Bnaco1 gene, protein, and use in regulating plant height of brassica napus

Abstract

Provided are a BnACO1 gene, a protein, and a use in regulating the plant height of Brassica napus. A plant height related gene BnACO1 is isolated from a leaf rolling mutant Bnuc1 of Brassica napus. A subcellular localization result indicates that BnACO1 is localized to the cytoplasm and the cell membrane. A BnACO1 overexpression vector is constructed, and Brassica napus wild-type Zhongshuang 11 (ZS11) is transformed. Compared with the control ZS11, a transgenic positive plant has a significantly reduced plant height, wrinkled leaves, and a 5- to 7-day delay in flowering time. BnACO1 can control the plant height of Brassica napus, and has an important application value in dwarf breeding of Brassica napus.

Description

BnACO1 gene, protein and its application in regulating plant height in Brassica napus Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving the BnACO1 gene, protein, and its application in regulating the growth and development of Brassica napus plants. Background Technology

[0002] Rapeseed (Brassica napus) originated in my country and is one of the country's important oil crops. Rapeseed seeds have a high oil content, generally around 40%. Plant height is an important agronomic trait and the foundation for excellent plant architecture, closely related to crop yield, light energy utilization efficiency, and mechanization efficiency. Currently, widely cultivated rapeseed varieties across the country are easy to cultivate and manage due to their tall stature; however, this characteristic also leads to reduced yield and increased harvesting difficulty. To effectively address these challenges, rapeseed breeding experts have actively invested in developing mutants of Brassica napus with more compact plant height. Their in-depth research aims to uncover and utilize the key genetic resources behind the dwarfing trait, hoping to achieve stable inheritance and application of this trait in rapeseed breeding practices. This process not only helps alleviate yield losses caused by excessive plant height but also significantly improves the efficiency of mechanized operations in rapeseed fields, thereby reducing production costs and improving overall economic benefits.

[0003] Plant height is a complex trait regulated by multiple genes. These genes may be distributed on different chromosomes, influencing plant height through quantitative trait loci (QTLs). For example, wheat plant height is regulated by multiple genes, with QTLs widely distributed across all 21 wheat chromosomes. Reduced plant height not only increases yield but also enhances wheat's resistance to lodging and fertilizer application, while also providing broad adaptability and yield stability. Apple's MdCo31 gene encodes a 2OG-Fe(II)-dependent oxygenase; overexpression of this gene leads to phenotypes such as slowed growth, shortened internodes, and reduced plant height in tobacco, demonstrating that MdCo31 controls dwarfing growth in apples. With the development of molecular biology techniques, an increasing number of genes controlling plant height are being cloned and functionally validated. The discovery of these genes provides important evidence for revealing the genetic mechanisms of plant height.

[0004] Plant height is also regulated by a variety of hormones. Several hormones have been identified as involved in the regulation of plant height, including gibberellin, brassinosteroid, and auxin. These hormones influence plant cell elongation and division through complex signal transduction pathways, thereby regulating plant height. Studies on dwarf plants have shown that the main plant hormones affecting plant height include gibberellin (GA), brassinosteroid (BR), and auxin (IAA).

[0005] As genomics research continues to deepen, more new genes controlling plant height will be discovered and cloned in the future. The discovery of these new genes will provide new perspectives and ideas for revealing the genetic mechanisms of plant height, and will help increase crop yields, enhance stress resistance, and promote sustainable agricultural development. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides the BnACO1 gene, protein, and their application in regulating the height of Brassica napus plants. Transgenic plants are shorter, have fewer branches, and have a delayed flowering period.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a Brassica napus BnACO1 protein, wherein the Brassica napus BnACO1 protein comprises any of the following amino acid sequences (1) and (2):

[0009] (1) The amino acid sequence as shown in SEQ ID NO.3;

[0010] (2) A sequence that has more than 90% homology with the amino acid sequence shown in SEQ ID NO.3.

[0011] The present invention also provides a Brassica napus BnACO1 gene, encoding the Brassica napus BnACO1 protein of claim 1, wherein the full-length genomic sequence of the Brassica napus BnACO1 gene is any one of the following nucleotide sequences (1) and (2):

[0012] (1) The nucleotide sequence as shown in SEQ ID NO.1;

[0013] (2) A DNA molecule whose nucleotide sequence is more than 90% homologous to the sequence shown in SEQ ID NO.1 and encodes the amino acid sequence shown in SEQ ID NO.3.

[0014] This invention also provides the application of the above-mentioned proteins or genes in regulating the plant height or flowering period of Brassica napus.

[0015] The present invention also provides a method for regulating the plant height of Brassica napus, wherein a BnACO1 overexpression vector is constructed to obtain dwarf Brassica napus plants; the full-length genome sequence of BnACO1 is shown in SEQ ID No. 1; the full-length cDNA sequence of BnACO1 is shown in SEQ ID No. 2.

[0016] The present invention also provides a method for regulating the flowering period of Brassica napus, wherein a BnACO1 overexpression vector is constructed to obtain late-flowering Brassica napus plants; the full-length genome sequence of BnACO1 is shown in SEQ ID No. 1; the full-length cDNA sequence of BnACO1 is shown in SEQ ID No. 2.

[0017] Furthermore, the BnACO1 overexpression vector is obtained by inserting the sequence shown in SEQ ID No. 2 between the CaMV35S promoter and the OCS terminator of the PBI121 vector.

[0018] Further, the method includes the following steps: inserting the sequence shown in SEQ ID No. 2 between the CaMV35S promoter and the OCS terminator of the PBI121 vector to obtain an overexpression vector, then transforming Agrobacterium tumefaciens GV3101, transforming the obtained transgenic engineered bacteria into Brassica napus recipient, and screening the progeny of the transformed plants.

[0019] Furthermore, new primers were constructed by introducing homologous sequences of two enzymes, Kpn I and BamHI, at both ends of the upstream and downstream primers of the gene clone, respectively.

[0020] Upstream primer P7: 5'-CGCGGATCCATGGAGAATCACACGAAA-3'; Downstream primer P8: 5'-ACGCGTCGACTCATATTTCTTGATTGTAAATG-3'.

[0021] Furthermore, the detection primers are:

[0022] Upstream primer P9: 7'-GAGGCTTACGCAGCAGGTCTCA-3';

[0023] Downstream primer P10: 5'-TGAGGCAATACATCAGAATCAG-3'.

[0024] Furthermore, the primer sequences for real-time quantitative PCR analysis are as follows:

[0025] Upstream primer P11: 5'-ATTACTGGCGTTAGCACTT-3';

[0026] Downstream primer P12: 5'-TATTCCTTTCGACGGATC-3'.

[0027] The BnACO1 described in this invention is located in the cytoplasm and cell membrane of tobacco leaf mesophyll cells.

[0028] This invention reduces plant height, decreases the number of branches, and delays flowering time by 5-7 days by overexpressing BnACO1.

[0029] The method for overexpressing BnACO1 described in this invention involves constructing the BnACO1 gene from the dwarf material M176 into the PBI121 overexpression vector, then transforming it into the Brassica napus variety Shuang 11, and finally screening for transgenic positive plants to obtain Brassica napus overexpressing BnACO1.

[0030] The plant overexpression vector of the present invention is obtained by inserting the sequence shown in SEQ ID No. 2 between the CaMV35S promoter and the OCS terminator of the PBI121 vector.

[0031] The Agrobacterium tumefaciens containing the plant overexpression vector described in this invention is GV3101.

[0032] Application of overexpression of BnACO1 in reducing the high blood pressure in Brassica napus plants.

[0033] A method for obtaining dwarf, late-flowering Brassica napus includes the following steps: inserting the sequence shown in SEQ ID No. 2 between the CaMV35S promoter and the OCS terminator of the PBI121 vector to obtain a vector overexpressing BnACO1, then transforming it into Agrobacterium tumefaciens GV3101, transforming the obtained transgenic engineered bacteria into the double 11 receptor in Brassica napus, and screening transgenic plants to obtain dwarf, late-flowering Brassica napus.

[0034] Preferably, the Brassica napus receptor is Shuang 11, a typical variety of Brassica napus. Beneficial effects

[0035] This invention provides the full-length genome, cDNA sequence, and protein sequence of the BnACO1 gene. Through transformation of the Zhongshuang 11 transgenic plant, it was confirmed that BnACO1 can affect plant height. The height of the transgenic plant decreased from 173cm to 35cm, a reduction of 80%; the stem diameter increased from 22.7cm to 27.6cm; the number of siliques per plant decreased from 290 to 94; the number of pods decreased from 31 to 12; and the flowering period was delayed by 5-7 days. Attached Figure Description

[0036] Figure 1 shows the electrophoresis diagram of the full-length DNA and cDNA amplification of the BnACO1 gene from Brassica napus.

[0037] Figure 2 shows the gene structure of BnACO1.

[0038] Figure 3 shows the domain distribution of BnACO1.

[0039] Figure 4 shows the multiple sequence alignment of BnACO1 with proteins such as Arabidopsis thaliana AT1G34390.1.

[0040] Figure 5 shows the subcellular localization of BnACO1.

[0041] Figure 6 shows the identification of DNA levels in transgenic plants.

[0042] Figure 7 shows the identification of transcriptional levels in transgenic plants.

[0043] Figure 8 shows the phenotype of the transgenic plant during the flowering period. Detailed Implementation

[0044] The preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Experimental methods in the preferred embodiments, where specific conditions are not specified, were performed according to the relevant product manufacturer's instructions or molecular cloning experimental guidelines. The plant material used in the embodiments: Zhongshuang 11, a type of Brassica napus, was provided by the Oil Crops Research Institute of the Chinese Academy of Sciences. Zhongshuang 11 is a publicly disclosed low-erucic acid, low-carbohydrate, high-quality conventional variety (approval number: Guoshenyou 2008030). The leaf-rolling mutant Bnuc1 was created in this experiment through EMS mutagenesis, and from this, the near-isogenic leaf-rolling line ZS11-uc1 was created through marker-assisted selection.

[0045] Example 1: Cloning of BnACO1 full-length DNA and cDNA

[0046] I. Extraction of total DNA and total RNA from Brassica napus

[0047] Young leaves of Brassica napus ZS11-uc1 were collected, and total DNA was extracted using a modified CTAB method. The concentration and quality of the DNA samples were evaluated using 1% agarose gel electrophoresis and UV spectrophotometry. DNA samples were stored at -20°C. Leaves of Zhongshuang 11 and BnACO1 overexpressing materials were collected during the seedling stage, and RNA was extracted using a plant total RNA extraction kit from TIANGEN and dissolved in pure water. The quality and purity of the RNA samples were assessed using 1% agarose gel electrophoresis and UV spectrophotometry. Electrophoresis results showed intact three bands of RNA (28S, 18S, 5S), with no significant degradation or DNA contamination, meeting the requirements for subsequent experiments. RNA samples were stored at -80°C.

[0048] II. Synthesis of the first strand of cDNA in Brassica napus

[0049] After mixing equal amounts of the RNA extracted in step 1 above, use Takara's PrimeScript... TM The 1st Strand cDNA Synthesis Kit was used for reverse transcription according to its instructions. The reverse transcription product was stored at -20°C for later use.

[0050] III. Cloning of the full-length BnACO1 DNA and cDNA of Brassica napus

[0051] Using extracted DNA as a template, the upstream primer P1 was 5'-CAGAGGTTGCTTTGTTGCCTGA-3' (SEQ ID NO.4) and the downstream primer P2 was 5'-CAGTGCAGTAGCAACTACACCA-3' (SEQ ID NO.5); using reverse-transcribed cDNA as a template, the upstream primer P3 was 5'-ATGGCGAGTGATCAAATCATG-3' (SEQ ID NO.6) and the downstream primer P4 was 5'-CTAATTGTTGTTGACATCTGGTGG-3' (SEQ ID NO.7) to amplify the full-length DNA and cDNA sequences of BnACO1, respectively. PCR amplification was performed using KOD enzyme (TOYOBO, Japan). The PCR reaction system was prepared according to the enzyme's instructions, with a reaction program of 94℃ pre-denaturation for 2 min, followed by 35 cycles of 94℃: 15 s; 52℃: 30 s; 68℃: 3 min. The PCR product was analyzed by agarose gel electrophoresis, showing that its size was consistent with the expected size. After gel recovery, ligation into the cloning vector, transformation into E. coli, PCR, and sequencing, the full-length DNA of BnACO1 was identified as 2054 bp (Figure 1), named BnACO1DNA, with the sequence shown in SEQ ID NO.1; the full-length cDNA of BnACO1, with a length of 1011 bp (Figure 1), was identified as BnACO1mRNA, with the sequence shown in SEQ ID NO.2, and the encoded amino acid sequence shown in SEQ ID NO.3.

[0052] Example 2: Bioinformatics Analysis of the BnACO1 Gene

[0053] The gene structure of BnACO1 was analyzed using the online website http: / / gsds.gao-lab.org / ; protein structure analysis was performed using the website https: / / blast.ncbi.nlm.nih.gov / Blast.cgi. The full-length BnACO1 DNA is 2054 bp, containing 11 exons and 10 introns (Figure 2). The full-length cDNA is 1011 bp, encoding 336 amino acid residues. Comparison of the BnACO1 protein with the homologous protein ACO from Arabidopsis thaliana and rice revealed that BnACO1 possesses two typical ACO protein domains: the N-terminal DIOX_N domain and the C-terminal 2OG-Fe(II)_Oxy domain (Figures 3 and 4).

[0054] Example 3: Subcellular localization analysis of BnACO1

[0055] Using the cloned full-length BnACO1 cDNA plasmid as a template, primers with restriction enzyme sites were designed to amplify the target fragment. The upstream primer was P5: 5'-GAACGATAGGGTACCATGGCGAGTGATCAAATCATG-3' (SEQ ID NO.8), and the downstream primer was P6: 5'-GCTCACCATGGATCCATTGTTGTTGACATCTGGTGG-3' (SEQ ID NO.9). Simultaneously, the plasmid pCAMBIA1305-GFP was digested with two enzymes (Kpn I and Sal I, Takara). The gel-recovered target gene fragment and the digested plasmid fragment were ligated using the homologous recombination ClonExpress II One Step Cloning Kit (Vazyme, Nanjing). The reaction system followed the enzyme's instructions. After transformation of *E. coli*, the recombinant plasmid was analyzed by bacterial culture and sequenced for verification.

[0056] Thaw one tube (100 μL) of Agrobacterium GV3101 (Weidi Biotechnology) competent cells on ice, add 4 μL of 35S::BnACO1-GFP recombinant plasmid, gently mix, place on ice for 5 min, freeze in liquid nitrogen for 5 min, then quickly place in a 37°C water bath for 5 min, followed by an ice bath for 2 min. Add 700 μL of antibiotic-free YEB liquid medium, and incubate at 28°C with shaking at 200 rpm for 3-4 h. Spread the bacterial culture evenly on YEB solid medium containing 50 μg / mL kanamycin and 50 μg / mL rifampin, and incubate upside down at 28°C for 48 h. Pick single clones for PCR detection. Store positive Agrobacterium cells for later use.

[0057] Add 200 μL of the above-mentioned positive Agrobacterium-positive bacterial suspension to 20 mL of YEB liquid medium containing 50 μg / mL kanamycin and 50 μg / mL rifampin, and incubate at 28°C and 200 rpm with shaking for 12-16 h; then add 1 mL of the bacterial suspension to 100 mL of fresh YEB liquid medium containing 50 μg / mL kanamycin and 50 μg / mL rifampin, and incubate homologously with shaking for 8-12 h until OD (Organic Difference) is reached. 600 The OD value should be around 0.8-1.2. Centrifuge at 4000 rpm for 5 min, discard the supernatant, and resuspend twice in a suspension prepared with 10 mM MES, 10 mM MgCl2, and 100 μM MS to adjust the OD value. 600=0.5, stand in the dark for 1-3 hours for later use. One day in advance, thoroughly water the tobacco plants. Inject the bacterial solution into the lower epidermis of the tobacco leaves using a syringe. Incubate at 23℃ in the dark for 24 hours, then remove and culture normally. In this experiment, the empty vector pCAMBIA1305-GFP Agrobacterium was used as a control. The location of fluorescence in the epidermal cells of the tobacco leaves in the control and experimental groups was observed and photographed using a laser confocal microscope. Figure 5 shows that the empty GFP vector in the control group fluoresces in the nucleus, cytoplasm, and cell membrane of tobacco mesophyll cells, while the BnACO1 of this invention is located in the cytoplasm and cell membrane of tobacco mesophyll cells.

[0058] Example 4: Construction and genetic transformation of BnACO1 gene overexpression vector

[0059] I. Construction of BnACO1 gene overexpression vector

[0060] The plant overexpression vector PBI121 was digested with restriction endonucleases Kpn I and BamHI (Takara), and the linearized vector fragment was recovered by 1% gel electrophoresis. Homologous sequences of Kpn I and BamHI were introduced at both ends of the upstream and downstream primers to construct new primers (upstream primer P7: 5'-CGCGGATCCATGGAGAATCACACGAAA-3' (SEQ ID NO.10), downstream primer P8: 5'-ACGCGTCGACTCATATTTCTTGATTGTAAATG-3' (SEQ ID NO.11)). PCR amplification yielded the target gene fragment homologous to both ends of the linearized vector. The target gene fragment was ligated to the linearized vector using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing), following the manufacturer's instructions. After incubation at 37°C for 30 min, the ligation product was transformed into *E. coli*, and single clones were picked for colony PCR detection. Positive single clones were sent for sequencing for further validation. The sequencing results were consistent with the gene cloning results, indicating that the BnACO1 gene was successfully inserted into the overexpression vector PBI121. The recombinant vector was named 35S::BnACO1.

[0061] II. Agrobacterium-mediated transformation of rapeseed

[0062] The 35S::BnACO1 vector constructed in Example 3 was transformed into Agrobacterium GV3101 competent cells using the freeze-thaw method. The cells were then cultured in 700 μL of YEB liquid medium with shaking for 3 hours. After brief centrifugation, the cells were collected and spread onto solid YEB plates containing Kan and Rif antibiotics. The plates were incubated at 28°C with inverted incubation for approximately 48 hours. Single Agrobacterium clones were picked for PCR detection and sequencing verification. The correctly sequenced Agrobacterium clones were transferred to 400 mL of YEB liquid medium containing 50 μg / mL rifampin and 50 μg / mL kanamycin, and cultured at 28°C with shaking until OD (out of 100°C). 600 Centrifuge at 5000 rpm for 15 min at 4℃ with a pH between 0.8 and 1.0, collect the precipitate, and suspend the bacterial cells in 1 / 2 MS liquid medium. Add 0.01 mg / L 6-BA and 0.05% Silwet-L-77 to the flowers during inoculation. Using Zhongshuang 11 as the recipient material, remove the fully opened flowers before inoculation, and place the remaining flower buds into the prepared Agrobacterium suspension. Inoculate every 5–7 days, for a total of 3 times, depending on temperature and weather conditions. Collect the seeds after the rapeseed matures, designated as generation T0.

[0063] III. Identification of Genetically Modified Rapeseed

[0064] Identification of transgenic positive plants: T0 generation transgenic seeds were sown at the Baima Base of Nanjing Agricultural University. DNA was extracted from rapeseed leaves at the 3-5 leaf stage. After electrophoresis to assess DNA quality, the DNA was used as a template for PCR detection. PCR was performed using 2×easy mix (Vazyme, Nanjing), and the system and procedure were designed according to the manufacturer's instructions. For DNA molecular detection of materials transformed with the overexpression vector, the upstream primer was derived from the 35S promoter sequence on the vector (upstream primer P9: 7'-GAGGCTTACGCAGCAGGTCTCA-3' (SEQ ID NO.12)), and the downstream primer was derived from the target gene sequence (primer sequence P10: 5'-TGAGGCAATACATCAGAATCAG-3' (SEQ ID NO.13)). The selected positive materials underwent routine field management (Figure 6), were bagged and self-pollinated at flowering time, and seeds were collected for later use, designated as the T1 generation. Generation experiments were conducted in an artificial climate incubator. Subsequent experiments were conducted using T3 generation positive plants with consistent phenotype and genotype.

[0065] Gene expression levels in transgenic materials were analyzed. Using quantitative primers (upstream primer P11: 5'-ATTACTGGCGTTAGCACTT-3' (SEQ ID NO.14) and downstream primer P12: 5'-TATTCCTTTCGACGGATC-3' (SEQ ID NO.15)) from the database https: / / brassica.biodb.org / , real-time quantitative PCR was performed to analyze the expression level of the BnACO1 gene in Zhongshuang 11 and the transgenic material. Before performing real-time PCR, the designed quantitative primers and the internal control gene BnActin primers (upstream primer P13: 5'-ATTCAGCCCCTTGTTTGTG-3' (SEQ ID NO.16) and downstream primer P14: 5'-GTAAGCGTCTTTTTGACCCAT-3' (SEQ ID NO.17)) were tested to determine the optimal annealing temperature (60℃) for the primers. Based on the Ct value, the template cDNA was diluted by the same factor. qRT-PCR was performed on a Bio-Rad CFX96 real-time quantitative PCR instrument using the SuperReal PreMix Plus (TIANGEN) kit, following the two-step qRT-PCR reaction procedure as per the manufacturer's instructions. The expression level of the BnACO1 gene in the transgenic material and the Zhongshuang 11 material was analyzed. qRT-PCR results showed that the expression level of the BnACO1 gene in the transgenic material was significantly higher than that in the control group (Figure 7). Both materials were cultured under the same conditions, and plant height differences were observed and photographed at the seedling, flowering, and maturity stages. Genetic transformation results showed that the height of the 35S-driven BnACO1 overexpression transgenic plants was significantly reduced, consistent with the expected phenotype (Figure 8). The transgenic plant height decreased from 172 cm to 37 cm, a reduction of 80%, while stem diameter increased from 22.7 cm to 27.6 cm; the number of siliques decreased from 290 to 94; the number of siliques decreased from 31 to 12; and the flowering period was delayed by 5-7 days.

[0066] The embodiments described above are merely preferred embodiments provided to fully illustrate the present invention. It should be noted that any equivalent substitutions or improvements made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

[0067] sequence list

[0068] SEQ ID NO.1

[0069] SEQ ID NO.2

[0070] SEQ ID NO.3

Claims

1. A Brassica napus BnACO1 protein, characterized in that, The Brassica napus BnACO1 protein comprises any of the following amino acid sequences (1) and (2): (1) The amino acid sequence as shown in SEQ ID NO.3; (2) A sequence that has more than 90% homology with the amino acid sequence shown in SEQ ID NO.

3.

2. A BnACO1 gene in Brassica napus, characterized in that, Encoding the Brassica napus BnACO1 protein of claim 1, wherein the full-length genomic sequence of the Brassica napus BnACO1 gene is any one of the following nucleotide sequences (1) and (2): (1) The nucleotide sequence as shown in SEQ ID NO.1; (2) A DNA molecule whose nucleotide sequence is more than 90% homologous to the sequence shown in SEQ ID NO.1 and encodes the amino acid sequence shown in SEQ ID NO.

3.

3. The application of the protein of claim 1 or the gene of claim 2 in regulating the plant height or flowering period of Brassica napus.

4. A method for regulating the plant height of Brassica napus, characterized in that, A BnACO1 overexpression vector was constructed to obtain dwarf Brassica napus plants; the full-length genome sequence of BnACO1 is shown in SEQ ID No. 1; the full-length cDNA sequence of BnACO1 is shown in SEQ ID No.

2.

5. A method for regulating the flowering period of Brassica napus, characterized in that, A BnACO1 overexpression vector was constructed to obtain late-flowering rapeseed plants of the Brassica napus type; the full-length genome sequence of BnACO1 is shown in SEQ ID No. 1; the full-length cDNA sequence of BnACO1 is shown in SEQ ID No.

2.

6. The method according to claim 4 or 5, characterized in that, The BnACO1 overexpression vector was obtained by inserting the sequence shown in SEQ ID No. 2 between the CaMV35S promoter and the OCS terminator of the PBI121 vector.

7. The method according to claim 6, characterized in that, The procedure includes the following steps: inserting the sequence shown in SEQ ID No. 2 between the CaMV35S promoter and the OCS terminator of the PBI121 vector to obtain an overexpression vector, then transforming Agrobacterium tumefaciens GV3101, transforming the obtained transgenic engineered bacteria into the Brassica napus recipient, and screening the progeny of the transformed plants.

8. The method according to claim 6, characterized in that, New primers were constructed by introducing homologous sequences of two enzymes, Kpn I and BamHI, at both ends of the upstream and downstream primers of the gene clone, respectively. Upstream primer P7: 5'-CGCGGATCCATGGAGAATCACACGAAA-3'; Downstream primer P8: 5'-ACGCGTCGACTCATATTTCTTGATTGTAAATG-3'.

9. The method according to claim 8, characterized in that, The detection primers are: Upstream primer P9: 7'-GAGGCTTACGCAGCAGGTCTCA-3'; Downstream primer P10: 5'-TGAGGCAATACATCAGAATCAG-3'.

10. The method according to claim 8, characterized in that, The primer sequences for real-time quantitative PCR analysis are: Upstream primer P11: 5'-ATTACTGGCGTTAGCACTT-3'; Downstream primer P12: 5'-TATTCCTTTCGACGGATC-3'.

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