Application of Tea Plant UGT708AC25 Gene

Abstract

The present invention discloses the application of the tea tree UGT708AC25 gene, belonging to the field of genetic engineering. The present invention provides the coding gene UGT708AC25 of UDP-glycosyltransferase in the tea tree, and its nucleotide sequence is as shown in SEQ ID NO.1. The present invention constructs an Arabidopsis thaliana line overexpressing the UGT708AC25 gene. Transgenic experiments prove that overexpressing the tea tree UGT708AC25 gene in Arabidopsis thaliana can significantly increase the plant height of Arabidopsis thaliana, and it can be applied to the directional trait genetic improvement of tea trees and the cultivation of new excellent germplasms. The present invention is of great significance for regulating the plant height of tea trees, promoting the popularization of mechanical tea picking, and promoting the upgrading and development of the tea industry.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and particularly to the application of the tea tree UGT708AC25 gene. Background Art

[0002] Tea production is a labor-intensive industry, and the picking of tea leaves requires a large amount of labor. With the continuous expansion of tea garden areas and the continuous increase in labor costs in China, the tea industry is facing a labor shortage. Mechanical picking is beneficial to the transformation and upgrading of the tea industry. However, currently commonly used large-scale mechanical picking and single-bud intelligent mechanical picking are restricted by the tree form of tea trees. Plant height is the key factor affecting the tree form and plant type of tea trees and is one of the important factors affecting mechanical picking. Therefore, it is urgent to establish a method for regulating the plant height of tea trees from the aspect of genetic engineering in order to facilitate the promotion of mechanical picking of tea leaves and promote the further development of the tea industry.

[0003] Glycosylation catalyzed by glycosyltransferases is an important modification that occurs in plants, which transfers an active sugar donor to a receptor aglycone to form a glycoside. Uridine diphosphate Glycosyltransferase (UGT) belongs to family 1 of the glycosyltransferase superfamily and plays an important role in regulating plant growth and development and stress responses. Tea tree UGT708AC25 is a UDP glycosyltransferase involved in the biosynthesis pathway of plant flavonoids. It has only been identified in previous studies and lacks relevant functional research, especially the research on the regulatory function between UDP glycosyltransferase and plant height. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of the tea tree UGT708AC25 gene to solve the problems existing in the above-mentioned prior art. Overexpressing the tea tree UGT708AC25 gene in plants can increase plant height.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] The present invention provides the application of the tea tree UGT708AC25 gene in regulating plant height, and the nucleotide sequence of the tea tree UGT708AC25 gene is shown as SEQ ID NO.1.

[0007] The present invention also provides the application of a recombinant vector containing the tea tree UGT708AC25 gene in regulating plant height, and the backbone vector of the recombinant vector includes the pK7FWG2.0 vector.

[0008] The present invention also provides the application of an engineering bacterium containing the recombinant vector in regulating plant height, and the starting bacterium of the engineering bacterium includes Agrobacterium.

[0009] The present invention also provides the application of the protein expressed by the tea tree UGT708AC25 gene in regulating the plant plant height, and the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0010] Preferably, the expression level of the tea tree UGT708AC25 gene is up-regulated to increase the plant plant height; the plants include tea trees and Arabidopsis thaliana.

[0011] The present invention also provides a method for increasing the plant plant height, including the step of up-regulating the expression level of the tea tree UGT708AC25 gene in the plant to make the plant plant height taller; the nucleotide sequence of the tea tree UGT708AC25 gene is shown in SEQ ID NO.1; the plants include tea trees and Arabidopsis thaliana.

[0012] Preferably, the method for up-regulating the expression level of the tea tree UGT708AC25 gene in the plant includes overexpressing the tea tree UGT708AC25 gene in the plant.

[0013] More preferably, the method for overexpressing the tea tree UGT708AC25 gene in the plant includes the following steps:

[0014] Construct an overexpression vector of the tea tree UGT708AC25 gene, transform the overexpression vector into Agrobacterium by heat shock method, and then infect the plant.

[0015] The present invention also provides a method for cultivating a transgenic plant with improved plant height, including the following steps:

[0016] Overexpress the tea tree UGT708AC25 gene in plant cells, then cultivate the plant cells, and regenerate plants using the plant cells to obtain the transgenic plant with improved plant height;

[0017] The nucleotide sequence of the tea tree UGT708AC25 gene is shown in SEQ ID NO.1.

[0018] Preferably, the plants include tea trees and Arabidopsis thaliana.

[0019] The present invention discloses the following technical effects:

[0020] The present invention for the first time constructs an Arabidopsis thaliana line overexpressing the tea tree UGT708AC25 gene. Through transgenic experiments, it is found that overexpressing the tea tree UGT708AC25 gene in Arabidopsis thaliana can significantly increase the plant height of Arabidopsis thaliana. The tea tree UGT708AC25 gene plays a positive regulatory role in increasing plant height and can be applied to the directional genetic improvement of tea trees and the cultivation of new excellent germplasms. The present invention is of great significance for regulating the plant height of tea trees, adjusting the tree posture of tea trees in tea gardens, promoting the popularization of mechanical tea picking, and promoting the transformation and upgrading of the tea industry. Brief Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0022] Figure 1 It is the vector map of the overexpression vector pK7FWG2.0 - UGT708AC25 of the UGT708AC25 gene;

[0023] Figure 2 It is the physical map of the overexpression line of the UGT708AC25 gene and the control line; Col: Untransformed wild - type Arabidopsis thaliana; Line1, Line2, Line3: Three Arabidopsis thaliana overexpression lines;

[0024] Figure 3 It is the statistical chart of the plant height of the overexpression line of the UGT708AC25 gene and the control line; Col: Untransformed wild - type Arabidopsis thaliana; Line1, Line2, Line3: Three Arabidopsis thaliana overexpression lines; The asterisk represents that there is a significant difference between the line and the control;

[0025] Figure 4 It is the statistical chart of the expression level of the UGT708AC25 gene in the overexpression line of the UGT708AC25 gene and the control line; Col: Untransformed wild - type Arabidopsis thaliana; Line1, Line2, Line3: Three Arabidopsis thaliana overexpression lines; The asterisk represents that there is a significant difference between the line and the control. Detailed Embodiments

[0026] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0027] It should be understood that the terms described in this invention are only for describing particular embodiments and are not intended to limit the invention. Additionally, for the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in this invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0028] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to those documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0029] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of this invention's specification, which are obvious to those skilled in the art. Other embodiments obtained from this invention's specification are obvious to those skilled in the art. This invention's specification and examples are merely exemplary.

[0030] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0031] Example 1 Obtaining the full-length sequence of the tea tree UGT708AC25 gene

[0032] 1. Extraction of total RNA from tea tree

[0033] Take the leaves of Shuchazao planted in the National Tea Germplasm Repository (Hangzhou), Xihu District, Hangzhou, Zhejiang Province, and extract RNA using the EASYspin Plus Polysaccharide and Polyphenol Complex Plant RNA Rapid Extraction Kit (Beijing Aidlab Biotechnologies Co., Ltd., RN53).

[0034] 2. Reverse transcription

[0035] Use the PrimeScript TM RT reagent Kit with gDNA Eraser (Perfect Real Time) kit (Takara, RR047Q) to reverse transcribe the extracted RNA into cDNA.

[0036] 3. PCR amplification

[0037] Using the tea plant cDNA obtained by reverse transcription as a template, primers for amplifying the entire UGT708AC25 gene were designed with Primer Premier 5, and specific primers UGT708AC25-F and UGT708AC25-R were obtained. The primer sequences are as follows:

[0038] UGT708AC25-F: 5’-ATGGGTGATCAAAAAATAGTTTCCG-3’ (SEQ ID NO.3),

[0039] UGT708AC25-R: 5’-TTAGGCAGTCTCTGCAAGCCTCAAC-3’ (SEQ ID NO.4).

[0040] Using the tea plant cDNA as a template, the UGT708AC25 fragment was amplified by PCR with KOD Plus-Neo high-fidelity enzyme.

[0041] PCR amplification reaction system: 32 μL ddH2O, 5 μL 10x PCR Buffer for KOD Plus-Neo, 5 μL 2 mM dNTPs, 3 μL 25 mM MgSO4, 1 μL KOD Plus-Neo high-fidelity enzyme, 1 μL cDNA, 1.5 μL primer UGT708AC25-F, 1.5 μL primer UGT708AC25-R, a total of 50 μL.

[0042] PCR reaction program: pre-denaturation at 94°C for 2 minutes; denaturation at 98°C for 10 seconds, annealing at 54°C for 30 seconds, extension at 68°C for 50 seconds, for 40 cycles; finally, final extension at 68°C for 5 minutes, and the PCR product was stored at 4°C.

[0043] The obtained PCR product was identified by electrophoresis on a 1% agarose gel by volume fraction, and then the amplified band was recovered and purified with the GelExtraction Kit kit. The recovered product was constructed into the Zero Cloning Kit vector to obtain a recombinant plasmid, and then the above recombinant plasmid was sent to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing confirmation.

[0044] The nucleotide sequence of the obtained UGT708AC25 gene is shown as SEQ ID NO.1;

[0045] SEQ ID NO.1:

[0046]

[0047] The amino acid sequence of the protein expressed by the UGT708AC25 gene is shown in SEQ ID NO.2.

[0048] SEQ ID NO.2:

[0049] MGDQKIVSASPIMANADDFSGKSPHVAVLPGAGMGHLTPALRLAAMLSSRGCLVTLIAVKPTVSAAESTTISNFFSTYPHINRLDFEILPRKPPSSTNDDPFFIQFEAISRSVVHLLKPLLLSSSPPLSAIFSDFTVSMTLTPILEELCIPNYVLSVTCARFLSLLAYLPYLINENSPKFGESDDYVHIPGLSPLPLSTIPPPFFNPTLFFTSFAISNALFLPKAKGILVNTFERFEPDTLAALNNGRVLSNLPPVHPIGPLEPYKLEKGEQLSWLDDQSVKSVVYVCFGNRTAMSKDQIRELGDGLERSGCPFLWVLKSSKVDKEDTEEIEDLLGDSFLERIKNRGIVVKAWVNQEQVLAHPAIGGFVSHAGWNSVMEAARYGVPLLAWPPHGDQRVISEVVENAGFGVWVRDWGWGGERVVKGEEIGGKVKQLMSDEKLRNRAMEVEEEARKAFQDANGSSEKALMGIIETLRLAETA.

[0050] Example 2 Construction of the overexpression vector of UGT708AC25 gene

[0051] The vector used in the present invention: the initial vector is Zero Cloning Kit, then ligated to the pQB-V3 vector, and the final vector is pK7FWG2.0. Finally, the overexpression vector pK7FWG2.0-UGT708AC25 with the 35S promoter was constructed.

[0052] 1. Restriction enzyme digestion of pQB-V3 vector

[0053] Restriction enzyme digestion reaction system: 7 μL ddH2O, 2 μL 10x Buffer R, 9 μL pQB-V3 vector, 2 μL EcoRV restriction enzyme, a total of 20 μL; the restriction enzyme digestion reaction program is: 37°C for 2 hours, 80°C for 20 minutes, and stored at 4°C. Recover the fragment with a size of about 3.3 KB, which is the blunt-ended pQB-V3 cloning vector after restriction enzyme digestion.

[0054] 2. Ligation of the target fragment to the pQB-V3 vector

[0055] The target fragment (the recombinant plasmid obtained in Example 1) was ligated to the digested pQB-V3 vector using DNALigation Kit Ver.2.1 SolutionⅠ ligase.

[0056] Ligation reaction system: 1 μL of digested pQB-V3 vector, 4 μL of target fragment, 5 μL of DNALigation Kit Ver.2.1 SolutionⅠ ligase, for a total of 10 μL.

[0057] React at 16 °C for more than 3 hours, and then send the ligation product to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing confirmation.

[0058] 3. LR reaction

[0059] LR reaction system: 1 μL of pQB-V3-UGT708AC25 recombinant plasmid, 1 μL of pK7FWG2.0 vector, 2 μL of ddH2O, 1 μL of LR Mix enzyme (Thermo Fisher Scientific), for a total of 5 μL.

[0060] React at 25 °C for 2 hours. After the reaction is completed, transfer the ligation product into Escherichia coli DH5α competent cells. Use spectinomycin resistance (50 μg / mL) for screening by the coating method, and use colony PCR to screen out the pK7FWG2.0 recombinant plasmid with the target fragment (the band with a size of about 1500 bp is the pK7FWG2.0 recombinant plasmid), and sequence and identify it.

[0061] Analyze the sequencing results using DNAMAN software. The correctly transformed clone was named pK7FWG2.0-UGT708AC25, and the vector map is as Figure 1 shown.

[0062] Example 3: Construction and detection of transgenic Arabidopsis thaliana

[0063] The overexpression vector pK7FWG2.0-UGT708AC25 was transferred into Agrobacterium tumefaciens GV3101 competent cells by the conventional 42°C water bath heat shock method, and then spread on LB solid medium containing spectinomycin (50 μg / mL) and rifampicin (50 μg / mL). The cells were cultured in an incubator at 28°C for 2 - 3 days, shaken, and identified by sequencing. The strains with correct sequencing were used for the Arabidopsis inflorescence infection experiment by the conventional method, and the T1 generation seeds were harvested. The T1 generation seeds were sown on 1 / 2 MS medium containing kanamycin (50 μg / mL) to screen for positive transgenic plants. The screened positive plants were transplanted into soil to continue growing, and the T2 generation seeds were harvested. The above process was repeated continuously until homozygous positive transgenic Arabidopsis was screened. Finally, 3 overexpression lines were obtained and named Line1, Line2, and Line3.

[0064] Screening conditions: Plants were considered overexpressing if the segregation ratio of normal germination to non-germination of seeds on 1 / 2 MS medium containing kanamycin (50 μg / mL) was in line with 3:1, and the gene expression level in the stems of the plants after bolting (sampled, ground into powder in liquid nitrogen for detection) was significantly increased as detected by RT-qPCR.

[0065] The specific RT-qPCR method was to use the TB green (Takara, RR420Q) dye premix and perform standard operations according to its instruction manual.

[0066] Example 4: Study on the plant height of transgenic Arabidopsis

[0067] Wild-type Arabidopsis and transgenic Arabidopsis lines were planted in the same artificial climate chamber of the Tea Research Institute, Chinese Academy of Agricultural Sciences. Photos were taken and recorded at the 30th day of growth, as Figure 2 shown.

[0068] The above-ground parts of wild-type Arabidopsis and transgenic Arabidopsis plants were cut, and the plant height was measured using a ruler software. Significance analysis was performed by SPSS and significant differences were marked with asterisks, as Figure 3 shown.

[0069] At the 30th day of growth, the expression level of the UGT708AC25 gene in the stems of Col, Line1, Line2, and Line3 plants was detected by the same RT-qPCR method as in Example 3. The detection results are as Figure 4 shown.

[0070] Results obtained: Compared with wild-type Arabidopsis, the plant heights of the three Arabidopsis overexpression lines Line1, Line2, and Line3 of the UGT708AC25 gene were significantly increased, and the expression level of the UGT708AC25 gene in the stems was significantly increased.

[0071] Therefore, increasing the expression level of the UGT708AC25 gene can significantly increase the plant height, that is, overexpressing the UGT708AC25 gene can positively regulate the plant height. By regulating the expression level of the UGT708AC25 gene in tea plants, the plant height of tea plants can be adjusted, and then the tree shape of tea plants can be corrected, making most tea plants more suitable for mechanical picking, and promoting the development and upgrading transformation of the tea industry.

[0072] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of the tea plant UGT708AC25 gene in regulating plant plant height, characterized in that, The nucleotide sequence of the tea plant UGT708AC25 gene is shown in SEQ ID NO.1; The regulation of plant plant height is to overexpress the tea plant UGT708AC25 gene in the plant, making the plant height of the plant increase; The plant is Arabidopsis thaliana.

2. Use of the recombinant vector containing the tea plant UGT708AC25 gene described in claim 1 in regulating plant plant height, characterized in that, The backbone vector of the recombinant vector includes the pK7FWG2.0 vector; The regulation of plant plant height is to overexpress the tea plant UGT708AC25 gene in the plant, making the plant height of the plant increase; The plant is Arabidopsis thaliana.

3. Use of the engineered bacterium containing the recombinant vector described in claim 2 in regulating plant plant height, characterized in that, The starting bacterium of the engineered bacterium includes Agrobacterium; The regulation of plant plant height is to overexpress the tea plant UGT708AC25 gene in the plant, making the plant height of the plant increase; The plant is Arabidopsis thaliana.

4. Use of the protein expressed by the tea tree UGT708AC25 gene as described in claim 1 in regulating the plant plant height, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.2; The regulation of plant plant height is to overexpress the tea plant UGT708AC25 gene in the plant, up-regulate the expression level of the protein, and make the plant height of the plant increase; The plant is Arabidopsis thaliana.

5. A method for increasing plant height, characterized in that, It includes the step of overexpressing the tea plant UGT708AC25 gene in the plant to make the plant height of the plant increase; The nucleotide sequence of the tea plant UGT708AC25 gene is shown in SEQ ID NO.1; The plant is Arabidopsis thaliana.

6. The method according to claim 5, characterized in that, The method for overexpressing the tea plant UGT708AC25 gene in the plant includes the following steps: Construct an overexpression vector of the tea plant UGT708AC25 gene, transform the overexpression vector into Agrobacterium by heat shock method, and then infect the plant.

7. A method for cultivating a transgenic plant with improved plant height, characterized in that, It includes the following steps: Overexpress the tea plant UGT708AC25 gene in plant cells, then cultivate the plant cells, and regenerate plants using the plant cells to obtain the transgenic plant with improved plant height; The nucleotide sequence of the tea plant UGT708AC25 gene is shown in SEQ ID NO.1; The plant is Arabidopsis thaliana.

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