CN113512550A

CN113512550A - Tea tree CsHAC1 gene and protein and application thereof

Info

Publication number: CN113512550A
Application number: CN202110654925.8A
Authority: CN
Inventors: 罗鸣; 吴华玲; 姚瑶; 姜晓辉
Original assignee: South China Botanical Garden of CAS; Tea Research Institute Guangdong Academy of Agricultural Sciences
Current assignee: South China Botanical Garden of CAS; Tea Research Institute Guangdong Academy of Agricultural Sciences
Priority date: 2021-06-11
Filing date: 2021-06-11
Publication date: 2021-10-19
Anticipated expiration: 2041-06-11
Also published as: CN113512550B

Abstract

The invention discloses a CsHAC1 gene of tea treeAnd the application of protein in changing the stress resistance of plants, wherein the nucleotide sequence of the tea tree CsHAC1 gene is shown as SEQ ID No. 1; the amino acid sequence of the encoded protein is shown as SEQ ID No. 2. The invention constructs the saccharomyces cerevisiae recombinant expression vector inserted with the CsHAC1 gene of the tea tree, transforms the recombinant expression vector into the saccharomyces cerevisiae, induces the over-expression of the gene in the saccharomyces cerevisiae through galactose, and can improve the H pair of the saccharomyces cerevisiae₂O₂And NaCl stress tolerance, and proves that the gene can improve the tolerance of the saccharomyces cerevisiae to hydrogen peroxide and/or sodium chloride stress. The tea tree CsHAC1 gene and the protein coded by the gene provide theoretical research foundation for subsequently utilizing the genetic engineering technology to cultivate stress-resistant tea trees, and have great application value for cultivating stress-resistant excellent tea tree varieties.

Description

Tea tree CsHAC1 gene and protein and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering and molecular biology, and particularly relates to a tea tree CsHAC1 gene and protein and application thereof.

Background

Tea trees originate from China, have a long planting history in China and are one of important economic crops in China. The tea made of tea leaves is popular due to its unique flavor, which not only has great influence on the health, medicine, culture, trade and the like of Chinese people, but also plays an important role in the daily life of many people in the world, and tea trees become important cultivated crops in many areas.

Tea plants are often subjected to various adverse environmental effects during the growth cycle, resulting in a reduction in the yield and quality of tea leaves and, in severe cases, even death of the tea plants. The stress resistance research of tea trees is urgent. Therefore, the potential resistance genes of the tea trees are screened by a biotechnology means, and the method has important significance for improving the tolerance capability of the tea trees under adversity stress and breeding novel stress-resistant tea tree varieties.

Disclosure of Invention

Based on the above, one of the purposes of the present invention is to provide an application of the tea tree CsHAC1 gene and the protein coded by the gene in improving the stress resistance of plants, wherein the gene and the protein coded by the gene have the function of improving the stress resistance of plants.

The specific technical scheme for realizing the aim of the invention is as follows:

an application of a tea tree CsHAC1 gene in improving plant stress resistance is disclosed, wherein the nucleotide sequence of the tea tree CsHAC1 gene is shown as SEQ ID No. 1; or the nucleotide sequence shown as SEQ ID No.1 is substituted, deleted and/or added with one or more nucleotides and can code the same functional protein; or a nucleotide sequence with the coding amino acid sequence shown as SEQ ID No. 2.

In some of these embodiments, the stress resistance is resistance to hydrogen peroxide and/or sodium chloride stress.

In some of these embodiments, the plant is tea, soybean, wheat, rice, or arabidopsis.

An application of protein coded by tea tree CsHAC1 gene in improving plant stress resistance, wherein the amino acid sequence of the protein coded by the tea tree CsHAC1 gene is shown as SEQ ID No. 2; or the amino acid sequence shown as SEQ ID No.2 is substituted, deleted and/or added with one or more amino acids, but the protein activity is the same.

The invention also aims to provide the application of the recombinant expression vector pCAMBIA1300(m) -CsHAC1 in improving the stress resistance of plants.

the recombinant expression vector pCAMBIA1300(m) -CsHAC1 is inserted with tea tree CsHAC1 gene or gene for expressing the protein coded by the tea tree CsHAC1 gene.

The invention also aims to provide application of the saccharomyces cerevisiae recombinant expression vector in improving the stress resistance of yeast.

the application of a saccharomyces cerevisiae recombinant expression vector in improving the stress resistance of saccharomyces cerevisiae is characterized in that the tea tree CsHAC1 gene is inserted into the saccharomyces cerevisiae recombinant expression vector, or a gene which can be used for expressing the protein coded by the tea tree CsHAC1 gene is inserted into the saccharomyces cerevisiae recombinant expression vector.

In some embodiments, the Saccharomyces cerevisiae recombinant expression vector is pYES2-CsHAC 1.

The invention also provides a method for improving the stress resistance of the plants.

A method for improving the stress resistance of plants comprises the step of improving the expression of the CsHAC1 gene of the tea trees in the plants.

Compared with the prior art, the invention has the following beneficial effects:

the invention clones the CsHAC1 gene of tea tree from tea tree, constructs the saccharomyces cerevisiae recombinant expression vector inserted with the CsHAC1 gene of tea tree, transforms the recombinant expression vector into saccharomyces cerevisiae, induces the overexpression of the gene in the saccharomyces cerevisiae through galactose, and can improve the H expression of the saccharomyces cerevisiae₂O₂And NaCl stress tolerance, and proves that the gene can improve the tolerance of the saccharomyces cerevisiae to the stress of hydrogen peroxide and/or sodium chlorideTherefore, the tea tree CsHAC1 gene and the protein coded by the gene provide theoretical research basis for subsequently utilizing the genetic engineering technology to cultivate stress-resistant tea trees, and have great application value for cultivating stress-resistant excellent tea tree varieties.

Drawings

FIG. 1 shows a fragment of the CsHAC1 gene of Camellia sinensis amplified by PCR in example 1 of the present invention; wherein: m: DNA molecular weight standard; CsHAC 1: an amplified CsHAC1 fragment;

FIG. 2 shows a recombinant expression vector pYES2-CsHAC1 of Saccharomyces cerevisiae constructed in example 1 of the present invention;

FIG. 3 shows that the transgenic yeast transformed with the CsHAC1 overexpression gene of the invention in example 2 can improve the tolerance of yeast to oxidative stress;

FIG. 4 shows that the transgenic yeast transformed to overexpress CsHAC1 gene in example 3 of the present invention can improve the tolerance of yeast to salt stress.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

To facilitate an understanding of the present technology, certain terms and phrases are defined below.

Throughout the specification and claims, the following terms have the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase "in one embodiment" as used in the present disclosure does not necessarily refer to the same embodiment, although it may. Moreover, the phrase "in another embodiment" as used in this disclosure does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined without departing from the scope or spirit of the invention.

Furthermore, as used herein, the term "or" is an inclusive "or" symbol and is equivalent to the term "and/or," unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. Furthermore, throughout the specification the meaning of "a", "an" and "the" include plural referents. The meaning of "in.

The nucleotide sequence of the CsHAC1 gene is shown in SEQ ID No.1, and the amino acid sequence of the coded protein is shown in SEQ ID No. 2.

SEQ ID NO.1

ATGAATGTGCAGGTACATATGTCAGGACAAATCTCAGGACAGGTACCTAATCAAGCAGGCACTCAGTTGCCTGGATTACCTCCCCAGAATGGAAGTTCTCTCTCTAACCATATACAAAATTTAGCTGGTCAGTGTAATACACTGAATATGGAGCCTGAGCTAGCGAAAGCTCGCAGATTTATGCAAGAGAAAATCTATGATTTTCTTATGCAGCGGCAGCAGCAAACTAATGATATACCACCAACGCGGGTTCTGGAAATTGTAAGACGCCTAGACGAGGGTCTATTCAGAAATGCTGCCACTAAGGAGGAGTATGTGAACCTGGAGACTTTGGAGAATCGTTTACGTGTTGTGATTGAGAGTTTACCAATGAGTACTCACAACCAAAAATATCCGCACCTTGTTAATGCTCCCTCTCCTATTGAGCCTGAGCTAGCGAAAGCTCGCAGATTTATGCAAGAGAAAATCTATGATTTTCTTATGCAGCGGCAGCAGCGAACTAATGATATACCACCAAAGCGGGTTCTGGAAATTGTAAGACGCCTAGACGAGGGTCTATTCAGAAATGCTGCCACTAAGGAGGAGTATTTGAACCTGGAGACTTTGGAGAATCGTTTACATGTTTTGATTAAGCGTTTACCAATGAGTACTCACAACCAACAATATCCGCGCCTTGTTAATGCTCCTTCTCCTATTGGTACAATGATACCAACCCCAGATAGTGCGAATTCAAGCGTGATGGTTGCATCGGCTGTAGATACTTCCATGATTGCTACCGGTGGTGGTAATAGCATGCCATCTGTGGCTGTCAATACTGGAAGCTTACTACCTCCTGCTAATGGGTCGTCTGTTGGGATACATGGTGGTTCTTTCAATTCTCAAGTACCAGCGAACTTTTTGCATGAGCAGAATATTCAAGAGGAATTCCATCAGAGAATAGCTGTGCAAGATGAAGCTCAGCTGAGCAGAATATTCAAGAGGCATTCCATCAGAGAATAA

SEQ ID NO.2

MNVQVHMSGQISGQVPNQAGTQLPGLPPQNGSSLSNHIQNLAGQCNTLNMEPELAKARRFMQEKIYDFLMQRQQQTNDIPPTRVLEIVRRLDEGLFRNAATKEEYVNLETLENRLRVVIESLPMSTHNQKYPHLVNAPSPIEPELAKARRFMQEKIYDFLMQRQQRTNDIPPKRVLEIVRRLDEGLFRNAATKEEYLNLETLENRLHVLIKRLPMSTHNQQYPRLVNAPSPIGTMIPTPDSANSSVMVASAVDTSMIATGGGNSMPSVAVNTGSLLPPANGSSVGIHGGSFNSQVPANFLHEQNIQEEFHQRIAVQDEAQLSRIFKRHSIRE

It is understood that modifications of the base sequences referred to in the following examples without changing the amino acid sequence, in view of the degeneracy of the codons, also fall within the scope of the present invention.

The present invention will be described in further detail with reference to the following specific embodiments and the accompanying drawings.

Example 1 cloning of the CsHAC1 Gene of tea Tree and construction of recombinant expression vector pYES2-CsHAC1 of Saccharomyces cerevisiae

Taking the tissue of tea tree 'Yinghong No. 9', extracting the total RNA of the tea tree by using a Plant RNAKit ultrafast Plant RNA extraction kit of Beijing Huayuyang biotechnology limited, and synthesizing Master Mix by using MightyScript first-chain cDNA for reverse transcription to obtain cDNA. Using cDNA as template to design homologous recombination primer of yeast expression vector pYES2 (upper case part of primer sequence is pYES2 vector homologous fragment, lower case part is CsHAC1 gene specific amplification primer)

pYES2-CsHAC1/F(SEQ ID NO.3)

5’-TACCGAGCTCGGATCCatgaatgtgcaggtacatatgtcaggac-3’

pYES2-CsHAC1/R(SEQ ID NO.4)

5’-TAGATGCATGCTCGAGttattctctgatggaatgcctcttgaatattctgctcag-3’

The full length of the cDNA reading frame of the CsHAC1 gene was amplified by PCR using KOD enzyme from Toyobo Biotech Co., Ltd, and the instructions for the enzyme were referred to in the PCR system. The amplified DNA fragments were recovered and sequenced according to the instructions of the reagent Kit for DNA recovery in agarose Gel by HiPure Gel Pure DNAMini Kit (FIG. 1). The resulting fragment was recovered for insertion into the yeast expression vector pYES 2.

The saccharomyces cerevisiae expression vector pYES2 is subjected to double enzyme digestion treatment by BamH I and Xho I, and a linearized vector is recovered. The recovered CsHAC1 fragment and the linearized pYES2 vector were ligated by homologous recombination of the DNA fragment and the vector using the Cloneexpress II One Step Cloning Kit from Nanjing Novowed Biotechnology Ltd. The reaction product was transformed into escherichia coli competent DH5 a according to the methods described in the specification. After colony PCR identification is carried out on the single clone, amplified culture is carried out on the positive colony to extract the plasmid, the sequencing identification is correct, the saccharomyces cerevisiae recombinant expression vector pYES2-CsHAC1 (shown in figure 2) is constructed, and the plasmid is stored for later use. After sequencing analysis, the nucleotide sequence of CsHAC1 is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 2.

Example 2 Saccharomyces cerevisiae recombinant expression vector pYES2-CsHAC1 vs. Saccharomyces cerevisiae H₂O₂Tolerance detection of stress

Saccharomyces cerevisiae oxidation-sensitive strains skn7 delta and yap1 delta (laboratory preservation) and a corresponding wild type yeast strain BY4741 (laboratory preservation) are cultured, and a pYES2 empty vector and a Saccharomyces cerevisiae recombinant expression vector pYES2-CsHAC1 plasmid are transformed into the yeast strains.

The method for yeast transformation is a lithium acetate transformation method, and the specific steps are as follows:

1) the oxidation-sensitive auxotrophic yeast strains skn7 delta and yap1 delta stored at-80 ℃ and the wild-type yeast strain BY4741 are taken out, streaked on a YPD medium plate, and placed in a constant temperature incubator at 30 ℃ for inverted culture.

2) Selecting single bacterial colony to 10mLYPD liquid culture medium, and culturing at 30 deg.C with constant temperature shaking table (200 rpm);

3) transferring the overnight-cultured yeast mother liquor into 20mLYPD liquid culture medium, and adjusting the concentration to OD₆₀₀0.2-0.3, placing in 30 deg.C constant temperature shaking table to culture to OD₆₀₀Is 0.4-0.6.

4) The yeast solution was transferred to a centrifuge tube, centrifuged at 3000rpm for 5 minutes, and the medium was discarded.

5) The cells were resuspended in 20mL of sterile water, centrifuged at 3000rpm for 5 minutes, and the supernatant was discarded.

6) The pellet was suspended by gently flicking the tube wall with an appropriate amount of TE/LiAc solution (100. mu.L per plasmid required for transformation of one strain). In this case, the yeast cells are in a competent state, and care should be taken to avoid damaging the cells.

7) The salmon sperm DNA was boiled for 5 minutes and cooled at 4 ℃ for 3 minutes. A1.5 mL centrifuge tube was prepared for sterilization and loading was started.

After addition, the mixture was inverted and mixed several times and incubated for 30 minutes at 30 ℃ on a constant temperature shaker (250 rpm).

8) After heat shock at 42 ℃ for 15 minutes, the cells were left at 4 ℃ for 5 minutes, centrifuged at 6000rpm at room temperature for 30 seconds, the supernatant was discarded, and the cells were then emptied for 30 seconds, dried with PEG by pipetting, and resuspended in 70. mu.L of 1 XTE.

9) The above-mentioned oxidative sensitive yeast containing the objective plasmid was smeared on YNB medium plates (YNB medium supplemented with histidine, leucine, methionine for BY4741, skn 7. delta., yap 1. delta.) supplemented with the corresponding amino acids, and cultured at 30 ℃ for 3 days in an inverted state until transformants appeared.

Respectively picking up skn7 delta, yap1 delta and BY4741 yeast monoclonals transferred with pYES2 empty vector and skn7 delta and yap1 delta yeast monoclonals transferred with saccharomyces cerevisiae pYES2-CsHAC1 recombinant plasmid, inoculating the monoclonals into 600 mu L YNB liquid culture medium containing amino acids required BY corresponding yeast strains, culturing for 2-3 days at 30 ℃ constant temperature shaking table (200rpm), and culturing until the bacterium liquid is turbid. Zeroing with YNB liquid culture medium, diluting with sterile water to obtain yeast liquid OD₆₀₀The value was adjusted to 1.0(± 0.05), and the bacterial suspension was mixed according to the ratio of 1: 1. 1:10, 1:100, 1:1000 dilution step by step (in FIG. 3, H for each concentration₂O₂On the plate, from left to right, represent 1: 1. 1:10, 1:100, 1: 1000). 2.5. mu.L of each of skn 7. delta., yap 1. delta., BY4741 bacteria solution was pipetted and dropped onAdding 0mmol/L, 0.5mmol/L, 0.75mmol/L, 1mmol/L H₂O₂YNB solution on solid medium plate. Culturing in 30 deg.C constant temperature incubator for 7 days, and observing yeast growth condition.

As shown in FIG. 3, the skn 7. delta. and yap 1. delta. yeast mutants transformed with a transgenic yeast overexpressing CsHAC1 gene (i.e., pYES2-CsHAC1 recombinant plasmid) were able to add 0.5mM, 0.75mM, and 1mM H₂O₂The mutant strains of skn 7. delta. and yap 1. delta. yeast transformed with yeast not expressing CsHAC1 gene (i.e., empty vector pYES2) were grown in YNB medium in which 0.5mM and 0.75mM H were added₂O₂YNB medium (YNB) showed very little or no growth, and 1mM H was added₂O₂The YNB medium showed little growth.

The above results show that: the CsHAC1 gene of tea tree can improve the tolerance of Saccharomyces cerevisiae oxidation-sensitive strains skn7 delta and yap1 delta to oxidation stress.

Example 3 tolerance assay for Saccharomyces cerevisiae salt stress in the recombinant expression vector pYES2-CsHAC1 of Saccharomyces cerevisiae

The saccharomyces cerevisiae salt sensitive strain AXT3 (stored in a laboratory) and the corresponding wild type yeast strain W303 (stored in a laboratory) are cultured, and the pYES2 empty vector and the saccharomyces cerevisiae recombinant expression plasmid pYES2-CsHAC1 are transformed into the yeast strain.

The method adopted for yeast transformation is a lithium acetate transformation method, and the specific steps are shown in example 2.

Sensitive yeasts AXT3 and W303 containing a target plasmid (Saccharomyces cerevisiae recombinant expression vector pYES2-CsHAC1) were applied to YNB medium plates supplemented with the corresponding amino acids (the YNB medium for AXT3 growth was supplemented with adenine; the YNB medium for W303 growth was supplemented with histidine, leucine, tryptophan, and adenine), and were subjected to inverted culture at 30 ℃ for 3 days until transformants appeared.

Respectively selecting AXT3 yeast monoclonal and W303 yeast monoclonal transferred with pYES2 empty vector and AXT3 yeast monoclonal and W303 yeast monoclonal transferred with PYES2-CsHAC1 recombinant plasmid, inoculating into 600 μ L YNB liquid culture medium containing amino acids required by corresponding yeast strains, culturing for 2-3 days at 30 deg.C constant temperature shaking table (200rpm), and culturing until the bacterial liquid is turbidTurbid. Zeroing with YNB liquid culture medium, diluting with sterile water to obtain yeast liquid OD₆₀₀The value was adjusted to 1.0(± 0.05), and the bacterial suspension was mixed according to the ratio of 1: 1. 1:10, 1:100 and 1:1000 (in FIG. 4, NaCl solution of each concentration represents 1:1, 1:10, 1:100 and 1:1000 in turn from left to right). Sucking 2.5 mu L of AXT3 bacterial liquid diluted step by step and respectively dripping the AXT3 bacterial liquid on YNB solid culture medium plates added with 0mmol/L, 50mmol/L, 75mmol/L and 100mmol/LNaCl solution; 2.5. mu.L of the W303 bacterial solution diluted stepwise was pipetted and dropped on YNB solid medium plates to which 0mol/L, 0.5mol/L, 0.75mol/L, and 1mol/L of NaCl solution was added. Culturing in 30 deg.C constant temperature incubator for 7 days, and observing yeast growth condition.

The results are shown in FIG. 4. On a culture medium plate added with 1mol/L NaCl, the transgenic yeast W303 which is transferred with the overexpression tea tree CsHAC1 gene (namely transferred with a saccharomyces cerevisiae recombinant expression vector pYES2-CsHAC1) has the concentration of 1:1 and 1:10 is capable of growing; while the yeast strain W303 transferred with the tea tree CsHAC1 gene not expressed (i.e. with pYES2 empty vector) only has the expression ratio of 1:1 concentration can grow.

Similarly, the AXT3 yeast mutant of a transgenic yeast transformed to overexpress the tea tree CsHAC1 gene (i.e., transformed with the Saccharomyces cerevisiae recombinant expression vector pYES2-CsHAC1) was cultured in the presence of 1mmol/L NaCl-supplemented medium plates at concentrations of 1 and 1:10, the bacterial liquid can grow, and the concentration is 1: the 100 bacteria solution also grows a little; the AXT3 yeast mutant strain which is transferred to the CsHAC1 gene of tea tree which is not expressed (namely, the pYES2 empty vector is transferred) has poor growth, and only the bacterium solution with the highest concentration (1:1) can grow. The yeast strains with the recombinant plasmids have better growth than the yeast strains with the empty vectors on other culture medium plates added with NaCl.

The results show that the tea tree CsHAC1 gene can improve the tolerance of the saccharomyces cerevisiae salt-sensitive mutant strain AXT3 to salt stress.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> institute of tea leaf of Guangdong province institute of agricultural sciences in south China plant Garden of China academy of sciences

<120> tea tree CsHAC1 gene and protein and application thereof

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Claims

1. An application of a tea tree CsHAC1 gene in improving plant stress resistance is characterized in that the nucleotide sequence of the tea tree CsHAC1 gene is shown as SEQ ID No. 1; or the nucleotide sequence shown as SEQ ID No.1 is substituted, deleted and/or added with one or more nucleotides and can code the same functional protein; or a nucleotide sequence with the coding amino acid sequence shown as SEQ ID No. 2.

2. The application of the protein coded by the tea tree CsHAC1 gene in improving the stress resistance of plants is characterized in that the amino acid sequence of the protein coded by the tea tree CsHAC1 gene is shown as SEQ ID No. 2; or the amino acid sequence shown as SEQ ID No.2 is substituted, deleted and/or added with one or more amino acids, but the protein activity is the same.

3. Use according to claim 1 or 2, wherein the stress resistance is resistance to hydrogen peroxide and/or sodium chloride stress.

4. Use according to claim 1 or 2, wherein the plant is tea, soybean, wheat, rice or arabidopsis.

5. The application of a saccharomyces cerevisiae recombinant expression vector in improving stress resistance of saccharomyces cerevisiae is characterized in that the gene of the tea tree CsHAC1 of claim 1 or the gene for expressing the protein coded by the tea tree CsHAC1 gene of claim 2 is inserted into the saccharomyces cerevisiae recombinant expression vector.

6. The use of claim 5, wherein the recombinant expression vector of Saccharomyces cerevisiae is pYES2-CsHAC 1.

7. Use according to claim 5 or 6, wherein the stress resistance is resistance to hydrogen peroxide and/or sodium chloride stress.

8. Use according to claim 5 or 6, wherein the plant is Camellia sinensis, Glycine max, Triticum aestivum or Arabidopsis thaliana.

9. The use of a recombinant expression vector for improving stress resistance in plants, wherein the recombinant expression vector has the tea tree CsHAC1 gene of claim 1 inserted therein, or has a gene for expressing a protein encoded by the tea tree CsHAC1 gene of claim 2 inserted therein.

10. The use according to claim 9, wherein the recombinant expression vector is pCAMBIA1300-CsHAC 1.