Method for increasing content of catechins and theanine in tea leaves

Abstract

The invention discloses a method for increasing the content of catechins and theanine in tea leaves, the content of catechins and theanine in tea leaves is increased by knocking out or inhibiting the expression of CsLBD38a genes in tea tree root systems and tender shoot leaves, and the gene sequence table of the CsLBD38a genes is shown as SEQ ID NO.1. The invention further discloses a method for increasing the content of catechins and theanine in tea leaves. According to the invention, transgenosis of CsLBD38a is inhibited, a CsLBD38a function deletion mutant is screened, and CsLBD38a is reduced and inhibited so as to improve the influence of nitrogen on synthesis of characteristic secondary metabolites.

Description

Technical Field

[0001] The present invention relates to the field of tea, and in particular to a method for increasing the contents of catechins and theanine in tea. Background Art

[0002] Tea is the most popular and widely consumed non-alcoholic beverage after water. Tea is cultivated in approximately 60 countries and regions worldwide, serving approximately 2 billion people and generating a market value of approximately $60 billion. Nitrogen is the primary nutrient for tea plants and a significant factor in their growth, yield, flavor, and quality. However, tea plants have a low nitrogen utilization efficiency of only approximately 23%, resulting in high annual nitrogen fertilizer usage of around 400-700 kg per hectare per year. Excessive nitrogen fertilizer application not only increases tea cultivation costs, but also pollutes the agricultural ecosystem by causing unabsorbed nitrogen to flow into lakes and groundwater systems. Furthermore, high levels of nitrate nitrogen in tea can be converted into nitrite, increasing tea food safety risks. Therefore, with the increasing emphasis on reducing nitrogen use and increasing efficiency in agriculture, as well as the implementation of environmental protection and sustainable development initiatives, improving nitrogen use efficiency in tea cultivation has become a pressing goal for innovative cultivation techniques and practices. In recent years, high nitrogen use efficiency has also been a goal of tea breeding and germplasm innovation.

[0003] The content of characteristic secondary metabolites of tea trees is the most important indicator of the flavor and health-promoting quality of tea trees. High content of catechins such as EGCG, high theanine, and low caffeine are often the main indicators for evaluating tea quality because they determine the flavor quality and health-promoting function of tea.

[0004] Nitrogen fertilizer significantly impacts the synthesis and accumulation of these characteristic secondary metabolites in tea plants. Studies have shown that nitrogen deficiency stimulates the synthesis and accumulation of catechins in leaves, but inhibits the synthesis and accumulation of theanine and caffeine. However, nitrogen fertilizer application often inhibits catechin synthesis in tea leaves, while increasing theanine to a certain extent. Therefore, how to increase the content of theanine, flavonoids, and theanine has become an urgent issue. Summary of the Invention

[0005] The object of the present invention is to provide a method for increasing the content of catechins and theanine in tea leaves, which can increase the content of catechins and theanine in tea leaves.

[0006] In one aspect, the present invention provides a method for increasing the content of catechins and theanine in tea leaves. According to an embodiment of the present invention, the content of catechins and theanine in tea leaves is increased by knocking out or inhibiting the expression of the CsLBD38a gene in the roots, buds, and leaves of tea plants. The gene sequence of the CsLBD38a gene is shown in SEQ ID NO. 1.

[0007] Compared with the prior art, the present invention has the following beneficial effects:

[0008] 1) The CsLBD38a gene in this invention is an important nitrogen-responsive transcription factor. Its expression is suppressed by nitrogen deficiency but upregulated by nitrogen fertilizer feeding. It is also inhibited by salt and osmotic stress. It negatively regulates root growth and elongation in tea plants. The CsLBD38a gene is positively activated by NLP1 / 7 / 9, key regulators of plant nitrogen use efficiency. NLP1 / 7 / 9 are activated under conditions of low nitrogen and nitrogen deficiency, regulating a series of nitrogen-responsive target genes.

[0009] 2) The CsLBD38a gene serves as a selection marker to improve nitrogen use efficiency without affecting tea quality.

[0010] 3) Through the suppression of CsLBD38a transgene, screening for CsLBD38a loss-of-function mutants, and other genetic engineering methods to down-regulate the inhibition of CsLBD38a, the effect of nitrogen on the synthesis of characteristic secondary metabolites can be improved.

[0011] 4) By utilizing the gene function of CsLBD38a, the nitrogen utilization efficiency of tea plants can be improved while maintaining the content and proportion of characteristic secondary metabolites of tea, the flavor quality and health functions of tea.

[0012] 5) The CsLBD38a gene binds to the promoter regions of the theanine synthase genes CsGS1 and CsTS1, inhibiting their expression; CsLBD38a binds to CsMYB75, CsTT2, or CsTT8 in the transcriptional complex MYB-bHLH-WD40 that regulates catechin and anthocyanin synthesis, thereby preventing the formation of the MBW complex, thereby inhibiting the transcriptional complex from activating the expression of several catechin and anthocyanin synthase genes such as CsANR and CsANS.

[0013] 6) Regulating the expression of CsLBD38a in tea plants can screen and cultivate new varieties with efficient nitrogen utilization and high levels of theanine and catechins. By screening tea germplasm resources with low expression of CsLBD38a and combining them with other genes such as NLP, tea varieties that are adapted to low nitrogen fertilizers but high in theanine, total amino acids, and catechins can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The nitrogen deficiency and 5 mM NH4 +The content of catechins, caffeine and theanine changes under nitrogen deficiency, where (a) is the content of caffeine (CAF) and catechins (EGC-epigallocatechin, EGCG-epigallocatechin gallate, ECG-epicatechin gallate) under nitrogen deficiency, (b) is the content of catechins (EC-epicatechin, GCG-epicatechin gallate), theanine (Thea), and glutamine (DL-C) under nitrogen deficiency, and (c) is the content of 5mMNH4 + Supply caffeine, catechin content, d is 5mMNH4 + catechins and theanine content;

[0015] Figure 2 Graphs showing transcript changes of the CsTT2b, CsMYB11, and CsGSTb genes under nitrogen deficiency in Example 2 of the present invention, wherein (a) is a graph showing the relative expression of CsTT2b under nitrogen deficiency, (b) is a graph showing the relative expression of CsMYB11 under nitrogen deficiency, and (c) is a graph showing the relative expression of CsGSTb under nitrogen deficiency;

[0016] Figure 3 Figure 2 is a graph showing the relative expression changes of the CsLBD37a, CsLBD37c, CsLBD38a, CsLBD38b, CsLBD39, and CsLBD16b genes under nitrogen deficiency in Example 2 of the present invention, wherein (a) is a heat map analysis of the relative expression changes of the CsLBD37a, CsLBD37c, CsLBD38a, CsLBD38b, CsLBD39, and CsLBD16b genes under nitrogen deficiency, (b) is a graph showing the relative expression of CsLBD37a by RT-qPCR analysis, and (c) is a graph showing the relative expression of CsLBD38a by RT-qPCR analysis;

[0017] Figure 4 In Example 2 of the present invention, 5mM NH4 + Supply, the relative expression changes of CsMYB11 / 12, CsTT2b, CsLBD37a / c, CsLBD38a / b, CsLBD39 genes, and the content changes of C and EC, total flavonols, K3G, and Q3G, where (a) is the heat map analysis under 5mM NH4 + Supply, relative expression changes of CsMYB11 / 12, CsTT2b, CsLBD37a / c, CsLBD38a / b, and CsLBD39 genes, (b) in 5 mM NH4 + Supply, C and EC content changes, C represents catechin, EC represents epicatechin, (c) in 5mM NH4 + Supply, the content changes of total flavonols, K3G, and Q3G;

[0018] Figure 5 Figure 3 shows that CsLBD38a regulates flavonoid biosynthesis by interfering with the ternary complex in Example 3 of the present invention, wherein a is a diagram of the interaction between CsLBD38a and the three components of the MBW complex detected by Y2H; b is a diagram of the nuclear localization of GFP-CsLBD38a and -CsLBD38b fusion proteins in tobacco leaf epidermal cells; c is a diagram of BiFC confirming the formation of homodimers or heterodimers between CsLBD38a and CsTT8a; d is a diagram of Y1H verifying the binding of CsLBD38 to the CsFLS, CsDFR and CsMYB12 gene promoters; e is a vector for constructing a promoter-driven luciferase reporter gene activation experiment Legend of the figure: The upper part shows the expression vectors for overexpressing transcription factors such as TT2, TT8, TTG1, MYB75, and LBD38a in tobacco; the lower part shows the expression vectors for the luciferase reporter gene (LUC) driven by the promoters of the CsANS1 and CsANR1 genes; f shows that CsLBD38a can inhibit the activation of the CsANS1 promoter by the transcriptional activation complex composed of the three transcription factors MYB75-TT8-TTG1; g shows that CsLBD38a can inhibit the activation of the CsANR1 promoter by the transcriptional activation complex composed of the three transcription factors TT2b-TT8-TTG1;

[0019] Figure 6 In Example 4 of the present invention, (a) a comparison chart of yeast one-hybrid assay results; (b) a chart showing the results of an EMSA assay; (c) a legend showing the construction of vectors for promoter-driven luciferase reporter gene activation experiments: an expression vector for CsLBD38a driven by the 35S promoter; and an expression vector for a luciferase reporter gene (LUC) driven by the promoters of the CsTS1 and CsGS1b genes; (d) a chart showing that CsLBD38a inhibits the expression of a luciferase reporter gene (LUC) driven by the promoters of the CsTS1 and CsGS1b genes.

[0020] Figure 7 Figure 5 is a graph showing the gene expression of the flavonoid biosynthesis metabolic pathway and theanine anabolism metabolic pathway in tea hairy roots affected by CsLBD38a gene knockdown in Example 5 of the present invention, wherein a1 represents the changes in the expression levels of CsFLS1 (a), CsDFR1 (b), CsANS1 (c), CsLAR1 (d), CsNAR1 (e), CsTT2a (f), CsTT8a (g) and nitrogen metabolism genes CsNIA1b (i), CsNRT1.1 (j), CsTS1 (k), CsGSIb (l), and SL gene CsD14 (h) in the roots of CsLBD38a-KD lines compared with the GUS control;

[0021] Figure 8 : This is an example of Example 6 of the present invention, in which CsLBD38a gene knockdown affects the contents of flavonoids, catechins and theanine in hairy roots of tea plants, wherein a is a diagram of hairy root chimeric tea seedlings expressing 35S::GUS and CsLBD38a-RNAi knockdown (CsLBD38a-KD) constructs, b is a qRT-PCR validation diagram of tea roots of CsLBD38a-KD line and GUS control line, c is a comparison diagram of insoluble PAs content in roots of CsLBD38a-KD line and GUS control line, d is a comparative analysis of major flavonols in roots of CsLBD38a-KD line and GUS control line, K3OG: kaempferol 3-o-glucoside, Q3OG: quercetin 3-o-glucoside -glucoside, e is a comparative analysis of the main catechins in the roots of CsLBD38a-KD line and GUS control line, C represents catechin, EC represents epicatechin, and f is a comparative graph of theanine content in the roots of CsLBD38a-KD line and GUS control;

[0022] Figure 9 In Example 6 of the present invention, knocking down CsLBD8a in tea leaves affects the content of catechins and theanine, and the expression of genes in the catechin anabolic pathway, wherein Figure a shows the expression pattern of CsLBD38a in different tea tissues, the first leaf (L1) to the fifth leaf (L5) and the stem internodes (S1-S4), and in MeJA treatment at different times (0, 12, 24, 48 h). Figure b is the qRT-PCR verification of the expression of the CsLBD38a gene (CsLBD38a-KD) in young tea leaves using antisense (asODN) technology, with sense oligonucleotide (sODN) as the control, monitoring the expression at different times. Figure ce shows the comparison of the catechin content in the young leaves of CsLBD38a-KD with that of the control, Figure f shows the comparison of theanine content in the stem tips of CsLBD38a-KD with that of the control, and Figure gm shows the expression of CsANS1 in the young leaves of CsLBD38a-KD. (g), expression changes of CsLAR1 (h), CsANR1 (i), CsTT8a (l), and CsTT2b (m) at different treatment times;

[0023] Figure 10In Example 7 of the present invention, from left to right in the first row are the relative expression graph of CsLBD38a gene at different incubation times, the EGCG content graph, the EGC content graph, and the ECG content graph; from left to right in the first row are the EC content graph, the total catechin content graph, and the theanine content graph at different incubation times; from left to right in the third row are the relative expression graph of CsLAR1 gene, the relative expression graph of CsANR1 gene, the relative expression graph of CsTT8α gene, and the relative expression graph of CsTT2α gene at different incubation times; and from left to right in the fourth row are the relative expression graph of CsNRT2.4 gene, the relative expression graph of CsTS1 gene, the relative expression graph of CsAlaDC1b gene, and the relative expression graph of CsGS1b gene at different incubation times. DETAILED DESCRIPTION

[0024] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0025] Example 1

[0026] The gene sequence of CsLBD38a is shown in SEQ ID NO.1:

[0027] ATGAGTTGCAATGGATGTCGAGTTCTTCGAAAGGGTTGCAGCGAAAACTGTATCCTCCGACCATGCCTTCAGTGGATCGACTCCCCTGAATCTCAAGGCCACGCCACCGTCTTCGTCGCCAAGTTCTTCGGTCGCGCCGGCCTCATGTCCTTCATCTCCTCCGTCCCCCCAAATCAACGTCCCTCTCTGTTTCAATCGCTTTTGTTCGAAGCGTGTGGCCGGACCGTGAACCCGGTTAACGGAGCGGTCGGGCTTCTCTGGACCGGAAACTGGCACCTCTGCCAGGCTGCGGTTCAGACCGTCCTCCGCGGCGGCGCGTTGCGGCCGATCCCGGAGTTCTTCGGCGGCGCCGTCCTGACTCCGCCGGACCTCGACGAGGCTTCCGAGGCTGAAGTCACCTGTTCAGACATGTTTAAACTTTATCACCATCAAGATCCGAACCCTGACTCACGATCGAAAGCGGCGCCCAAGCGCCGCCGTATCGGCGACGAATTAGAGAACCTCGGGCTCAGCTTGACGCCATCATCATCGGAAGCAGTTGTCGGAAGAGTACTGAATTTTTGGAGTTCGCCGGAAAAGCTGAGACCTGGGACGCCGTCGATGAATTCGGAGGAATCTGGGACGACGACTTTACATGAAAGTGGTGGTTTTGGAGATCAAACGAAACTTCTAAACTTGTTCACCTGA

[0028] The amino acid sequence of CsLBD38a is shown in SEQ ID NO.2:

[0029] MSCNGCRVLRKGCSENCILRPCLQWIDSPESQGHATVFVAKFFGRAGLMSFISSVPPNQRPSLFQSLLFEACGRTVNPVNGAVGLLWTGNWHLCQAAVQTVLRGGALRPIPEFFGGAVLTPPDLDEASEAEVTCSDMFKLYHHQDPNPDSRSKAAPKRRRIGDELENLGLSLTPSSSEAVVGRVLNFWSSPEKLRPGTPSMNSEESGTTTLHESGGFGDQTKLLNLFT

[0030] Example 2

[0031] Shucha was subjected to nitrogen deficiency and nitrogen supply experiments:

[0032] The Chinese tea plant Camellia sinensis (L) O. Kuntze cv. Shucha Zao was used to harvest tissues and seeds and to propagate additional tea seedlings. Two-year-old Shucha Zao tea seedlings were hydroponically cultured in a greenhouse at 20-25°C until new roots grew. Then, healthy tea seedlings were transferred to a hydroponic solution containing 0 mM nitrogen (Shigeki Konishi solution without any nitrate or ammonium nitrogen and calcium chloride) for nitrogen deficiency testing. At the same time, they were transferred to ordinary SK medium supplemented with 5 mM NH4 + A nitrogen supply experiment was conducted using a hydroponic solution containing 100 μg of water. Roots and young leaves of tea plants from both the nitrogen-deficient and nitrogen-supplemented experiments were then collected at 0, 6, 12, 24, 48, 72, and 240 hours after treatment. The collected root or tea leaf samples were immediately placed in liquid nitrogen and then stored at -80°C for metabolite analysis and RNA extraction.

[0033] Transcriptome analysis was performed by BGI-Shenzhen Biotech on an Illumina HiSeq 2500 platform, including total RNA isolation, quality assessment, cDNA library construction, and RNA sequencing. Based on the tea plant genome sequence, approximately 7 GB of data were generated for each biological sample for genome assembly and annotation analysis. Reads per Kilobase per Million mapped reads (RPKM) and read counts were calculated using eXpress. Subsequent analysis of the DGE data was similar to published methods. The data were used to quantify RPKM values ​​for tea plant genes.

[0034] The content of flavonoids and amino acids in tea root or tea leaf samples was determined using HPLC instrument. Figure 1 As shown in Figure 2, tea plants under nitrogen deficiency / low nitrogen conditions showed increased accumulation of catechins and flavonols, and decreased accumulation of theanine and glutamine; tea plants fed with 5 mM NH4 + After nitrogen fertilization, the accumulation of catechins and flavonols was reduced, and the accumulation of theanine and glutamine was reduced.

[0035] Calculation of relative gene expression changes under nitrogen deficiency using qRT-PCR: For qRT-PCR, total RNA was extracted from tea plant tissues using an RNA extraction kit (Tiangen Biotechnology Co., Ltd., Beijing, China) according to the manufacturer's instructions. It was converted into cDNA using the PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, Dalian, China) according to the manufacturer's instructions. For conventional qRT-PCR, CsACTIN was used as a standard for normalization. The primer pairs used for qRT-PCR are shown in Table 1. qRT-PCR was performed on the Bio-Rad iQ5 fluorescent quantitative PCR platform using the SYBR Premix Ex Taq kit (TaKaRa, Dalian, China). The fold change of genes was calculated using the comparative CT method: the fold change was calculated as 2 −∆∆CT , each sample was performed in triplicate.

[0036] like Figure 2 As shown, it was found that nitrogen deficiency altered the expression of flavonoid regulatory factors and transporter genes related to nitrogen-responsive genes, such as the transcript change diagram of CsTT2b (a), CsMYB11 (b), and CsGSTb (c). The relative expression increase trend under nitrogen deficiency was opposite to the expression trend of CSLBD8a ( Figure 3 ).

[0037] like Figure 3 As shown, under nitrogen deficiency, CsLBD38a expression was downregulated, which was associated with increased accumulation of catechins and flavonols and decreased accumulation of theanine and glutamine.

[0038] like Figure 4 As shown, in 5 mM NH4 + Under nitrogen feeding, CsLBD38a expression was upregulated, the expression of flavonoid regulatory factors was reduced, and the accumulation of catechins and flavonols was reduced.

[0039] In summary, CsLBD38a is a key regulatory factor mediating N response, theanine and flavonoid synthesis in tea roots.

[0040] Example 3

[0041] Perform yeast hybridization, subcellular localization, and BiFC analysis:

[0042] Yeast one-hybrid (Y1H) assay: The ORF cassettes of CsLBD38a, CsHRS1, CsNLP7 / 9, and CsS40 were recombined as effector vectors into the pGADT7 vector (Clontech, Palo Alto, USA). The approximately 2 kb promoters of CsMYB75 / CsTT2b / CsFLS1 / CsDFR1 / CsGS1b / CsGS1b / CsS40 / CsLBD38a or CsTCS8 were inserted into the pHIS2.1 vector. The pGADT7 and pHIS2.1 recombinant plasmids were co-transformed into yeast strain Y187 cells and selected in SD-Trp-Leu (-TL) medium. The interaction between the effector and promoter was detected in medium lacking Trp, Leu, and His (-TLH).

[0043] In a yeast two-hybrid (Y2H) assay, the ORFs of CsLBD38a, CsTT8a, CsTT2b, CsMYB12, and CsMYB75 were constructed in-frame into the pGADT7 or pGBKT7 vectors (Clontech, Palo Alto, USA). The constructed plasmids were co-transformed into the yeast strain AH109 and cultured and selected on medium lacking tryptophan and leucine (-TL). Transformed yeast cells were cultured on medium lacking Trp, Leu, His, and Ade (-TLHA) with or without the addition of X-gal. Various combinations of pGADT7 or pGBKT7 vectors served as negative controls. For subcellular localization experiments, the ORF frames of CsLBD38a and CsTT8a were seamlessly cloned into the pCAMBIA1300 vector (CAMBIA, Canberra, Australia). The correct construction of the recombinant plasmid was confirmed by sequencing and used for subcellular localization experiments. The recombinant plasmid was fused to the N-terminus of GFP and transformed into Agrobacterium tumefaciens GV3101. The Agrobacterium carrying the constructed recombinant plasmid was infiltrated into tobacco leaves.

[0044] Bimolecular fluorescence complementation (BiFC) experiments were performed in tobacco cells. For BiFC experiments, the ORFs of the CsLBD38a and CsTT8a genes were cloned into the pCAMBIA1300-eYFPN vector (for the N-terminal portion of YFP) and the pCAMBIA1300-eYFPC vector (for the C-terminal portion of YFP) (CAMBIA, Canberra, Australia). The correct construction of the recombinant plasmids was confirmed by sequencing and then transformed into A. tumefaciens strain GV3101. The recombinant plasmids were infiltrated into tobacco leaves individually or in various combinations. Fluorescence observations were performed using a Leica DMi8 M laser scanning confocal microscope (Leica, Germany). GFP fluorescence was excited with a 488 nm laser, and fluorescence was recorded in the 500–520 nm range. EYFP fluorescence was excited with a 510 nm laser, and emission was recorded in the 530–550 nm range. A green pseudocolor was used in all images, rather than a true yellow fluorescence color.

[0045] like Figure 5 As shown, CsLBD38a interacts with CsTT2b, CsMYB75, and CsTT8a, components of the MBW transcriptional complex that specifically regulates flavonoid genes, inhibiting the formation and activation activity of the transcriptional activation complex, thereby inhibiting the expression of downstream genes CsANR1 and CsANS1. Figure 5 As shown in a, CsLBD38a interacts with CsTT2b, CsMYB75, CsMYB12, and CsTT8a. Figure 5 b confirmed that the GFP fusion proteins of CsLBD38a and CsTT8a are both transcription factors localized in the cell nucleus. Figure 5 As shown in c, it was confirmed that CsLBD38a-C-YFP interacted with CsTT8a-N-YFP. Yeast one-hybrid (Y1H) experiments showed that CsLBD38a did not bind to the promoters of CsFLS1 and CsDFR1, but bound to the promoters of CsTT2b, CsMYB12, and CsMYB75 ( Figure 5 d). Figure 5 e shows the legend of constructing vectors for promoter-driven luciferase reporter gene activation experiments: the upper part shows expression vectors for overexpressing transcription factors such as CsTT2, CsTT8, CsTTG1, CsMYB75, CsLBD38a, etc. in tobacco; the lower part shows expression vectors for luciferase reporter genes (LUC) driven by the promoters of CsANS1 and CsANR1 genes. Figure 5 f shows that CsLBD38a can inhibit the activation of the CsANS1 promoter by the transcription activation complex composed of three transcription factors MYB75-TT8-TTG1, confirming that CsLBD38a can negatively regulate the synthesis of anthocyanins. Figure 5 g shows that CsLBD38a can inhibit the activation of the CsANR1 promoter by the transcription activation complex composed of three transcription factors TT2b-TT8-TTG1, confirming that CsLBD38a can negatively regulate the synthesis of catechins.

[0046] Example 4

[0047] Transinhibition assay of CsLBD38a transcription factor on the transcription of CsANS1, CsANR1, CsGS1b and CsTSI:

[0048] The approximately 2 kb promoter region of the CsANR1 / CsANS1 / CsGS1b / CsTSI genes (Table 1) was amplified using primers and subcloned into the p2GWL7 or pGreenII 0800 vectors to generate a promoter:luciferase (LUC) reporter construct. The effector genes CsTT8a, CsMYB75, CsMYB12, CsTTG1, CsTT2b, and CsLBD38a were cloned into the p2GW7 or pGreenII62-SK vectors to generate constructs for expression of 35S-driven transcription factor effector proteins. Tobacco leaves were infiltrated with Agrobacterium-borne reporter genes pGreenII-proCsANS1-LUC, -proCsGSI-LUC, -proCsTSI-LUC, -proCsANR1-LUC, and pGreenII 62-SK 35S-CsTT8a, 35S-CsMYB75, 35S-CsLBD38a, 35S-CsTT2b, 35S-TTG1, or vectors pGreenII 0800-LUC and pGreenII 62-SK in various combinations. Firefly LUC activity was normalized using Renilla LUC. Luciferase activity was quantified using a microplate luminometer using a dual-luciferase reporter assay kit (Promega). Figure 5 The promoter deactivation experiment showed that the addition of CsLBD38a to the MBW complex activation system further inhibited the activity of the CsANS1 and CsANR1 promoters.

[0049] CsLBD38a can also bind to the promoter region of theanine synthase genes CsTSI and CsGS1b and inhibit their expression, thereby inhibiting the synthesis and accumulation of theanine. Figure 6As shown, CsLBD38a binds to the promoter regions of the theanine synthase genes CsTSI and CsGS1b and represses their expression, thereby inhibiting theanine synthesis and accumulation. (a) Yeast one-hybrid assays confirm that CsLBD38a binds to the promoter regions of the theanine synthase gene CsTSI, as well as the promoter region of another gene involved in theanine biosynthesis, CsGSIb. (b) DNA gel mobility shift assays (EMSA) demonstrate that CsLBD38a protein binds to conserved sequence elements in the promoter regions of the theanine synthase genes CsTSI and CsGS1b. (c) Legend for constructing vectors for promoter-driven luciferase reporter gene activation experiments: expression vector for CsLBD38a driven by the 35S promoter; and expression vectors for the luciferase reporter gene (LUC) driven by the promoters of the CsTS1 and CsGS1b genes. (d) CsLBD38a inhibited the expression of luciferase reporter gene (LUC) driven by the promoters of CsTSI and CsGS1b, confirming that CsLBD38a can inhibit theanine synthesis.

[0050] Example 5

[0051] Inhibition of CsLBD38a in tea leaves using asODN technology can promote the synthesis of flavonoids (e.g. Figure 7 ):

[0052] Because tea plant transformation technology is still underdeveloped, we used a candidate gene knockout method (asODN) containing a complementary fragment of the target gene to examine changes in CsLBD38a and catechin expression in tea leaves. Antisense oligonucleotides targeting CsLBD38a were designed using Soligo software (http: / / sfold.wadsworth.com / soligo.pl) as input sequences (Table 1). To achieve antisense knockdown of CsLBD38a, freshly excised apical buds and first leaves of the tea cultivar "Bai Hao Zao" were incubated in 2 ml centrifuge tubes containing 30 μM asODN solution and 10 mM sucrose solution for varying periods of time. Leaves incubated with 10 mM sucrose solution (sODN) served as controls. Samples were collected at different time points for RNA and catechin analysis.

[0053] Determination of theanine, several and total catechins in tea plants:

[0054] Amino acids and theanine were extracted and quantified using a high-speed amino acid analyzer (L-8900, Hitachi). Flavonols were analyzed by high-performance liquid chromatography (HPLC). The contents of several catechins and theanine in tea plant tissues were determined by HPLC. Reference standards EC, EGC, EGCG, C, ECG, GC, GCG, theanine, and caffeine were purchased from Sigma-Aldrich (Steinheim, Germany). Soluble and insoluble proanthocyanidin polymers (PAs) were determined in tea plant roots and leaves using DMACA (4-(dimethylamino)-cinnamaldehyde) staining.

[0055] like Figure 7 As shown, after tea buds and leaves were treated with antisense oligonucleotide inhibition (asODN) technology, the expression of CsLBD38a was significantly reduced (i.e., CsLBD38a-KD knockout mutant). The transcription level of CsLBD38a gradually decreased (Figure d); at the same time, the content of total catechins decreased (Figure c); theanine content increased (Figure f); and catechin-related synthesis genes and regulatory genes were upregulated (Figure g).

[0056] Example 6

[0057] A method for increasing the content of catechins and theanine in tea leaves by knocking down the CsLBD38a gene to alter the expression of flavonoid and nitrogen-responsive genes in tea roots.

[0058] Specifically, a gene-specific fragment of approximately 300 bp was amplified from CsLBD38a using BP and LR cloning (Invitrogen, USA) and subcloned into the final RNA interference (RNAi) destination vector, pB7GWIWG. The resulting recombinant vector, pB7GWIWG-CsLBD38a, was transformed into Agrobacterium tumefaciens ATCC 15834 by electroporation. Positive transformants carrying pB7GWIWG-CsLBD38a were selected and transformed into three-month-old tea seedlings after pretreatment with acetosyringone. Transgene expression was detected by qRT-PCR, confirming the presence of transgene-positive hairy root lines in the hypocotyl region following needle puncture. At least three independent hairy root lines were selected for RNA and flavonoid analysis.

[0059] The 300 bp sequence of CsLBD38a was replaced by a fragment expressing the GUS gene, and pB7GWIWG-GUS of the same destination vector pB7GWIWG was constructed and transformed into plant roots as a control.

[0060] like Figure 8As shown, transgenic hairy roots of tea plants expressing CsLBD38a were generated using RNAi (RNAi) knockdown (KD) (Figure a). qRT-PCR analysis revealed that CsLBD38a was significantly knocked down in CsLBD38a-RNAi (CsLBD38a-KD) hairy roots (Figure b). Correspondingly, the content of insoluble polyacrylic acid (PAs) in transgenic hairy roots was higher than that in the GUS control (Figure c). Furthermore, compared with the GUS control, the CsLBD38a-KD line showed elevated levels of total flavonols (K-3-OG and Q-3-OG), catechins (primarily EC), and theanine (Figures d to f). Consistent with these findings, genes involved in flavonoid biosynthesis and regulation, including CsFLS1, CsDFR1, CsANS1, CsLAR1, CsANR1, CsTT2b, and CsTT8a, were upregulated to varying degrees.

[0061] The expression of N-responsive genes and SL signaling genes in CsLBD38a-KD hairy roots was examined. Compared with the GUS control line, the expression of CsNIA1b, CsNRT1.1b, CsTS1, and CsGSIb in all CsLBD38a-KD lines was upregulated. Figure 9 As shown, CsD14 was also upregulated in the CsLBD38a-KD line compared with GUS hairy roots, that is, overexpression of LBD37 / 38 / 39 in Arabidopsis repressed NIA1, NRT2.5, and GLN1.4 genes, while knockdown of Arabidopsis LBD38a upregulated NIA1, NRT2.5, and GLN1.4 genes.

[0062] Analysis of the promoter regions of these nitrogen metabolism genes and flavonoid biosynthesis regulatory factor genes revealed that they all contained one or several LBD38a-binding cis-acting elements, such as GCGGCG, CACGTG, TGGG or TGGGC[C / T], TGTCTC, GAGACA, CCGG or CCGGXTTTXXXG (Table 2).

[0063] Example 7

[0064] Using asODN oligonucleotide fragments specific to the CsLBD8a gene sequence to inhibit the growth of one-bud-one-leaf tea buds and alleviate the inhibition of nitrogen fertilizer on the synthesis of catechins and theanine:

[0065] In order to verify whether tea plants with the CsLBD38a gene knocked out can change their response to nitrogen, whether they can alleviate the inhibition of catechin synthesis by nitrogen fertilizer, or whether they can lift the inhibition of theanine synthesis and accumulation, we further carried out experiments to inhibit the expression of the CsLBD8a gene in tea buds and leaves, and applied nitrogen fertilizer to the young tea buds and leaves after the gene was inhibited, and tested the content of catechins and theanine in the tea buds and leaves.

[0066] The experiment found that inhibiting the CsLBD38a gene in young tea shoots changed the response to nitrogen fertilizer, significantly alleviated the inhibition of catechin synthesis caused by nitrogen fertilizer feeding, and promoted the accumulation of theanine synthesis induced by nitrogen fertilizer stimulation ( Figure 10 ).

[0067] As shown in the asODN gene expression inhibition technology in Example 5 above, feeding young tea buds with asODN oligonucleotide fragments specific for the CsLBD8a gene sequence can inhibit the expression of CsLBD38a in tea buds and leaves ( Figure 10 ) . Incubation in 10 mM sucrose solution of asODN and 10 mM sucrose solution (sODN) with one bud and one leaf apical bud as control was added with 5 mM NH4NO3 (as Figure 10 ) and measured the contents of catechins in tea plant tissues, including the major catechins EGCG, EGC, ECG, and EC, as well as the total catechin content. The results showed that applying 5 mM ammonium nitrate nitrogen fertilizer to detached tea buds also significantly inhibited the contents of EGCG, EGC, ECG, EC, and total catechins in control tea buds. However, the contents of these catechins in the single-bud, single-leaf apical bud of CsLBD8a-KD plants, where CsLBD8a expression was suppressed, were significantly higher than those in the wild-type control plants with normal CsLBD8a expression ( Figure 10 At the same time, feeding nitrogen fertilizer stimulated the synthesis of theanine in young shoots and leaves; however, the theanine content in the one-bud-one-leaf apical bud of CsLBD8a-KD tea plants was significantly higher than that in the young shoots and leaves of the wild-type control ( Figure 10 Fluorescence real-time quantitative PCR detection found that the expression of several major catechin synthase genes or regulatory factors CsANR1, CsLAR1, CsTT8a, and CsTT2a, which were inhibited by nitrogen fertilizer feeding, in the young shoots of CsLBD8a-KD were significantly higher than those in the young shoots of the wild type control ( Figure 10 This suggests that the inhibition of CsLBD8a on theanine synthase genes such as CsTSI and CsGS1b, or on the expression of catechin synthase genes or regulatory factors CsANR1, CsLAR1, CsTT8a, and CsTT2a, is relieved, leading to greater accumulation of theanine and catechins in the young shoots of CsLBD8a-KD.

[0068] Table 1 List of primers used for gene cloning, vector construction, qRT-PCR detection, etc.

[0069]

[0070] Table 2 Promoter region sequences of CsLBD38a regulatory targets predicted by bioinformatics for binding to various nitrogen metabolism assimilation and flavonoid biosynthesis regulatory factors

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[0075] The above contents are merely examples and explanations of the present invention. Those skilled in the art may make various modifications or additions to the described specific embodiments or replace them in similar ways. As long as they do not deviate from the structure of the present invention or exceed the scope defined by the claims, they shall fall within the scope of protection of the present invention.

Claims

1. A method for increasing the content of catechins and theanine in tea, characterized by: By knocking out or inhibiting the expression of the CsLBD38a gene in the roots and young shoots and leaves of tea plants, the content of catechins and theanine in tea leaves is increased. The gene sequence of the CsLBD38a gene is shown in SEQ ID NO.1.

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