Application of CsDOF3 gene in regulation and control of theanine synthesis

Abstract

The invention provides application of a CsDOF3 gene in regulation and control of theanine synthesis, and belongs to the technical field of gene engineering. The nucleotide sequence of the CsDOF3 gene is as shown in SEQ ID NO. 1. The invention finds that the CsDOF3 gene can negatively regulate and control the synthesis of theanine through a transient silence technology (VIGS). The method disclosed by the invention has important significance in regulating and controlling synthesis of theanine in the existing tea tree variety, and lays a theoretical foundation for cultivating a new tea tree variety with high theanine yield.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to the application of CsDOF3 gene in regulating theanine synthesis. Background Art

[0002] Theanine is a unique biochemical component in tea plants. It is an important secondary metabolite in tea plants along with tea polyphenols, catechins and caffeine, and is also an important criterion for judging the quality of tea. The diversity and complexity of tea plant secondary metabolites not only determine the special flavor of tea, but also have many benefits for human health. Theanine gives tea its umami taste and has a positive impact on human neuroprotection, liver protection, cardiovascular disease and immune regulation.

[0003] Gene expression regulation is one of the central topics in modern molecular biology research and one of the hot topics in current tea research. At present, the regulatory role of various transcription factors in material metabolism has been confirmed, but for the biosynthesis of theanine, there are few reports on the regulatory mechanism of the interaction between promoters and transcription factors at the gene level, which causes the activation or inhibition of gene transcription effects.

[0004] Transcription factors play an important role in plant growth and development. DNA-binding zinc finger (Dof) proteins are a subfamily of the zinc finger protein family, which is plant-specific and has not yet been found in yeast or nematodes. Dof proteins are composed of a conserved N-terminal single zinc finger DNA binding domain (Dof domain) and a C-terminal domain. Studies have found that the length of Dof protein is about 200 to 400 amino acids, and its domain consists of 50 to 52 amino acids. It has a classic four-cysteine ​​zinc finger that specifically binds to the core sequence (A / T)AAAG of the target gene promoter.

[0005] The inventors discovered that the CsDOF3 gene has the function of regulating theanine biosynthesis. This discovery is of great importance for enriching the mechanism of theanine regulation and further utilizing this mechanism to regulate the synthesis of theanine in existing tea varieties. It also lays the foundation for enriching the theory of theanine metabolism regulation in tea trees and cultivating new high-quality tea varieties. Summary of the invention

[0006] The purpose of the present invention is to provide an application of the CsDOF3 gene in regulating theanine synthesis, so as to lay a foundation for the tea tree theanine metabolism regulation theory and the cultivation of high-quality new tea tree varieties.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides application of CsDOF3 gene in regulating theanine synthesis, and the nucleotide sequence of the CsDOF3 gene is shown as SEQ ID NO.1.

[0009] SEQ ID NO.1: ATGCAAGACCCATCAACGTATCAACAGATCCAACC CCAATTCCCCGAACACGAGCACTTGAAATGCCCAAGATGTGATTCAACAAACACCAAATTTTGCTACTACAACAACTACAATCTCTCTCAGCCTCGCCACTTCTGCAAGAACTGCCGGAGATACTGGACCAAAGGCGGAGCTCTTCGCAACATCCCAATCGGCGGTGGGAGTCGCAAGAACACGAAGCGAAATTCGAACCCTAAACGCTCTTCATCGTCGTCTTCTTCGTCCTCCACCACCACAGCAACAACCACCCAAGTCGCCGGAAAACCAGAACCCTCCTCTGCCATGTACGGTTTCGCTGCGGCGGCGTTCGATCAGGAGCAGCATCGGATGATGGATATCAATGGGAGTTTTAGCTCGCTTCTGGCTTCGAGTGGGCAGTTTGGGAATTTGATGGAGGGTTTGAATCCAAATGGATCGACGGTTCGATTGGGGGAGTTTGGCGAGAATAATTTGAATTTGAATTCGAATTTGGAGGCGAATTTGATGAGAAACTCACAAGTTGAGTTACAGAGTAGTAATAATTCAGAGAGCTTTTTGGGTGTTCAGAGTGGTGGTGGTGGTGGTGGTGGTGATTGGGGTGGTGGCAATGGGTGGCCCGATCTTGCTATTTACACACCAGGTTCGACTTTTCAGTAA

[0010] Preferably, the amino acid sequence of the protein encoded by the CsDOF3 gene is as shown in SEQ ID NO.2.

[0011] SEQ ID NO.2:MQDPSTYQQIQPQFPEHEHLKCPRCDSTNTKFCYYN NYNLSQPRHFCKNCRRYWTKGGALRNIPIGGGSRKNTKRNSNPKRSSSSSSSTTTATTTQVAGKPEPSSAMYGFAAAAFDQEQHRMMDINGSFSSLLASSGQFGNLMEGLNPNGSTVRLGEFGENNLNLNSNLEANLMRNSQVELQSSNNSESFLGVQSGGGGGGGDWGGGNGWPDLAIYTPGSTFQ

[0012] Preferably, the application is to silence or knock out the CsDOF3 gene in tea plants, thereby increasing the synthesis of theanine.

[0013] The present invention also provides a method for increasing theanine synthesis in tea trees, comprising recombining the non-translated region of the CsDOF3 gene with a linear plasmid vector after enzyme cutting to obtain a VIGS silencing vector of the CsDOF3 gene; transferring the VIGS silencing vector of the CsDOF3 gene into Agrobacterium, and infecting tea trees with the obtained Agrobacterium to obtain gene-silenced tea trees with increased theanine synthesis.

[0014] Preferably, the nucleotide sequence of the untranslated region of the CsDOF3 gene is shown as SEQ ID NO.3.

[0015] SEQ ID NO.3:AAATGAAATACGATGAGCGTTTGAAGAATTTGATTT TGGCCGTTCGCTACAGGGTCTTTCGAGAAATTTTATGCTAGACTCTGGTTTTGCACAATAAAGTTATGGAAGCATTTTGGTTGCTGGAGATTCAGAAGATGCACACGTGATCCACGAGTGGTGAATTCATCTGTTTTGGGTCTATCAACG CTGTAAATAGAAGAATCTTGCGAGTTAAGTGGGTTGTTTTTGGTACTTGGCCTATTTCAGGTTTGTCTAATCCAGAATTTTGTAAATTGCAGTTCTTTTGCATAACAAGGTTTATTTTTTATTAAATCTTTAATGAATGCAATATTACA

[0016] Preferably, the plasmid vector is pTRV2, and the restriction sites are EcoRI and BamHI.

[0017] Preferably, the method for transferring the VIGS silencing vector of the CsDOF3 gene into Agrobacterium is the freeze-thaw method.

[0018] Preferably, the Agrobacterium is GV3101.

[0019] Preferably, the method of infecting the tea tree is injection or vacuum infection.

[0020] Preferably, the part of the tea tree that is infected is the tea tree leaves.

[0021] The present invention provides an application of the CsDOF3 gene in regulating theanine synthesis. Through transient silencing technology (VIGS), it is found that the CsDOF3 gene can negatively regulate theanine synthesis. The present invention is of great significance for regulating theanine synthesis in existing tea tree varieties, and lays a theoretical foundation for cultivating new tea tree varieties with high theanine production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0023] Figure 1 The subcellular localization result of CsDOF3 gene in Example 1, the scale bar in the figure is 20 μm;

[0024] Figure 2 is a schematic diagram of an overexpression vector containing the CsDOF3 gene in Example 2;

[0025] Figure 3 The results of measuring theanine content in leaves of plants transiently overexpressing the CsDOF3 gene and leaves of original plants in Example 2;

[0026] Figure 4 The results of measuring the expression levels of the CsDOF3 gene and its downstream TS1 gene in the leaves of the plant transiently overexpressing the CsDOF3 gene and the leaves of the original plant in Example 2;

[0027] Figure 5 Schematic diagram of the VIGS silencing vector of the CsDOF3 gene in Example 3;

[0028] Figure 6 The results of the determination of theanine content in leaves of plants with VIGS silenced CsDOF3 gene and leaves of original plants in Example 3;

[0029] Figure 7The expression level measurement results of the CsDOF3 gene and its downstream TS1 gene in the leaves of the plant with VIGS silenced CsDOF3 gene and the leaves of the original plant in Example 3;

[0030] Figure 8 These are the fluorescence intensity measurement results of different injection areas in Example 4. DETAILED DESCRIPTION

[0031] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0032] Example 1 Subcellular localization of CsDOF3 gene

[0033] The CsDOF3-GFP fusion expression vector was constructed using the homology arm method. CsDOF3-GFP, RFP and GFP were transferred into Agrobacterium GV3101 by freeze-thaw method and injected into 4-week-old tobacco leaves for transient expression. The fluorescent site was observed using a confocal microscope to determine the location of the CsDOF3 gene. Figure 1 As shown, 35S:GFP represents an empty vector, DOF3-GFP represents CsDOF3-GFP, and the red dots are nuclear markers.

[0034] Depend on Figure 1 It can be seen that the green fluorescence of the empty vector is distributed throughout the cell membrane, cell nucleus, cytoskeleton and other structures, indicating that the empty vector can be successfully expressed in tobacco cells; the CsDOF3 gene is located in the cell nucleus.

[0035] Example 2

[0036] The CsDOF3 gene was inserted between the Xba I and Kpn I restriction sites on the original vector pSH737-5S to obtain an overexpression vector containing the CsDOF3 gene. The schematic diagram of the overexpression vector containing the CsDOF3 gene is shown in FIG. Figure 2 The overexpression vector containing the CsDOF3 gene was transformed into Agrobacterium GV3101 by freeze-thaw method to prepare transgenic Agrobacterium.

[0037] The prepared transgenic Agrobacterium was cultured until the OD600 of the bacterial solution was 1.0-1.2, and the solution was centrifuged at 6000 rpm for 15 min. The obtained bacterial precipitate was resuspended with a resuspension solution (MS liquid culture medium) to an OD600 of 1.0 to obtain a bacterial solution for infecting tea leaves. The tea leaf infection bacterial solution was injected into the 4th and 5th leaves of tea tree cuttings (Camellia sinensis (L.) O. Kuntze) with a syringe, and the entire leaf was injected. The solution was cultured at 20°C in the dark for 3 days to obtain plants transiently overexpressing the CsDOF3 gene. The leaves of the plants transiently overexpressing the CsDOF3 gene were sampled and stored in a -80°C refrigerator.

[0038] The method specified in GB / T 23193-2017 was used to determine the content of theanine in the leaves of plants transiently overexpressing the CsDOF3 gene and the leaves of the original plants, and the expression levels of the CsDOF3 gene and its downstream TS1 gene (key gene for theanine synthesis) in the two leaves were determined at the same time. The primer sequences used to determine the expression levels of the CsDOF3 gene and its downstream TS1 gene are shown in Table 1.

[0039] Table 1 Primers used to quantify the tea plant gene CsDOF3

[0040] Name Sequence(5'-3') Actin-F CAGACCGTATGAGCAAGGAAAT SEQ ID NO.4 Actin-R GTGCTTAGGGATGCAAGGATAG SEQ ID NO.5 DOF3-F CTACAGCGTCTTCCTCTTCTACTTC SEQ ID NO.6 DOF3-R TACACTCTCGTTCAGATTCACATCA SEQ ID NO.7 TS1-F ATCAGTTGTGGATGGCTCG SEQ ID NO.8 TS1-R CACTTCGCATGGACTTGGTAC SEQ ID NO.9

[0041] The results of the determination of theanine content in the leaves of plants transiently overexpressing the CsDOF3 gene and the leaves of original plants are as follows: Figure 3 The expression levels of CsDOF3 gene and its downstream TS1 gene were measured as shown in Figure 4 As shown in Figure 2, it can be seen that after overexpression of the CsDOF3 gene, the expression level of the TS1 gene was significantly downregulated, and the synthesis of theanine in the leaves of the plant was reduced, indicating that overexpression of the CsDOF3 gene inhibited the synthesis of theanine in tea plants.

[0042] Example 3

[0043] The untranslated region (UTR region) of the CsDOF3 gene shown in SEQ ID NO.3 was inserted into the linear pTRV2 empty vector after digestion with EcoRI and BamHI to obtain the VIGS silencing vector of the CsDOF3 gene. The schematic diagram of the VIGS silencing vector of the CsDOF3 gene is shown in Figure 5 The VIGS silencing vector of CsDOF3 gene was transformed into Agrobacterium GV3101 by freeze-thaw method to prepare VIGS-silenced transgenic Agrobacterium.

[0044] The VIGS-silenced transgenic Agrobacterium and pTRV1 Agrobacterium were cultured at 28°C until the OD600 of the bacterial solution was 1.0-1.2, and centrifuged at 6000rpm for 15min. The obtained bacterial precipitate was resuspended with a resuspension solution (MS liquid culture medium) to an OD600 of about 0.7. The VIGS-silenced transgenic Agrobacterium resuspension and the pTRV1 Agrobacterium resuspension were mixed at a volume ratio of 1:1 to obtain a tea leaf infection bacterial solution; tea tree cuttings (Camellia sinensis (L.) O. Kuntze) were infected by vacuum infection and cultured at 25°C in the dark for 40d to obtain plants with VIGS-silenced CsDOF3 gene; leaves of plants with VIGS-silenced CsDOF3 gene were sampled and stored in a -80°C refrigerator.

[0045] The content of theanine in the leaves of the plants with VIGS silenced CsDOF3 gene and the leaves of the original plants was determined by the method specified in GB / T 23193-2017, and the expression levels of the CsDOF3 gene and its downstream TS1 gene (key gene for theanine synthesis) in the two leaves were determined at the same time. The primer sequences used to determine the expression levels of the CsDOF3 gene and its downstream TS1 gene are shown in Table 1 in Example 2.

[0046] The results of the determination of theanine content in the leaves of the plants with VIGS silencing of the CsDOF3 gene and the leaves of the original plants are as follows: Figure 6 The expression levels of CsDOF3 gene and its downstream TS1 gene were measured as shown in Figure 7 As shown, DOF3-1, DOF3-2, and DOF3-3 represent different strains of CsDOF3 gene treated with VIGS. It can be seen that when the CsDOF3 gene was silenced, the expression level of the CsDOF3 gene was significantly downregulated, the expression level of the TS1 gene was significantly upregulated, and the production of theanine increased significantly, proving that the CsDOF3 gene negatively regulates the synthesis of theanine.

[0047] Example 4

[0048] The purpose of this example is to study the specific regulatory mechanism of the CsDOF3 gene on its downstream TS1 gene. Using the dual fluorescein reporter system, Agrobacterium liquid containing different vectors was injected into the upper grain area of ​​tobacco leaves. After 48 hours of incubation under dark conditions, the fluorescence intensity was observed using a live imaging microscope, and the regulatory effect of the CsDOF3 gene on its downstream TS1 was determined by calculating the fluorescence value.

[0049] The results are as follows Figure 8As shown, Region1 to 4 represent 4 different injection regions; Region1: 0800 empty vector + 62-SK empty vector; Region2: 0800 empty vector + 62-SK-CsDOF3; Region3: 0800-TS1 promoter + 62-SK-empty vector; Region4: 0800-TS1 promoter + 62-SK-CsDOF3. Figure 8 It can be seen that the fluorescence value of Region 3 is significantly higher than that of Region 4, indicating that the CsDOF3 gene specifically binds to the promoter sequence of TS1 and inhibits the transcription of TS1.

[0050] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of CsDOF3 gene in regulating theanine synthesis, characterized in that: The nucleotide sequence of the CsDOF3 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The amino acid sequence of the protein encoded by the CsDOF3 gene is shown in SEQ ID NO.

2.

3. The use according to claim 2, characterized in that The application is to silence or knock out the CsDOF3 gene in tea plants, thereby increasing the synthesis of theanine.

4. A method for increasing theanine synthesis in tea plants, characterized in that: The non-translated region of the CsDOF3 gene is recombined with the linear plasmid vector after enzyme cutting to obtain the VIGS silencing vector of the CsDOF3 gene; the VIGS silencing vector of the CsDOF3 gene is transferred into Agrobacterium, and the obtained Agrobacterium is used to infect tea trees to obtain gene-silenced tea plants with increased theanine synthesis.

5. The method according to claim 4, characterized in that The nucleotide sequence of the untranslated region of the CsDOF3 gene is shown in SEQ ID NO.

3.

6. The method according to claim 5, characterized in that The plasmid vector is pTRV2, and the restriction sites are EcoRI and BamHI.

7. The method according to claim 6, characterized in that The method for transferring the VIGS silencing vector of CsDOF3 gene into Agrobacterium is freeze-thaw method.

8. The method according to claim 7, characterized in that The Agrobacterium is GV3101.

9. The method according to claim 8, characterized in that The methods of infecting tea trees are injection or vacuum infection.

10. The method according to any one of claims 4 to 9, characterized in that: The parts of the tea tree that are infected are the leaves.

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