development is cultured to be a transgenic plant with enhanced root development or a transgenic plant with reduced root development.

4. A method for breeding transgenic plant A comprises the following steps: increasing the expression level and/or activity of the protein GhBRK7 in claim 1 in a starting plant to obtain a transgenic plant A; the transgenic plant A has improved heat tolerance and/or enhanced root development compared to the starting plant.

5. The method of claim 4, wherein: the expression level and/or activity of the protein GhBRK7 in the starting plant is/are improved by introducing a nucleic acid molecule encoding the protein GhBRK7 into the starting plant.

6. A method for breeding a transgenic plant B comprises the following steps: inhibiting the expression level and/or activity of the protein GhBRK7 in claim 1 in a starting plant to obtain a transgenic plant B; the transgenic plant B has a reduced heat tolerance and/or a reduced root development compared to the starting plant.

7. The method of claim 6, wherein: the expression level and/or activity of the protein GhBRK7 in the starting plant is inhibited by introducing a substance that inhibits the expression of the protein GhBRK7 into the starting plant.

8. A plant breeding method A or a plant breeding method B;

the plant breeding method A comprises the following steps: increasing the content and/or activity of the protein GhBRK7 in claim 1 in a plant, thereby increasing heat tolerance and/or enhancing root development;

the plant breeding method B comprises the following steps: reducing the content and/or activity of the protein GhBRK7 in claim 1 in a plant, thereby reducing heat tolerance and/or reducing root development.

9. The use according to claim 3 or the method according to any of claims 4 to 8, wherein:

the promoting plant root development or enhancing root development is manifested by an increase in at least one of main root length, lateral root number, root hair density, and root hair length;

the reduced heat resistance is expressed by reduced plant height, H₂O₂And an increase in the number of dead cells.

10. The use of claim 1, 2, 3 or 9, or the method of any one of claims 4 to 9, wherein: the plant is any one 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.