A tea tree transcription factor CsATH1 and its application in improving tea quality

Abstract

The invention discloses a tea tree transcription factor CsATH1 in the field of gene regulation technology and an application thereof to improve tea quality. The nucleotide sequence of the transcription factor CsATH1 is derived from the CsTGY14G0000594 sequence of the tea tree genome. The invention analyzes the tea tree genome sequence, adopts specific primers to amplify CsTGY14G0000594, and successfully clones its coding sequence. CsATH1 has a biological function similar to that of Arabidopsis thaliana ATH1, and can inhibit the flowering process by regulating a gene network related to flowering.

Description

Technical Field

[0001] The present invention relates to the technical field of gene regulation, and in particular to a tea tree transcription factor CsATH1 and an application thereof in improving tea quality. Background Art

[0002] Tea tree (Camellia sinensis) is one of the important economic crops in the world, and its leaves are the main raw material for making tea. The quality of tea is determined by its chemical composition and sensory properties, which are regulated by both genetics and the environment. In the study of tea quality improvement, tea transcription factors (TFs) have become a research field that has attracted much attention due to their importance in regulating gene expression. In recent years, studies have found that a tea transcription factor CsATH1 plays an important role in regulating the expression of genes related to tea quality, providing new ideas and technical directions for the molecular improvement of tea quality; the quality of tea is closely related to its secondary metabolites, including polyphenols, caffeine, amino acids (especially theanine), and aromatic substances. Among them, the ratio of tea polyphenols (such as catechins) to theanine has a key influence on the taste, aroma, and nutritional value of tea. In addition, alkaloids (such as caffeine) and aromatic compounds in tea also directly affect consumers' sensory experience. These secondary metabolites of tea plants are encoded by a series of specific genes and are regulated at multiple levels, including the participation of transcription factors; transcription factors are a class of proteins in plants that can bind to specific DNA sequences and regulate gene expression, and are involved in a variety of biological processes such as plant growth and development, secondary metabolism, and stress response. In tea plants, multiple transcription factors such as MYB, bHLH, NAC, and AP2 / ERF have been identified as key factors regulating the synthesis of tea polyphenols, caffeine, and aromatic substances.

[0003] As a perennial woody plant, the vegetative growth and reproductive growth of tea trees are carried out alternately. Tea trees have the characteristics of frequent flowering and long flowering period. The reproductive growth of tea trees absorbs a large amount of nutrients, which inhibits the vegetative growth of tea trees and affects the yield and quality of tea leaves. Extensive flowering will lead to changes in the content of sugar substances, delaying the germination of new shoots in the following year. The types and proportions of amino acids in tea flowers and leaves are relatively close. To a certain extent, extensive flowering will affect the amino acid content of tea leaves, and then affect the quality of tea leaves. Studies have shown that flower picking can effectively increase the yield and quality of spring tea. Reducing the differentiation of flower buds and the consumption of nutrients by flowers and fruits can improve the utilization rate of water and fertilizer by tea trees, thereby increasing tea yield. Therefore, reducing the nutrient consumption caused by the flowering of tea trees and accumulating more energy for the germination of new shoots and leaf buds in spring is a new idea to increase the yield and quality of spring tea in a green, safe and efficient way. Summary of the invention

[0004] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a tea transcription factor CsATH1 and its application in improving tea quality. By analyzing the genome sequence of the tea tree, specific primers were used to amplify CsTGY14G0000594, and its coding sequence was successfully cloned. CsATH1 has biological functions similar to Arabidopsis ATH1, and can inhibit the flowering process by regulating the gene network related to flowering.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The present invention proposes a tea plant transcription factor CsATH1, the nucleotide sequence of the transcription factor CsATH1 is shown in SEQ ID NO: 1;

[0006] Preferably, the nucleotide sequence of the transcription factor CsATH1 is derived from the CsTGY14G0000594 sequence of the tea plant genome;

[0007] Preferably, the nucleotide sequence of the transcription factor CsATH1 is derived from the coding sequence of the CsTGY14G0000594 sequence of the tea plant genome, the upstream sequence of the primer sequence for cloning the nucleotide sequence of the transcription factor CsATH1 is shown as SEQ ID NO: 2, and the downstream sequence of the primer sequence for cloning the nucleotide sequence of the transcription factor CsATH1 is shown as SEQ ID NO. 3;

[0008] The present invention also provides an application of tea tree transcription factor CsATH1 in improving tea quality of tea trees;

[0009] Preferably, the application of the tea tree transcription factor CsATH1 in improving the quality of tea leaves from tea trees includes delaying the flowering of tea trees;

[0010] Delaying the flowering of tea trees can effectively delay the reproductive growth in the growth cycle of tea trees, avoid the content of sugars and amino acids caused by the flowering of tea trees, and thus increase the yield and quality of tea.

[0011] The beneficial effects achieved by the present invention are as follows:

[0012] The present invention provides an application of tea tree transcription factor CsATH1 in improving tea quality of tea leaves. By analyzing the tea tree genome sequence, CsTGY14G0000594 is amplified using specific primers, and its coding sequence is successfully cloned. After comparison with the Arabidopsis genome, it is found that it has a high homology with the Arabidopsis ATH1 gene, so the gene is named CsATH1. In the Arabidopsis model plant, through the transformation of the 35S:CsATH1:GFP recombinant vector, the experiment shows that the overexpression of CsATH1 leads to a significant delay in the flowering time of Arabidopsis plants. Compared with the control group, the delayed flowering of the transgenic plants shows that CsATH1 has a biological function similar to that of Arabidopsis ATH1, and can inhibit the flowering process by regulating the gene network related to flowering. As an important flowering regulatory factor, CsATH1 can significantly affect the flowering time of plants. The flowering time of tea trees directly affects their growth cycle and economic traits. The functional validation results of CsATH1 showed that the gene can delay flowering, which provides a potential molecular breeding tool for tea cultivation. By regulating the expression of CsATH1, the vegetative growth stage of tea trees can be extended, avoiding the impact of premature flowering on yield and quality, thereby improving the economic benefits of tea trees. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a diagram showing the comparison of the CsATH1 sequence of the present invention with Arabidopsis thaliana;

[0014] Figure 2 This is the result of gel electrophoresis of PCR identification of 35S: CsATH1: GFP overexpression transgenic positive plant seedlings;

[0015] Figure 3 This is the result diagram of the expression level of CsAHT1 in the leaves of Arabidopsis plants in Example 4;

[0016] Figure 4 The results of flowering time of Arabidopsis strains in each treatment are shown in Fig.

[0017] Figure 5 The flowering inhibition phenotypes of each treated line under the same conditions.

[0018] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0020] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0021] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials and test strains used in the following examples are purchased from commercial channels unless otherwise specified.

[0022] Example 1: Method for cloning the tea plant CsATH1 gene:

[0023] (1) Extraction of RNA from tea leaves:

[0024] Tea leaves were collected, and leaf RNA was extracted using the Tiangen RNA extraction kit (DP452). Young leaves of Yinghong No. 9 tea tree were placed in a mortar, and liquid nitrogen was added to grind them into powder. 100 mg of Yinghong No. 9 tea leaf powder was added with 600 μL lysis solution SG and 10 μL Proteinase K, and placed on a vortex to shake and mix. After leaving at room temperature for 5 minutes, centrifuged at 12000 rpm for 2 minutes, 500 μL of the supernatant was taken and added to the genomic DNA removal column, and centrifuged at 12000 rpm for 0.5 min. The filtrate was retained, 250 μL of anhydrous ethanol was added, mixed, and transferred to the RNase-Free adsorption column CR4, centrifuged at 12000 rpm for 0.5 min, the waste liquid was discarded, 700 μL of deproteinization solution RW3 was added, and 12000 rpm for 0.5 min, discard the waste liquid, and put the adsorption column back into the collection tube; add 500 μL of rinsing solution RW to the RNase-Free adsorption column CR4, let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 30-60 sec, discard the waste liquid, put the adsorption column back into the collection tube, repeat the steps of adding rinsing solution RW for washing once, let the RNase-Free adsorption column CR4 stand at room temperature for 2 min to completely dry the residual rinsing solution in the adsorption material, transfer the RNase-Free adsorption column CR4 into a new RNase-Free centrifuge tube, and drop 30-100 mL of RNase-Free ddH 2O, place at room temperature for 2 min, centrifuge at 12000 rpm for 2 min to obtain RNA solution;

[0025] (2) Synthesis of cDNA:

[0026] Takara reverse transcription kit RR047A was used to synthesize cDNA. According to the instructions of the reverse transcription kit, 1 μg of RNA in step (1) was added to 2 μL of 5×gDNA Eraser Buffer and 1 μL of gDNA Eraser. RNase-Free ddH 2 Prepare a 10 μL system in 42°C in a PCR instrument and incubate for 2 minutes. Remove gDNA, take out the PCR tube, and immediately place it in a 4°C environment. Add 4 μL of 5× PrimeScript Buffer II (for Real Time), 1 μL of RTPrimer Mix, and 1 μL of PrimeScriptRT Enzyme Mix to the tube. Add RNase-Free ddHO 2 O was prepared into a 10 μL system, mixed evenly, and placed in a PCR instrument for reverse transcription. The program was set as follows: 37°C / 15 min, 85°C / 5 s, and stored at 4°C to obtain cDNA;

[0027] (3) PCR amplification:

[0028] Using the cDNA in step (2) as a template, design specific primer sequences based on the target gene:

[0029] Upstream primer sequence: 5′-ATGACTGAGAATGATGCATTC-3′ (SEQ ID NO: 2);

[0030] Downstream primer sequence: 5′-CTTTTTGCAGTGTTACTTCATA-3′ (SEQ ID NO: 3);

[0031] Furthermore, specific primer sequences with sticky ends are designed according to the target gene:

[0032] Upstream sticky primer sequence: 5′-ggccgcccccttcaccATGACTGAGAATGATGCATTC-3′;

[0033] Downstream sticky primer sequence: 5′-cggcgcgcccacccttCTTTTTGCAGTGTTACTTCATA-3′;

[0034] The PCR amplification system includes:

[0035] Upstream sticky primer: 0.5 µL;

[0036] Downstream sticky primer: 0.5µL;

[0037] Template cDNA: 1µL;

[0038] 2X DNA polymerase: 10 µL;

[0039] dH 2 O:8µL:

[0040] Total system: 20µL;

[0041] The PCR amplification procedure includes:

[0042] 98℃ 30s;

[0043] 58℃ 1.5min;

[0044] 72℃ 5min;

[0045] Extension at 72°C for 10 min;

[0046] 30 cycles.

[0047] Example 2: Ligation, transformation and sequencing;

[0048] (4) Ligation: Use In-Flusion ligase to connect the linear target gene fragment with sticky ends and the linearized entry vector pENTR to obtain a circular entry vector containing the target gene. The ligation system includes:

[0049] 5×In-Fusion HD Enzyme Premix 2μL;

[0050] pENTR vector 2 μL;

[0051] PCR Fragment 2 μL;

[0052] Cloning Enhancer 1 μL;

[0053] dH 2 O 5 μL;

[0054] Total system 10 μL;

[0055] Positive clones were screened and sequenced. After sequence analysis, the nucleotide sequence was shown in SEQ ID NO: 1. The sequence was homologously compared with Arabidopsis thaliana. The comparison results were as follows: Figure 1 As shown, the sequence is homologous to Arabidopsis ATH1, and the sequence is named CsATH1. Figure 1 This is a diagram showing the comparison of the CsATH1 sequence of the present invention with Arabidopsis thaliana;

[0056] (5) Transformation: Add the circular entry vector prepared in step (4) to 100 µL of E. coli DH5α competent cells, place on ice for 30 min, in a 42°C water bath for 45 s, and immediately place back on ice for 2 min. Add 600 µL of blank LB liquid culture medium to the clean bench, place in a 37°C shaker for 1 h, collect the bacteria, centrifuge at 3000 g for 1 min, remove the supernatant in the clean bench, spread on LB solid culture medium containing the corresponding resistance, seal, and invert in a 37°C incubator for overnight culture.

[0057] (6) Sequencing: A single colony on the culture medium was picked up with a sterile pipette tip in a clean bench and inoculated into LB liquid culture medium. The culture was shaken at 37°C for 5 h, followed by PCR identification, screening of positive clones and sequencing. After sequence analysis, the nucleotide sequence was shown in SEQ ID NO: 1. The sequence was homologously compared with Arabidopsis thaliana. The comparison results were shown in Figure 1 As shown, the sequence is homologous to Arabidopsis ATH1, and the sequence is named CsATH1. Figure 1 This is a diagram showing the comparison of the CsATH1 sequence of the present invention with Arabidopsis thaliana.

[0058] Example 3: Gateway system construction of recombinant expression vector:

[0059] (7) Extract the recombinant plasmid, use the phage site recombination system attB*attP-attL*attR, use 35S as the promoter and GFP as the reporter gene, construct the vector 35S: CsATH1:GFP, perform secondary transformation of the recipient cells, screen and identify the transformants, and extract the plasmid;

[0060] (8) Add the plasmid prepared in step (7) to the competent GV3101 strain, place it in an electroporator, apply a high voltage electric field at both poles to create gaps between the bacterial cell wall and the cell membrane, and allow the plasmid molecules to enter the cell. After the electric shock, quickly add 600 µL of blank LB, place it in a 28°C shaker for 2 hours, collect the bacteria, centrifuge at 3000g for 1 minute, remove the supernatant solution in a clean bench, spread it on the LB solid culture medium containing the corresponding resistance, seal it, and invert it in a 28°C incubator for 3 days; pick a single colony, inoculate it into LB liquid culture medium, culture it in a 28°C shaker overnight, and then perform PCR identification;

[0061] Example 4: Functional study of CsAHT1 gene

[0062] (9) Infection experiment: Infection of Arabidopsis thaliana was carried out by immersing the flowering wild-type Arabidopsis thaliana buds into the infection solution (OD = 0.8) containing the 35S:CsATH1:GFP construct of Agrobacterium (GV3101) for 1 minute and then continuing to culture in the greenhouse at 22°C, 16 h light / 8 h dark;

[0063] (10) Screening of transgenic plants: BASTA herbicide (10% glufosinate, CB2471, Coolab) was diluted 1:500 and sprayed on one-week-old Arabidopsis seedlings to screen positive plants and conduct further PCR identification;

[0064] The DNA of the leaves of positive plants was extracted by CTAB method. The leaves were ground in liquid nitrogen, 600 μL of 2% CTAB and 100 μL of β-mercaptoethanol were added, and after mixing, they were placed in a 65°C water bath for 6 min, immediately transferred to ice water and allowed to stand for 2 min, centrifuged at 4°C and 12000 rpm for 2 min, the supernatant was taken, 400 μL of water-saturated phenol and 200 μL of chloroform were added, shaken for 5 min, and immediately transferred to ice water and allowed to stand for 2 min, centrifuged at 4°C and 12000 rpm for 5 min, the supernatant was taken, 600 μL of chloroform was added, shaken for 5 min, and immediately transferred to ice water and allowed to stand for 2 min. min, centrifuge at 4℃, 12000rpm for 5min; take the supernatant, add 400µL lithium chloride solution, 250µL anhydrous ethanol and 100µL sodium acetate solution, mix well, transfer to ice water immediately and let stand for 15min, centrifuge at 4℃, 12000rpm for 10min; remove the supernatant, add 1mL 75vol% ethanol to rinse for 2min, centrifuge at 4℃, 12000rpm for 5min; remove the supernatant, let stand on the clean bench, add 1mL ddH 2 O dissolves;

[0065] PCR identification system: Specific primers F and R were designed according to the CsAH1 gene sequence, and Arabidopsis DNA was used as a template for amplification;

[0066] Primer F: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3';

[0067] Primer R: 5′-CTTTTTGCAGTGTTAC TTCATA-3′;

[0068] The amplification reaction system is as follows:

[0069] Primer F 0.5 µL;

[0070] Primer R 0.5 µL;

[0071] DNA template 1 µL;

[0072] EvaGreen 2×qPCR MasterMix 5 µL;

[0073] dH 2 O 3 µL;

[0074] Total system 10µL;

[0075] The PCR reaction procedure is as follows:

[0076] 94°C for 5 min;

[0077] 94 °C for 30 s;

[0078] 56 °C for 30 s;

[0079] 72 °C for 20 s;

[0080] 72 °C for 7 min;

[0081] 30 cycles.

[0082] The results of agarose gel electrophoresis analysis were as follows: Figure 2 As shown, Figure 2 This is the result of gel electrophoresis of PCR identification of 35S:CsATH1:GFP overexpressing transgenic positive plants. The WT band is wild-type Arabidopsis, and bands 1-10 are positive plants. It can be seen that the wild-type band position does not show a corresponding band, while the positive plants show obvious bands, indicating that CsAHT1 can be transferred into Arabidopsis plants.

[0083] Example 4: Detection of CsAHT1 expression in positive Arabidopsis plants:

[0084] Positive Arabidopsis plant leaves and wild-type Arabidopsis plant leaves were collected, and the leaves of Arabidopsis plants in each group were extracted using the Tiangen RNA extraction kit (DP452). The RNA was reversely transcribed into cDNA using the TAKARA reverse transcription kit (RR047A), and qPCR primers were designed to detect the relative expression of CsATH1 in each positive plant by qPCR:

[0085] The qPCR primer sequences are as follows:

[0086] q-CsATH1-F: 5'-TCCAGCAGCTACGAAGTAAAG-3';

[0087] q-CsATH1-R: 5'-CGTCTTTAGGATACGGGTGAAG-3';

[0088] PCR amplification procedure:

[0089] 95℃, 30s;

[0090] 95℃, 10s;

[0091] 68℃, 10;

[0092] 40 cycles;

[0093] Figure 3 The result diagram of the expression level of CsAHT1 in the leaves of Arabidopsis plants in Example 4, wherein the expression level of wild-type Arabidopsis (WT) is relatively low, and strains CsATH1-2 and CsATH-18 with medium expression levels were selected for subsequent phenotypic observation experiments;

[0094] Example 5: Phenotypic observation of positive Arabidopsis plants:

[0095] The medium expression strain CsATH1-2, the high expression strain CsATH1-18 and the wild-type Arabidopsis strain were cultured at 22°C, 16 h light / 8 h dark. Figure 4 The results of flowering time of Arabidopsis thaliana strains in each treatment are shown in Figure 2. Figure 5 The flowering inhibition phenotype of each treatment line under the same conditions; as shown in the figure, the flowering time of the medium expression line CsATH1-2 and the high expression line CsATH1 is significantly prolonged, indicating that the CsATH1 transcription factor of the present invention can significantly regulate the flowering time of Arabidopsis thaliana, and prolong the plant's vegetative growth time by prolonging the flowering time.

[0096] While the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that many changes, modifications, substitutions and variations can be made to the embodiments without departing from the principles and spirit of the invention.

[0097] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. An application of tea tree transcription factor CsATH1 in delaying flowering of Arabidopsis thaliana, characterized in that: The nucleotide sequence of the transcription factor CsATH1 is shown in SEQ ID NO:

1.

2. The use of a tea tree transcription factor CsATH1 in delaying flowering of Arabidopsis thaliana according to claim 1, characterized in that: The nucleotide sequence of the transcription factor CsATH1 is derived from the CsTGY14G0000594 sequence of the tea plant genome.

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