Application of transcription factor csdof2 in regulating catechin synthesis in camellia sinensis

Abstract

The application provides application of a transcription factor CsDof2 in regulation of catechin synthesis in a tea tree, and belongs to the technical field of plant molecular biology and genetic engineering.The application provides application of a transcription factor CsDof2 with an amino acid sequence as shown in SEQ ID NO.1 in regulation of catechin synthesis in a tea tree, and it is found that the expression amount of CsF3'5'H1 on a catechin synthesis pathway can be reduced by silencing the CsDof2 gene, and then the content of catechin is reduced; the expression amount of CsF3'5'H1 on the catechin synthesis pathway can be up-regulated by overexpressing the CsDof2 gene, and then the content of catechin is increased.

Description

Technical Field

[0001] This invention relates to the fields of plant molecular biotechnology and genetic engineering, and in particular to the application of transcription factor CsDof2 in regulating the synthesis of catechins in tea plants. Background Technology

[0002] tea tree[ Camellia sinensis ( L .) O. Kuntze ] Belongs to the Camellia family ( Theaceae Camellia genus ( Camellia L. Tea leaves are shade-loving, moisture-loving, and warm-loving plants that are sensitive to cold. Tea contains abundant polyphenols, vitamins, amino acids, and other components, possessing various medicinal functions such as antibacterial, antioxidant, blood circulation-promoting, cholesterol-lowering, and anti-cancer properties. Flavonoids participate in the formation and development of flowers, fruits, and seeds in plants, as well as other functions such as antioxidant activity, ultraviolet protection, and resistance to biotic and abiotic attacks from plant pathogens. Currently, the most studied flavonoids include rutin, catechins, quercetin, taxanein, and citrus flavonoids. Therefore, identifying transcription factors involved in the regulation of flavonoid synthesis has significant biological and industrial implications.

[0003] The flavonoid synthesis pathway involves multiple enzymes, but the enzyme involved in the B-ring hydroxylation of flavonoids is tea phenylalanine ammonia-lyase (PAL). The PAL gene catalyzes the direct removal of ammonia from L-phenylalanine to produce trans-cinnamic acid. The expression level of this gene is regulated by transcription factors. Transcription factors are a class of trans-acting factors that typically bind to cis-acting elements on the promoters of target genes, regulating their expression levels. Currently, research has found that MYB-type, bHLH-type, WD40-type, and WRKY-type transcription factors are mainly involved in the regulation of flavonoid synthesis, while literature on the regulation of tea catechin synthesis by members of the Dof gene family is relatively rare. Summary of the Invention

[0004] The purpose of this invention is to provide an application of transcription factor CsDof2 in regulating the synthesis of catechins in tea plants, through overexpression... CsDof2 Genes can increase the catechin content in tea, thereby improving the quality of the tea.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides an application of transcription factor CsDof2 in regulating the synthesis of catechins in tea trees, wherein the amino acid sequence of transcription factor CsDof2 is shown in SEQ ID NO.1.

[0007] Preferably, the transcription factor CsDof2 CsDof2The nucleotide sequence of the gene is shown as SEQ ID NO. 2.

[0008] Preferably, the gene is silenced CsDof2 The gene can reduce the content of catechin, and the overexpression CsDof2 The gene can increase the content of catechin.

[0009] Preferably, the gene is amplified CsDof2 The specific primer pair of the gene is shown as SEQ ID NO. 3 and SEQ ID NO. 4.

[0010] Preferably, the gene is silenced CsDof2 The silencing vector of the gene is pTRV2-CsDof2.

[0011] Preferably, the gene is overexpressed CsDof2 The overexpression vector of the gene is 35s-pCAMBIA2301.

[0012] The present application can reduce the expression of CsF3'5'H1 in the catechin synthesis pathway by silencing CsDof2 The expression of the gene, thereby reducing the content of catechin; by overexpressing CsDof2 The gene can significantly up-regulate the expression of CsF3'5'H1 in the catechin synthesis pathway, thereby increasing the content of catechin; the present application finds CsDof2 The gene can positively regulate the synthesis of catechin and increase the content of catechin in tea. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 The subcellular localization result of the gene in Example 1 is shown in the figure, and the scale is 20 μm; CsDof2

[0014] The total catechin content, TRIC (trihydroxy catechin) content and catechin (EGCG) content after silencing of the gene in Example 2 are shown in the figure, and the scale is 20 μm; Figure 2 The relative expression level of the gene and CsDof2 The relative expression level of the gene is shown in the figure; CsDof2 CsF3'5'H1 The total catechin content, TRIC (trihydroxy catechin) content and catechin (EGCG) content after overexpression of the gene in Example 3 are shown in the figure, and the scale is 20 μm;

[0015] The relative expression level of the gene and Figure 3 The relative expression level of the gene is shown in the figure; CsDof2 CsDof2 CsF3'5'H1

[0016] Figure 4 A is the fluorescence detection result in Example 4, and B is the average fluorescence intensity statistical result in Example 4. DETAILED DESCRIPTION​​​

[0017] The technical solutions provided by the application will be described in detail below in combination with the embodiments, but they should not be understood as limiting the scope of protection of the application.

[0018] The amino acid sequence of the transcription factor CsDof2 is shown in SEQ ID NO. 1 (CSS0048616):

[0019] MSEAKDPAIKLFGKTIPLPDIPPPPSAGPPPLLSDSESLNLDRSSSPEDDKSSRVGEDHYPDNKVDGARPIPPEEIADPTTVVNENPKTPSVDNEAATLKASKTEEEQSETSNSQEKTLKKPDKILPCPRCNSMDTKFCYYNNYNVNQPRHFCKNCQRYWTAGGTMRNVPVGAGRRKNKNSASHYRHIAVSEALQNARADLPNGIHHSTLKPNGTVLTFGSSDTPLCESMASVLNIAEKTMRNCAQTGFRKPEKLIIPVSYVGGENGDDHSSGSSVTAANSKDDVGSKTGLQDPVMQNCHGFPPQVPCFPGTPWPYPWNSAQWSGPVPVPAFCPPGFPMPFYPPPYWGCTIPGTWNVPWVTPPSSSPNHIAPSSGPNSPTLGKHSRDENMLKQTKSGEEDPQRENNPERSLWIPKTLRIDDPGEAAKSSIWATLGIKNDKVDSVSGGGLFKPFQPKGDDKSHVSETSPVLQANPAALSRSLDFHESS

[0020] The nucleotide sequence of the gene encoding the transcription factor CsDof2 is shown in SEQ ID NO. 2: CsDof2

[0021] ​

[0022] Specific primer SEQ ID NO.3:

[0023] TTTTCACTGTCTATAACTTTTTCAACC

[0024] Specific primer SEQ ID NO.4:

[0025] CTTGACTTCCATGAAAGCTCATAA

[0026] Example 1 CsDof2 Subcellular localization of genes

[0027] according to CsDof2 Gene-specific primers were designed, and amplification PCR without stop codons was performed using high-fidelity enzymes with the specific primers (SEQ ID NO.3 and SEQ ID NO.4) according to the primer annealing temperature. CsDof2 The coding sequence was amplified and then digested with Xba I and Kpn I enzymes and inserted into the green fluorescent protein (GFP) fusion expression vector PCAMBIA1300 to obtain the fusion expression vector CsDof2-PCAMBIA1300-35S-GFP. The empty vector was used as a positive control. Transformation was performed via Agrobacterium-mediated transformation, using the following method: ① Place competent Agrobacterium on ice, add 1g of plasmid DNA (volume should not exceed 10 L), mix thoroughly, and incubate on ice for 30 min; ② Rapidly cool in liquid nitrogen for about 1 min, then quickly transfer to a 37℃ water bath to thaw; ③ Add 1 mL of antibiotic-free YM liquid culture medium and incubate at 28℃, 230 r / min for 4 h (or directly use 1 mL, leaving 500 L in the tube); ④ Centrifuge at 3000 r / min for 2 min to collect the bacterial cells, aspirate the supernatant, and directly spread the cultured cells onto a YM plate containing antibiotics; ⑤ Resuspend the bacterial cells and spread them onto a YM plate containing appropriate antibiotics, then air-dry and incubate at 28℃ for 48 h; ⑥ Pick a single colony and inoculate it into screening liquid YM medium, incubate at 28℃, 250 r / min for 48 h, and use the bacterial solution for preservation or transformation; ③ Inject the fusion expression vector into tobacco leaf epidermal cells for transient expression. After 24 hours of immersion, the fluorescence signal was observed using a confocal microscope, and the results showed that... CsDof2 Subcellular genes are located on the cell nucleus, such as Figure 1 As shown.

[0028] Example 2 CsDof2 VIGS validation of gene silencing

[0029] The amplified CsDof2The gene sequence was loaded into the pTRV2 vector to construct the pTRV2-CsDof2 vector. Using the empty pTRV2 vector as a negative control, the transformation of tea plants to silence gene expression was performed. Tea leaves were sampled on day 32 for preservation, and the expression levels of related genes were measured. Results are as follows: Figure 2 As shown, the total catechin content, Tric (trihydroxycatechin) content, and catechin content all decreased significantly. CsDof2 Gene expression levels also decreased significantly. CsDof2 The catechin synthesis pathway after gene silencing CsF3'5'H1 The expression level of [something] decreased significantly, indicating that... CsDof2 Genes may be positively regulated CsF3'5'H1 The expression of these substances regulates the synthesis and accumulation of catechins in tea leaves.

[0030] Example 3 CsDof2 Gene transient overexpression verification

[0031] Transformed CsDof2 Agrobacterium gene expression was cultured in liquid YPE medium and then resuspended in an Agrobacterium suspension containing 10 mM MES, 10 mM MgCl2, and 150 μM AS until the OD value reached 1.0. As a control, Agrobacterium containing the pCAMBIA2301 vector with the 35S promoter was injected into the interior of the 4th or 5th leaf (through the abaxial surface). Each experiment was repeated three times, with each replicate including six leaves. Samples were collected three days after injection to obtain three groups of transient overexpression. CsDof2 Tea tree leaves containing genes. The expression levels of relevant genes were measured, and the results are as follows: Figure 3 As shown in the results, transient overexpression of tea leaves increased the total catechin content, increased Tric (trihydroxycatechin) content, significantly increased catechin content, and significantly upregulated Dof2 expression, indicating an increase in the catechin synthesis pathway. CsF3'5'H1 The expression level was significantly upregulated, further proving that... CsDof2 Through positive regulation CsF3'5'H1 The expression of these substances regulates the synthesis and accumulation of catechins in tea leaves.

[0032] Example 4 Dual-luciferase verification

[0033] Using the dual reporter vector 0800-LUC+35S-62-SK containing the pGreenll 62-SK vector and the internal control pGreenll 0800-LUC vector, the... CsDof2 The CDS sequence was inserted into the 35S-62-SK vector, CsDof2 and CsF3'5'H1Promoters of the genes were cloned into 0800-LUC vectors, respectively. All recombinant plasmids were transformed into Agrobacterium tumefaciens GV3101 strain, respectively, and then transiently expressed in GV3101-infected leaf cells by agroinfiltration. After 4 days, the infected leaves were sprayed with 0.2 mg·mL -1 of D-luciferin sodium salt. Fluorescent images were created and the fluorescent values were calculated using a chemiluminescence device named Fusion FX7 (VILBER, France), and the results are shown in Figure 4 It was found that CsDof2 directly binding CsF3'5'H1 enhanced the expression of the promoters, thereby increasing the content of catechins.

[0034] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Application of a transcription factor CsDof2 in regulating catechin synthesis in tea plants, characterized in that, The amino acid sequence of the transcription factor CsDof2 is shown as SEQ ID NO.

1.

2. Use according to claim 1, wherein The nucleotide sequence of the gene of the transcription factor CsDof2 is shown as SEQ ID NO.

2. CsDof2 The nucleotide sequence of the gene of the transcription factor CsDof2 is shown as SEQ ID NO.

2.

3. The use according to claim 1, wherein Silencing CsDof2 The gene can reduce the content of catechin, and overexpression of CsDof2 gene can increase the content of catechin.

4. The use according to claim 3, wherein the compound is ###0002### Specific primer pairs for amplifying CsDof2 the gene are shown as SEQ ID NO. 3 and SEQ ID NO.

4.

5. The use according to claim 3, wherein the compound is ###0002### Silencing CsDof2 The silencing vector for the gene is: pTRV2-CsDof2.

6. The use according to claim 3, wherein the compound is ###0002### The overexpression CsDof2 CsDof2 The overexpression vector of the gene is: 35s-pCAMBIA2301.

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