Application of tea tree CsCIPK20 gene in improving plant cold resistance

Abstract

The application of tea tree CsCIPK20 gene in improving plant cold resistance belongs to the technical field of biological gene engineering and plant genetic breeding. The tea tree CsCIPK20 gene is positively regulated to plant cold resistance. The biological function verification is carried out by overexpressing the gene in Arabidopsis thaliana and silencing the gene in tea tree, and it is proved that the CsCIPK20 gene can improve the low temperature resistance of plants. The application provides a gene resource for cultivating new plant varieties with low temperature resistance, and has good potential application value.

Description

Technical Field

[0001] This invention belongs to the fields of bioengineering and plant genetics breeding technology, specifically involving the application of the CsCIPK20 gene in tea trees to improve plant cold resistance. Background Technology

[0002] Low temperature is a severe environmental stressor and a significant limiting factor for plant growth and development. Temperature stress has seriously threatened the yield and quality of economic crops. Tea is an important economic crop primarily grown for its leaves, originating in tropical or subtropical regions and is particularly sensitive to low temperatures. The tea plant's tolerance to low temperatures is closely related to tea yield, quality, and economic benefits, especially during the spring harvest season. Cold waves can damage newly sprouted tea shoots, leading to substantial economic losses. Therefore, researching how tea plants respond to, adapt to, and resist low-temperature environments, and breeding and obtaining low-temperature-resistant varieties, is of great significance for ensuring the development of the tea industry.

[0003] Calcium ions, acting as second messengers, participate in numerous biological processes in plants. The initial response of plants to low-temperature signals may involve calcium ion channels on the cell membrane. Upon exposure to low temperatures, the intracellular calcium ion concentration rapidly increases within a short period, thereby triggering downstream cold-resistance defense responses. CIPK (Calcineurin B-like interacting kinase) protein kinases are a class of plant-specific protein kinases that transmit calcium ion signals by binding to the calcium ion signaling receptor protein CBL (Calcineurin B-like). Previous studies have shown that the expression of CsCIPK20, as described in this invention, is significantly induced by low-temperature stress, but its specific function under low-temperature stress remains unknown.

[0004] Transgenic technology allows the introduction of endogenous or exogenous stress-resistance genes into plants requiring improvement, resulting in offspring exhibiting stable, inherited stress resistance. Currently, transgenic systems for tea trees are still immature, while Arabidopsis thaliana is a model plant in plant biology research. Agrobacterium-mediated transgenic technology can be used to overexpress candidate genes in Arabidopsis, allowing for the study of their biological functions. Overexpression of the CsCIPK20 gene in Arabidopsis using transgenic technology significantly increased the frost resistance of transgenic plants. Simultaneously, reducing CsCIPK20 gene expression in tea leaves using antisense oligonucleotide silencing technology significantly reduced the frost resistance of tea leaves with the CsCIPK20 gene silenced. Therefore, cloning and functional verification of the CsCIPK20 gene in tea trees can provide genetic resources for cold-resistance breeding in tea trees. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of this invention is to design and provide a technical solution for the application of the tea tree CsCIPK20 gene in improving the cold resistance of plants.

[0006] The concept of this invention is as follows: Through research on the gene CsCIPK20, which encodes the protein kinase interacting with calcineurin B in tea, this application found that transgenic plants overexpressing this gene have a significant frost-resistant phenotype under low-temperature stress compared to wild-type plants, and their electrical conductivity values ​​decrease.

[0007] This invention is specifically achieved through the following technical solutions:

[0008] This invention provides, in one aspect, the application of the tea plant calcineurin B protein-interacting protein kinase gene CsCIPK20 in improving plant cold resistance, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] Furthermore, the plants include woody plants and herbaceous plants.

[0010] In another aspect, the present invention provides the application of biomaterials containing the tea plant calcineurin B protein-interacting protein kinase gene CsCIPK20 in improving plant cold resistance, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0011] Furthermore, the biomaterial is recombinant DNA, expression cassette, plasmid vector, viral vector, or engineered bacteria.

[0012] Furthermore, the plants include woody plants and herbaceous plants.

[0013] Another aspect of the present invention provides a method for obtaining cold-resistant plants, comprising the following steps:

[0014] 1) Infuse the plant with the tea tree cold-resistant gene CsCIPK20; or

[0015] 2) Overexpress the cold resistance gene CsCIPK20 in tea plants.

[0016] Furthermore, in method 2), the gene CsCIPK20 is introduced into plants to obtain transgenic plants overexpressing the CsCIPK20 gene.

[0017] Furthermore, the specific steps include:

[0018] 1) Extract total RNA from tea tree leaves, reverse transcribe to obtain cDNA, design primers F and R, use tea tree cDNA as template for PCR amplification, obtain the amplified product CsCIPK20 gene, construct the amplified product into the pCambia 1300 plant expression vector with a super promoter promoter, and obtain the recombinant expression vector.

[0019] 2) Agrobacterium was transformed using the above recombinant expression vector, and the transformed Agrobacterium was used to infect Arabidopsis inflorescences to obtain transgenic Arabidopsis strains.

[0020] Furthermore, the nucleotide sequence of primer F is shown in SEQ ID NO.3, and the nucleotide sequence of primer R is shown in SEQ ID NO.4.

[0021] This application has the following beneficial effects:

[0022] This invention, through overexpression experiments, validated the biological function of CsCIPK20 for the first time and revealed for the first time that CsCIPK20 positively regulates cold resistance in plants. Overexpression of the CsCIPK20 gene in Arabidopsis thaliana enhances the plant's cold tolerance. Therefore, this invention provides an important gene resource that can be used to cultivate cold-resistant plants and has potential application value. Attached Figure Description

[0023] Figure 1 This is a graph showing the expression of the CsCIPK20 gene in the overexpression lines OE-1, OE-2, OE-3, and OE-4 in Example 2 of this invention.

[0024] Figure 2 The phenotypes of the overexpression lines OE-1, OE-2, OE-3 and OE-4 in Example 3 of this invention under low temperature stress;

[0025] Figure 3 The conductivity of the overexpression lines OE-1, OE-2, OE-3 and OE-4 in Example 3 of this invention under normal culture and low temperature stress;

[0026] Figure 4 This describes the silencing effect of CsCIPK20 in tea plants after antisense oligonucleotide injection in Example 4 of this invention.

[0027] Figure 5 This is the phenotype of tea plants with CsCIPK20 silenced under low temperature stress in Example 4 of the present invention;

[0028] Figure 6 The electrical conductivity of the CsCIPK20-silenced tea plant under low-temperature stress in Example 4 of this invention is shown. Detailed Implementation

[0029] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions (see Sambrook J. & Russell DW, Molecular cloning: a laboratory manual, 2001) or according to the manufacturer's instructions.

[0030] In the following examples, the pEASYbluntzero vector is a commonly used cloning vector that is commercially available; the Super promoter-pCambia1300 vector is a vector preserved by our research group; the Arabidopsis thaliana variety is the Columbia ecotype; and the Agrobacterium GV3101 strain is a commonly used strain that is preserved in most molecular biology laboratories.

[0031] The main reagents used in the following examples were: pEASYbluntzero cloning vector kit purchased from Beijing TransGen Biotech Co., Ltd.; restriction endonucleases, Taq polymerase, dNTPs, reverse transcription kits, etc. purchased from TAKARA; plasmid miniprep kit and agarose gel extraction kit purchased from Axygen; plant RNA extraction kit purchased from Tiangen Biotech Co., Ltd.; hygromycin, SYBR green, etc. purchased from Roche; agarose, kanamycin, rifampin, etc. purchased from Sigma; and all other chemical reagents used in the examples were imported or domestically produced analytical grade reagents.

[0032] The primers used in the examples were synthesized by Shanghai Huajin Biotechnology Co., Ltd., and the relevant sequencing was performed.

[0033] Example 1: Construction of an overexpression vector for the CsCIPK20 gene in tea plants

[0034] Based on the transcriptome database of our research group, the nucleotide sequence of the tea tree CsCIPK20 gene is shown in SEQ ID NO: 1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO: 2. Primers F and R were designed, and the gene CsCIPK20 was amplified by PCR using tea leaf cDNA as a template. The amplified sequence was ligated into the pEASYbluntzero vector, and finally ligated into the Super promoter-pCambia1300 plant expression vector through a double enzyme digestion and ligation reaction.

[0035] The primer used was F: 5'- CGGGGCCC ATGGGCTATGAAAAATTATC-3' (as shown in SEQ ID NO:3); R: 5'- GCGTCGAC CTAGAAATGTTGTTCATCATCAT-3' (as shown in SEQ ID NO:4).

[0036] Example 2: Construction and screening of plants overexpressing the CsCIPK20 gene

[0037] The correctly sequenced vector constructed in Example 1 was transformed into Agrobacterium GV3101 strain, and then into wild-type Arabidopsis plants to obtain seeds of the transgenic Arabidopsis seedlings. The super promoter-pCambia1300 vector carries a hygromycin-containing resistance gene. Hygromycin was used to screen the transgenic Arabidopsis seedlings. The T1 generation of positive seedlings with hygromycin resistance were harvested individually, and the T2 generation seeds were then screened for hygromycin resistance. Lines with a segregation ratio of approximately 3:1 between resistant and non-resistant seedlings were selected, indicating that the vector sequence containing the target gene was inserted as a single copy in these lines. The hygromycin-resistant plants from these lines were removed, and individual seedlings were harvested again for hygromycin resistance screening. If no segregation occurred, the transgenic line was considered homozygous, and this homozygous line could be used for propagation and physiological experiments.

[0038] Overexpression lines OE-1, OE-2, OE-3, and OE-4 were obtained through screening. Total RNA was extracted from wild-type, OE-1, OE-2, OE-3, and OE-4, and cDNA was synthesized by reverse transcription. The expression level of CsCIPK20 in the obtained overexpression lines OE-1, OE-2, OE-3, and OE-4 was detected by qRT-PCR. The results are shown in [Figure 1]. Figure 1 It can be seen that overexpression of the CsCIPK20 gene can be detected in OE-1, OE-2, OE-3 and OE-4.

[0039] Example 3: Low-temperature tolerance of plants overexpressing CsCIPK20

[0040] After sterilization, Arabidopsis seeds were sown on half MS medium supplemented with 1.5% sucrose and cultured normally (22°C daytime / 20°C nighttime, 10 h light / 14 h darkness, 10000 Lux light intensity) for 8 days. Afterward, they were transplanted into seedling blocks. When the seedlings reached 23 days old, they were subjected to a 7-day low-temperature treatment at 4°C, followed by a 7.5-hour treatment at -10°C. After the low-temperature treatment, they were returned to normal culture for 6 days. Photographs were taken for record-keeping. Figure 2 Wild-type (WT) tea plants exhibited curled and wilted leaves, while the CsCIPK20-OE strain retained upright and extended leaves, indicating that overexpression of CsCIPK20 in tea plants enhances their low-temperature resistance. After the low-temperature treatment, the relative conductivity of the entire aboveground portion was measured. The results are shown below. Figure 3 The results showed that, compared with the wild type, the relative conductivity content of OE-1, OE-2, OE-3 and OE-4 overexpressing plants was significantly reduced and their cold tolerance was increased under low temperature stress.

[0041] Example 4: Low-temperature tolerance of tea plants with silenced CsCIPK20 gene

[0042] To further verify the role of the CsCIPK20 gene in plant cold tolerance, antisense oligonucleotide injection was used to transiently silence the CsCIPK20 gene in tea trees. Five sense oligonucleotides (sODN) and five antisense oligonucleotides (AsODN) were mixed separately, and the injected oligonucleotide concentration was 30 μM. After 15 h, CsCIPK20 gene expression was measured to determine the silencing effect. The experimental results are as follows: Figure 4 As shown, compared with plants injected with the sense strand as a control, the expression of CsCIPK20 in tea leaves injected with the antisense strand was significantly downregulated, downregulated to 36% of the control. Fifteen hours after injection, the plants were subjected to a -5°C low-temperature treatment for 2 hours. After the treatment, as shown... Figure 5 As shown, the tea leaves injected with antisense chains curled. Relative conductivity of the leaves was measured, see... Figure 6 The relative electrical conductivity of CsCIPK20-silenced plants was significantly higher than that of the control, indicating that silencing CsCIPK20 reduces the plant's low-temperature tolerance. This demonstrates that CsCIPK20 positively regulates the low-temperature response of tea plants, consistent with the results of functional identification using CsCIPK20-OE Arabidopsis materials.

[0043] Antisense oligonucleotide (AsODN) sequence:

[0044] 5'-CCTTCTCTTTGTTGATGACTTTGAC-3' (as shown in SEQ ID NO:5),

[0045] 5'-CAAATATCTGCCTTCTCTCCGTCGT-3' (as shown in SEQ ID NO:6).

[0046] 5'-GGGCTTTATGGTGGTGAACCGGGCT-3' (as shown in SEQ ID NO:7),

[0047] 5'-AGCGGGCTTTATGGTGGTGAACCGG-3' (as shown in SEQ ID NO:8),

[0048] 5'-CTTCTTTGCTCCCTTGCATGTTCAA-3' (as shown in SEQ ID NO:9);

[0049] The sequence of the positive oligonucleotide (sODN):

[0050] 5'-GGAAGAGAAACAACTACTGAAACTG-3' (as shown in SEQ ID NO:10).

[0051] 5'-GTTTATAGACGGAAGAGAGGCAGCA-3' (as shown in SEQ ID NO:11).

[0052] 5'-CCCGAAATACCACCACTTGGCCCGA-3' (as shown in SEQ ID NO:12).

[0053] 5'-TCGCCCGAAATACCACCACTTGGCC-3' (as shown in SEQ ID NO:13).

[0054] 5'-GAAGAAACGAGGGAACGTACAAGTT-3' (as shown in SEQ ID NO:14).

[0055] The above results demonstrate that the CsCIPK20 gene positively regulates the cold resistance of plants.

[0056] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. sequence list <110> Tea Research Institute, Chinese Academy of Agricultural Sciences <120> Application of the CsCIPK20 gene in tea plants to improve cold resistance <160> 14 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1470 <212> DNA <213> Tea tree (tea) <400> 1 atgggctatg aaaaattatc aataattgtt ctcttgatat acttattgat tatgttgaga 60 gcgagaagca catcgaatat ggagaagaag gggactatgt tgttggggaa gtacgagctc 120 gggaggattc taggtcaagg cacattcgcc aaggtgtacc atggccggaa cataaaatcc 180 ggccaaaccg tggctgtcaa agtcatcaac aaagaagaagg tgatgaaggt cggtttgatt 240 gatcaaatca aacgagagat ctcggtgatg aggctagtca gacaccctaa tgtggtccaa 300 ttatacgagg tcatggcgag caagaccaag atctatttcg ccatggagta tgtgagaggc 360 ggagagcttt tcaacaaggt agccaaaggg cgtctcaagg aagatgttgc aaagaagtac 420 ttccagcagc tggtcgcagc ggtagacttc tgccatagca gaggcgtcta ccaccgtgac 480 ctgaagccgg aaaatctcct cctcgatgag cacggaaacc taaaagttc cgatttcggt 540 ttgagcgcct tgcacgagtc aagggggcaa gacggcctcc tccacacgac atgtggaacc 600 ccggcctatg ttgcccccga agtcatcaac aagagaggct acgacggaga gaaggcagat 660 atttggtcat gtggggttgt cctattcgtc ctcttagccg gttatcttcc attccatgat 720 780 ttcccaccgg aggtccgaaa gcttctctca agaattctgg atccgaatcc atgcacaaga 840 atcacagtag ccaagctcat ggacaattca tggttcaaga aagaattcaa acaaattgaa 900 gtcccattgc ccattcaaga cgaacacgac aacacacctc gcagccttct agacttcaac 960 gaagcatttg aatccgattc agaaatcata gagaagaaga gaggagaggc gaccacaagt 1020 tccatgatca tgaagccaac ttgcatgaat gcctttgata tcatctctct ctcgcaagga 1080 tttgatctct cgggcttgtt tgagaatgac aacgatcaaa agtctgaagc ccggttcacc 1140 accataaagc ccgcttcaga cattgtgtca aaattagaag agatagccat gacggagagt 1200 ttcaaagtga agaagaaaaa tgggactttg aacatgcaag ggagcaaaga aggtaggaag 1260 gggcacttgg ccataaacgc ggagatattc gaggtggcgc cgtcgtttca catggtggag 1320 gtgaagaaag ttgccggaga cacgctggaa tacaaagagt tttgcaacca gggtttgaag 1380 ccttccctca aggacatagt ttggacttgg caaggttgtg agcaacaaaa acaaaaacaa 1440 cagcagcatg atgatgaaca acatttctag 1470 <210> 2 <211> 489 <212> PRT <213> Tea plant <400> 2 Met Gly Tyr Glu Lys Leu Ser Ile Ile Val Leu Leu Ile Tyr Leu Leu 1 5 10 15 Ile Met Leu Arg Ala Arg Ser Thr Ser Asn Met Glu Lys Lys Gly Thr 20 25 30 Met Leu Leu Gly Lys Tyr Glu Leu Gly Arg Ile Leu Gly Gln Gly Thr 35 40 45 Phe Ala Lys Val Tyr His Gly Arg Asn Ile Lys Ser Gly Gln Thr Val 50 55 60 Ala Val Lys Val Ile Asn Lys Glu Lys Val Met Lys Val Gly Leu Ile 65 70 75 80 Asp Gln Ile Lys Arg Glu Ile Ser Val Met Arg Leu Val Arg His Pro 85 90 95 Asn Val Val Gln Leu Tyr Glu Val Met Ala Ser Lys Thr Lys Ile Tyr 100 105 110 Phe Ala Met Glu Tyr Val Arg Gly Gly Glu Leu Phe Asn Lys Val Ala 115 120 125 Lys Gly Arg Leu Lys Glu Asp Val Ala Arg Lys Tyr Phe Gln Gln Leu 130 135 140 Val Ala Ala Val Asp Phe Cys His Ser Arg Gly Val Tyr His Arg Asp 145 150 155 160 Leu Lys Pro Glu Asn Leu Leu Leu Asp Glu His Gly Asn Leu Lys Val 165 170 175 Ser Asp Phe Gly Leu Ser Ala Leu His Glu Ser Arg Gly Gln Asp Gly 180 185 190 Leu Leu His Thr Thr Cys Gly Thr Pro Ala Tyr Val Ala Pro Glu Val 195 200 205 Ile Asn Lys Arg Gly Tyr Asp Gly Glu Lys Ala Asp Ile Trp Ser Cys 210 215 220 Gly Val Val Leu Phe Val Leu Leu Ala Gly Tyr Leu Pro Phe His Asp 225 230 235 240 Ser Asn Leu Met Glu Met Tyr Arg Lys Ile Ser Arg Gly Val Phe Lys 245 250 255 Cys Pro Gln Trp Phe Pro Pro Glu Val Arg Lys Leu Leu Ser Arg Ile 260 265 270 Leu Asp Pro Asn Pro Cys Thr Arg Ile Thr Val Ala Lys Leu Met Asp 275 280 285 Asn Ser Trp Phe Lys Lys Glu Phe Lys Gln Ile Glu Val Pro Leu Pro 290 295 300 Ile Gln Asp Glu His Asp Asn Thr Pro Arg Ser Leu Leu Asp Phe Asn 305 310 315 320 Glu Ala Phe Glu Ser Asp Ser Glu Ile Ile Glu Lys Lys Arg Gly Glu 325 330 335 Ala Thr Thr Ser Ser Met Ile Met Lys Pro Thr Cys Met Asn Ala Phe 340 345 350 Asp Ile Ile Ser Leu Ser Gln Gly Phe Asp Leu Ser Gly Leu Phe Glu 355 360 365 Asn Asp Asn Asp Gln Lys Ser Glu Ala Arg Phe Thr Thr Ile Lys Pro 370 375 380 Ala Ser Asp Ile Val Ser Lys Leu Glu Glu Ile Ala Met Thr Glu Ser 385 390 395 400 Phe Lys Val Lys Lys Lys Asn Gly Thr Leu Asn Met Gln Gly Ser Lys 405 410 415 Glu Gly Arg Lys Gly His Leu Ala Ile Asn Ala Glu Ile Phe Glu Val 420 425 430 Ala Pro Ser Phe His Met Val Glu Val Lys Lys Val Ala Gly Asp Thr 435 440 445 Leu Glu Tyr Lys Glu Phe Cys Asn Gln Gly Leu Lys Pro Ser Leu Lys 450 455 460 Asp Ile Val Trp Thr Trp Gln Gly Cys Glu Gln Gln Lys Gln Lys Gln 465 470 475 480 Gln Gln His Asp Asp Glu Gln His Phe 485 <210> 3 <211> 28 <212> DNA <213> Primer <400> 3 cggggcccat gggctatgaa aaattatc 28 <210> 4 <211> 31 <212> DNA <213> Primer <400> 4 gcgtcgacct agaaatgttg ttcatcatca t 31 <210> 5 <211> 25 <212> DNA <213> Antisense oligonucleotides (AsODN) <400> 5 ccttctcttt gttgatgact ttgac 25 <210> 6 <211> 25 <212> DNA <213> Antisense oligonucleotides (AsODN) <400> 6 caaatatctg ccttctctcc gtcgt 25 <210> 7 <211> 25 <212> DNA <213> Antisense oligonucleotides (AsODN) <400> 7 gggctttatg gtggtgaacc gggct 25 <210> 8 <211> 25 <212> DNA <213> Antisense oligonucleotides (AsODN) <400> 8 agcgggcttt atggtggtga accgg 25 <210> 9 <211> 25 <212> DNA <213> Antisense oligonucleotides (AsODN) <400> 9 cttctttgct cccttgcatg ttcaa 25 <210> 10 <211> 25 <212> DNA <213> positive oligonucleotides (sODN) <400> 10 ggaagagaaa caactactga aactg 25 <210> 11 <211> 25 <212> DNA <213> positive oligonucleotides (sODN) <400> 11 gtttatagac ggaagagagg cagca 25 <210> 12 <211> 25 <212> DNA <213> positive oligonucleotides (sODN) <400> 12 cccgaaatac caccacttgg cccga 25 <210> 13 <211> 25 <212> DNA <213> positive oligonucleotides (sODN) <400> 13 tcgcccgaaa taccaccact tggcc 25 <210> 14 <211> 25 <212> DNA <213> positive oligonucleotides (sODN) <400> 14 gaagaaacga gggaacgtac aagtt 25

Claims

1. Application of the tea plant calcineurin B protein-protein interaction kinase gene CsCIPK20 in improving plant cold resistance. The nucleotide sequence of the gene is shown in SEQ ID NO.

1. The plant is tea plant or Arabidopsis thaliana.

2. Application of biomaterials containing the tea plant calcineurin B protein-protein interaction kinase gene CsCIPK20 in improving plant cold resistance, the nucleotide sequence of which is shown in SEQ ID NO.1, wherein the plant is tea plant or Arabidopsis thaliana.

3. The application as described in claim 2, characterized in that... The biomaterial is recombinant DNA, expression cassette, plasmid vector, viral vector, or engineered bacteria.

4. A method for obtaining a cold-resistant plant, wherein the plant is a tea tree or Arabidopsis thaliana, characterized in that... Including the following methods: 1) Infuse the plant with the tea tree cold-resistant gene CsCIPK20; or 2) Overexpress the cold resistance gene CsCIPK20 in tea plants.

5. The method as described in claim 4, characterized in that... In method 2), the gene CsCIPK20 is introduced into plants to obtain transgenic plants overexpressing the CsCIPK20 gene.

6. The method as described in claim 5, characterized in that... Specifically, the following steps are included: 1) Extract total RNA from tea tree leaves, reverse transcribe to obtain cDNA, design primers F and R, use tea tree cDNA as template for PCR amplification, obtain the amplified product CsCIPK20 gene, construct the amplified product into the pCambia1300 plant expression vector with a super promoter promoter, and obtain the recombinant expression vector. 2) Agrobacterium was transformed using the above recombinant expression vector, and the transformed Agrobacterium was used to infect Arabidopsis inflorescences to obtain transgenic Arabidopsis strains.

7. The method as described in claim 6, characterized in that... The nucleotide sequence of primer F is shown in SEQ ID NO.3, and the nucleotide sequence of primer R is shown in SEQ ID NO.4.

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