Method for synergistically regulating accumulation and quality of gamma-aminobutyric acid through tea tree CsBAT transport protein and flavonoid metabolism key gene and application

Abstract

A method for synergistically regulating accumulation and quality of gamma-aminobutyric acid through tea tree CsBAT transport protein and flavonoid metabolism key genes is characterized in that the tea tree CsBAT transport protein is utilized to transport gamma-aminobutyric acid from mature leaves of a mediated tea tree to young shoots, and the tea tree CsBAT transport protein and flavonoid metabolism key enzymes synergistically participate in secondary metabolism regulation; the accumulation of gamma-aminobutyric acid in the tea tree shoots is dynamically regulated and controlled; the process causes the increase of the amino acid content of the tender shoots and leaves and the reduction of the amino acid content of the mature leaves, so that the formation of the tea quality is influenced and optimized. According to the method, the amino acid content of the tender shoots and leaves can be remarkably increased, gamma-aminobutyric acid is most remarkably increased, meanwhile, the amino acid content of mature leaves is effectively reduced, the method has high amino acid transfer and redistribution efficiency, the flavor of the tea leaves can be improved, and the quality of the tea leaves is improved; meanwhile, a new way is provided for cultivating high-stress-resistance and high-quality tea tree varieties.

Description

Technical Field

[0001] This invention relates to a method and application of synergistic regulation of transport proteins and key enzymes in flavonoid metabolism, and more particularly to a method and application of synergistic regulation of γ-aminobutyric acid accumulation and quality by tea plant CsBAT transport protein and key genes in flavonoid metabolism, belonging to the field of biotechnology. Background Technology

[0002] As a widely consumed beverage globally, tea's quality is of paramount importance to consumers. Amino acids are crucial quality components in tea, not only contributing to its refreshing taste but also offering various health benefits. For instance, theanine can alleviate anxiety, promote sleep, and enhance memory, playing a key role in the quality and economic value of tea. However, the amino acid content in tea currently generally needs improvement, failing to fully meet consumer demand for high-quality tea.

[0003] In existing technologies, the amino acid content in tea is generally increased through single gene overexpression. This method has several drawbacks. First, it is inefficient; the effect of a single gene is limited, making it difficult to significantly increase amino acid content. Second, it can easily lead to metabolic imbalance in tea plants, as tea plant metabolism is a complex network, and altering a single gene may disrupt the existing metabolic balance, affecting the normal growth and development of the tea plant. Furthermore, this method may also affect the flavor and resilience of tea. For example, it may alter the originally balanced proportions of flavor compounds, resulting in a deterioration in flavor; simultaneously, the tea plant's resistance to pests, diseases, drought, low temperatures, and other adverse conditions may decrease, increasing cultivation risks. Therefore, how to efficiently and rationally increase the amino acid content in young buds and leaves has long been a technical challenge in this field, and this invention addresses that challenge. Summary of the Invention

[0004] The purpose of this invention is to address the need to increase the content of γ-aminobutyric acid (GABA) in tea. Existing technologies generally increase the GABA content in tea through single gene overexpression, which is not only inefficient but also prone to causing metabolic imbalance in tea trees, and may even affect the flavor and stress resistance of tea, thus hindering the improvement of tea quality. This invention provides a method that can effectively regulate GABA accumulation and quality by utilizing the tea tree CsBAT transporter to mediate the transport of GABA from mature leaves to new shoots. The tea tree CsBAT transporter works synergistically with key enzymes in flavonoid metabolism to participate in secondary metabolic regulation, which can significantly increase the amino acid content in young buds and leaves and improve tea quality.

[0005] To achieve the aforementioned objectives, the technical solution of this invention is: a method for synergistic regulation of γ-aminobutyric acid (GABA) accumulation and quality by tea plant CsBAT transporter and key genes in flavonoid metabolism, characterized in that: the tea plant CsBAT transporter mediates the transport of GABA from mature leaves to new shoots of the tea plant; the tea plant CsBAT transporter and key enzymes in flavonoid metabolism synergistically participate in secondary metabolic regulation to dynamically regulate the accumulation of GABA in the new shoots of the tea plant; this process leads to an increase in the amino acid content of young buds and leaves, while the amino acid content of mature leaves decreases, thereby affecting and optimizing the formation of tea quality.

[0006] Furthermore, the tea plant CsBAT transporter protein mediates amino acid allocation bias towards young buds and leaves, and is specifically highly expressed in the vascular tissues of tea plants, especially in one bud with three leaves and one bud with five leaves.

[0007] Furthermore, in the mature leaves of the tea plant, the high expression of the tea plant CsBAT transporter protein participates in the redistribution of senescence-related amino acids, transporting the amino acids, mainly GABA, stored in the mature leaves to the vigorous new shoots.

[0008] Furthermore, the key genes for flavonoid metabolism include LAR, 4CL, or C4H.

[0009] Furthermore, the amino acid content of the young buds and leaves can be significantly increased by 30%-63%, while the amino acid content of mature leaves can be reduced by 8%-35%.

[0010] The application of a method for synergistic regulation of γ-aminobutyric acid (GABA) accumulation and quality by tea plant CsBAT transporter and key genes of flavonoid metabolism in tea cultivation is characterized by applying the method of synergistic regulation of GABA accumulation and quality by tea plant CsBAT transporter and key genes of flavonoid metabolism to tea cultivation.

[0011] The beneficial effects of this invention are:

[0012] 1. This invention breaks through the traditional technology by utilizing the CsBAT transporter protein of tea tree and the key enzymes of flavonoid metabolism to participate in the secondary metabolic regulation, thereby realizing the dynamic regulation of the accumulation of γ-aminobutyric acid in the new shoots of tea tree. This is conducive to the directional transport of amino acids such as γ-aminobutyric acid from old leaves to young buds and leaves, thus promoting the enrichment of γ-aminobutyric acid in young buds and leaves.

[0013] 2. This invention enhances the transport efficiency of γ-aminobutyric acid (GABA) to new shoots and leaves by overexpressing CsBAT transporter protein, thus avoiding the metabolic burden caused by excessive accumulation in mature leaves. By combining CsBAT transporter protein with flavonoid metabolism genes, the limitations of a single metabolic pathway are overcome, and the synergistic enhancement of GABA, tea polyphenols and other components is achieved.

[0014] 3. This invention has high amino acid transport and redistribution efficiency, which can significantly increase the amino acid content of young buds and leaves, while significantly reducing the amino acid content of mature leaves. It is not only highly efficient, but also improves the flavor and quality of tea. At the same time, it provides a new way to cultivate highly resistant and high-quality tea varieties, and has high industrial application prospects. Attached Figure Description

[0015] Figure 1 This is a diagram showing the functional verification and kinetic analysis of the CsBAT transporter protein of this invention in a 22Δ10α yeast mutant strain.

[0016] Figure 2 This is a phylogenetic tree analysis result of the CsBAT transporter protein of this invention.

[0017] Figure 3 This is a three-dimensional structural model of the CsBAT transporter protein of this invention.

[0018] Figure 4 This invention provides an analysis of the expression pattern and subcellular localization map of the CsBAT transporter protein.

[0019] Figure 5 This is a comprehensive analysis diagram of the metabolic regulation of tea plants by CsBAT-RNAi of this invention.

[0020] Figure 6 This is an information diagram of the pYES2 carrier of the present invention.

[0021] Figure 7 This is an information diagram of the carrier pK7FWG2 of this invention. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0023] A method for synergistically regulating γ-aminobutyric acid (GABA) accumulation and quality in tea plants using CsBAT transporter and key genes involved in flavonoid metabolism is characterized by: utilizing CsBAT transporter to mediate the transport of GABA from mature leaves to new shoots; and having CsBAT transporter synergistically participate in secondary metabolic regulation with key enzymes involved in flavonoid metabolism to dynamically regulate GABA accumulation in new shoots. This process leads to an increase in amino acid content in young buds and leaves, especially a significant increase in GABA, while the amino acid content in mature leaves decreases, thereby influencing and optimizing tea quality formation.

[0024] The tea plant CsBAT transporter protein mediates amino acid allocation bias towards young buds and leaves, and is specifically highly expressed in the vascular tissues of tea plants, especially in one bud with three leaves and one bud with five leaves.

[0025] In the mature leaves of the tea plant, the high expression of the tea plant CsBAT transporter protein participates in the redistribution of senescence-related amino acids, transporting the amino acids, mainly GABA, stored in the mature leaves to the vigorous new shoots.

[0026] The key genes for flavonoid metabolism include LAR, 4CL, or C4H.

[0027] The amino acid content of the young buds and leaves can be significantly increased by 30%-63%, while the amino acid content of mature leaves can be decreased by 8%-35%.

[0028] The application of a method for synergistic regulation of γ-aminobutyric acid (GABA) accumulation and quality by tea plant CsBAT transporter and key genes of flavonoid metabolism in tea cultivation is characterized by applying the method of synergistic regulation of GABA accumulation and quality by tea plant CsBAT transporter and key genes of flavonoid metabolism to tea cultivation.

[0029] The principle and main process of this invention are as follows: Utilizing the CsBAT transporter protein-mediated amino acid allocation bias towards young buds and leaves, the CsBAT transporter protein is specifically and highly expressed in the vascular tissues of tea trees (especially in one bud with three leaves and one bud with five leaves), indicating its preferential involvement in nutrient allocation in young tissues. Verification using tea tree hair root system and RNAi technology revealed that when BAT function was inhibited, the amino acid content in older leaves (five or six leaves) decreased by 8%-15%, while the amino acid content in young buds and leaves (new shoots) significantly increased by 30%-63%. This result confirms that BAT mediates the directional transport of amino acids from older leaves to young buds and leaves, thereby promoting the accumulation of γ-aminobutyric acid (GABA) in young buds and leaves. This result is consistent with the physiological strategy of tea trees to preferentially allocate nutrients to metabolically active tissues.

[0030] This invention utilizes tissue-specific regulation of nitrogen redistribution. Silencing the CsBAT transporter leads to a significant accumulation of amino acids in roots, while increasing amino acid content in new shoots and decreasing it in older leaves, revealing the differentiated regulatory function of the CsBAT transporter in different tissues of the tea plant. In roots, the CsBAT transporter mediates the unloading and long-distance transport of amino acids to the phloem by promoting the expression of GABA-T (γ-aminobutyrate transaminase) and GGT1 (γ-glutamyltransferase 1). Silencing this process is blocked, resulting in amino acid retention in the roots. In older, mature leaves, high expression of the CsBAT transporter may participate in senescence-related amino acid redistribution, transporting amino acids stored in older leaves to new shoots. Silencing this process reduces amino acid output from older leaves, thus explaining the dynamic changes in the "source-sink" relationship of amino acids in new shoots. New shoots of tea plants, where the "sink" function is dominant, mainly rely on roots and mature leaves for nitrogen transport. Amino acids are the most important form of nitrogen transport from "source" to "sink" in plants. This result is consistent with the physiological characteristic of tea shoot growth relying on the redistribution of nutrients from older leaves, and this differential regulation highlights the important role of CsBAT transporters in maintaining inter-tissue nitrogen homeostasis.

[0031] The following is a detailed explanation of the specific implementation:

[0032] Example 1: Full-length cloning, gene transcription expression analysis, and sequence analysis of the CsBAT transporter protein

[0033] Total RNA was extracted from Arabidopsis thaliana and tea plant tissue samples using an RNA extraction kit. RNA was then measured using A... 260 / A 280 RNA quality was verified by ratios (1.9-2.1) and agarose gel electrophoresis. Approximately 1 μg of total RNA was used to synthesize first-strand cDNA using the PrimeScript RT kit (Takara, Japan). Gene-specific primers (see Appendix SX) were designed for qRT-PCR analysis. GAPDH was used as an internal control gene. -ΔCt The method calculates the relative expression changes of the target gene mRNA level. For the validation of Arabidopsis mutants and homozygotes, DNA was extracted using a plant DNA extraction kit, and primers were used for validation. Bioinformatics software such as DNAman, ProtParam, ProtScale, TMHMM services v, and Phyre2 were used for gene sequence analysis.

[0034] This invention cloned the CsBAT transporter gene from tea plants (accession number: KY709678.1), which encodes a protein containing 533 amino acids. Domain analysis revealed the presence of the 2A0303 domain (PFAMcl45918), which is associated with amino acid transport. 2A0303 is a domain involved in transmembrane and ammonium transport, primarily transporting and binding proteins, amino acids, peptides, and amines. Phylogenetic analysis showed that CsBAT is most closely related to the plant cationic amino acid transporter family (see appendix). Figure 3 The three-dimensional structural model predicted by Phyre2 (99% confidence level) further supports its transmembrane transport properties; see appendix. Figure 4 .

[0035] Example 2: Analysis of CsBAT transporter expression patterns and subcellular localization

[0036] CsBAT transporter expression pattern analysis: 1) Different tissues of tea seedlings, including roots, stems, leaves, and buds, were selected, flash-frozen in liquid nitrogen, and stored at -80℃. 2) RNA extraction from tea seedling roots. The procedure was performed according to the Simgen RNA extraction kit instructions. 3) cDNA reverse transcription. The procedure was performed according to the Simgen reverse transcription kit instructions. 4) Quantitative real-time PCR. The reaction procedure was described in the 2×Q3SYBR qPCR Master Mix (Universal) instructions. The relative expression levels are shown in the attached figure. Figure 4 As shown in Part A of the document. (Attached) Figure 4 Section A shows the expression pattern analysis of CsBAT transporter in different tissue sites. The expression levels of CsBAT transporter were detected in bud, first leaf and its midrib (1st MV), third leaf and its midrib (3rd MV), fifth leaf and its midrib (5th MV), stem vascular bundle (VB), stem, and root.

[0037] Subcellular localization of CsBAT transporter: Fluorescence was observed using laser confocal microscopy to detect the subcellular localization of the CsBAT protein fused with GFP expressed in *Nicotiana benthamiana* epidermal cells. From left to right: bright field, empty vector or gene, Dil-stained membrane, GFP+Dil fusion, as shown in the attached image. Figure 4 As shown in Part B. (Attached) Figure 4 Part B shows the subcellular localization of the CsBAT transporter. The CsBAT protein fused with GFP is expressed in the epidermal cells of *Nicotiana benthamiana*. From left to right: bright field, empty vector or gene, Dil-stained membrane, GFP+Dil fusion.

[0038] We found that the CsBAT transporter is constitutively expressed, present in various tissues of the tea plant at high levels. BAT is primarily expressed in the vascular tissue of leaves, with higher expression levels in highly mature leaves than in tender new shoots. The highest expression levels were observed in the veins of one bud with five leaves and one bud with three leaves. Expression in the vascular bundles of roots, stems, and leaves further demonstrates the important role of the CsBAT transporter in the unloading, loading, distribution, and balance of amino acids between the aboveground and underground parts of the plant. Expression in the veins of highly mature leaves promotes the unloading and transport of CsBAT from mature leaves to new shoots, thus contributing to the accumulation of umami substances such as GABA and thea in the new shoots.

[0039] Example 3: Construction of CsBAT-RNAi vector and verification of positive hair root function

[0040] CsBAT was introduced into Agrobacterium rhizogenes A4 culture, and then spread onto a selection culture containing kanamycin to screen for positive transformants. After removing all roots from tea seedlings with 3-4 true leaves, the seedlings were incised with a blade and placed in Agrobacterium for overnight infection. Once new roots grew from the wounds, PCR screening was performed. Roots with different gene expression levels were selected, and the gene expression levels in different root cells were detected using GUS staining and in situ hybridization. Amino acid content was determined using the Waters AccQ-Tag amino acid analysis method.

[0041] After infection using the tea tree hair root system, a total of 8 positive hair roots with relative BAT expression levels between 0.17 and 0.47 were obtained, as shown in the attached figure. Figure 5 As shown in Part A of the diagram. In the roots, GABA, Glu, Gln, Thea, and other amino acids showed a significant accumulation trend in different positive hair roots, while the total free amino acid content increased by 20%-76%. Furthermore, we found that the total free amino acid content in new shoots (one or two leaves) increased by 30%-63%, while that in older leaves (five or six leaves) decreased by 8.3%-15%. Combined with the high expression of CsBAT in the veins of five and six leaves and the results of in situ hybridization, we hypothesize that the CsBAT transporter in the roots mainly unloads amino acids into the phloem and then transports them to the aboveground tissues for growth. In the aboveground parts, the CsBAT transporter mainly functions by unloading amino acids from older leaves and transporting them to new shoots, as shown in the attached diagram. Figure 5 As shown in Part B.

[0042] By detecting the expression of key genes in the metabolic pathways of CsBAT transporter, theanine metabolism, and flavonoid metabolism, we found that in BAT-RNAi positive hairy roots, the expression level of the BAT gene in the roots of BAT-RNAi lines was significantly negatively correlated with the accumulation of all free amino acids including GABA. The correlations between BAT and GABA, Glu, Thea, Gln were as high as -0.999, -0.998, -0.980, -0.917 respectively. In the roots of control tea seedlings, BAT was positively correlated with the accumulation of all amino acids. The key enzymes in the amino acid metabolic pathway such as GABA-T (<r<), GGT, GOGAT, and ADC, as well as C4H, PAL, and CHS in the flavonoid metabolic pathway were significantly positively correlated with the contents of amino acids such as GABA, thea, gln, and glu. Whether in BAT-RNAi or control tea seedlings, the correlations between GABA-T, GOGAT, ADC, SSADH, F3H, PAL, and CHS and amino acids were consistent. Meanwhile, in BAT-RNAi tea seedlings, the expression levels of TS, GAD, GDH, ANR, and 4CL were negatively correlated with the contents of various free amino acids, while GGT and C4H were positively correlated; unexpectedly and non-obviously: in control tea seedlings, the expression levels of TS, GAD, GDH, ANR, and 4CL were positively correlated with the contents of various free amino acids, while GGT and C4H were negatively correlated. The completely opposite correlation patterns indicate that knocking out the BAT gene affects and even subverts the expression of key genes and enzymes in some amino acid and flavonoid metabolic pathways, as shown in Figure 5 Part D in

[0043] In BAT-RNAi positive hair roots, LAR (r=0.973), GAD (r=0.961), ANR (r=0.949), 4CL (r=0.939) and GDH (r=0.890) were co-expressed with BAT, while C4H (r=-0.999), PAL (r=-0.960), and GGT (r=-0.913) were antagonistically expressed. In the control group, the co-expressing genes with BAT were ADC (r=0.999), 4CL (r=0.937), and GS (r=0.926), while the antagonistic genes were GGT (r=-1), C4H (r=-0.999), CHS (r=-0.969), PAL (r=-0.969), LAR (r=-0.967), GDH (r=-0.895), and DFR (r=-0.739). In both the control and positive hair root groups, 4CL was consistently co-expressed with BAT, while C4H and GDH were consistently antagonistic to BAT. Unexpectedly and unobviously, LAR was co-expressed in the positive hair root group but antagonistically expressed in the control group. Based on these results, it can be boldly inferred that LAR, 4CL, and C4H in the flavonoid metabolic pathway may mediate BAT's involvement in the transport, distribution, and metabolism of γ-aminobutyric acid (GABA) in tea plants, as shown in the attached diagram. Figure 5 As shown in section C.

[0044] Figure 5 This is a comprehensive analysis diagram of the metabolic regulation of tea plants by CsBAT-RNAi of this invention, with attached... Figure 5 Part A shows the detection of CsBAT gene expression level in transgenic hairy roots, with empty vector-transformed plants as controls; Appendix Figure 5 Part B shows a comparison of amino acid content in different tissues (young leaves, mature leaves, and roots) between the RNAi lines and the control; Appendix Figure 5 Section C shows the co-expression network analysis of CsBAT and metabolic genes; red and blue indicate positive and negative correlations, respectively. Figure 5 Section D shows the correlation analysis between metabolic gene expression and amino acid content. All data are the mean ± standard deviation of three biological replicates.

[0045] The nucleotide sequence of the CsBAT transporter protein of this invention:

[0046]

[0047] The amino acid sequence corresponding to the CsBAT transporter protein of this invention is as follows:

[0048] MGWHSHSHVQNGDSSLDTGHTRLHELGYKQELKRDLSVVSNFAFFSIISVLTGITTLYNTGLNYGGPVVLVYGWLIAGVFTMIVGLSMAEICSSYPTSGGLYYWSAKLAGPSWAPFASWLTGWFNIVGQ WAVTTSVDFSLAQLIQVIILLSTGGKNGGGYQASKYVVIGIHGGILLLHAIINSLPITWLSLFGQFAALWNLIGVFVLMILIPTVAKEKASAKFVFTFFNTDNGDGINSKVYIFVLGLLMSQYTLTGYDA SAHMTEETKSADKNGPKGIISAIGISVIVGWGYLLGVTFAVTKIPNLLSENNDAGGYAIAEVFYLAFKNRYGNGIGGIICLGVVAVAIFFCCMSSITTSNSRMAYAFSRDGAMPLSSVWHKVNKHEVPLNA VWLSAIIAFCMALTSLGSIVAFQAMVSIATIGLYIAYALPIFFRVTLARKSFTPGPFNLGRYGVLVGWIAVLWVATISVLFSLPVSYPITDQTLNYTPVAVGGLLILTVASWIFSARHWFKGPITNIDDVL

[0049] In summary, through extensive research and experimentation, this invention significantly increases the amino acid content of young buds and leaves while significantly decreasing the amino acid content of mature leaves. It exhibits high efficiency in amino acid transport and redistribution without causing metabolic imbalance in tea trees. Not only is it highly efficient, but it also improves the flavor of tea, resulting in a mellow taste, better nutritional value, and enhanced tea quality.

[0050] The above description is a further detailed explanation of the present invention in conjunction with specific embodiments. It should not be considered that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, any simple modifications and substitutions made without departing from the concept of the present invention should be considered to fall within the protection scope of the present invention.

Claims

1. A method for synergistic regulation of γ-aminobutyric acid accumulation and quality by the CsBAT transporter protein and key genes in flavonoid metabolism in tea plants, characterized in that: By utilizing the CsBAT transporter in tea trees to mediate the transport of γ-aminobutyric acid (GABA) from mature leaves to new shoots, and by having the CsBAT transporter synergistically participate in secondary metabolic regulation with key enzymes in flavonoid metabolism, the accumulation of GABA in new shoots of tea trees can be dynamically regulated. This process leads to an increase in the amino acid content of young buds and leaves, while the amino acid content of mature leaves decreases, thereby influencing and optimizing the formation of tea quality.

2. The method for synergistic regulation of γ-aminobutyric acid accumulation and quality by the CsBAT transporter protein and key genes of flavonoid metabolism in tea plants according to claim 1, characterized in that: The tea plant CsBAT transporter protein mediates amino acid allocation bias towards young buds and leaves, and is specifically highly expressed in the vascular tissues of tea plants, especially in one bud with three leaves and one bud with five leaves.

3. The method for synergistic regulation of γ-aminobutyric acid accumulation and quality by the CsBAT transporter protein and key genes of flavonoid metabolism in tea plants according to claim 1, characterized in that: In the mature leaves of the tea plant, the high expression of the tea plant CsBAT transporter protein participates in the redistribution of senescence-related amino acids, transporting the amino acids, mainly GABA, stored in the mature leaves to the vigorous new shoots.

4. The method for synergistic regulation of γ-aminobutyric acid accumulation and quality by the CsBAT transporter protein and key genes of flavonoid metabolism in tea plants according to claim 1, characterized in that: The key genes for flavonoid metabolism include LAR, 4CL, or C4H.

5. The method for synergistic regulation of γ-aminobutyric acid accumulation and quality by the CsBAT transporter protein and key genes of flavonoid metabolism in tea plants according to claim 1, characterized in that: The amino acid content of the young buds and leaves can be significantly increased by 30%-63%, while the amino acid content of mature leaves can be decreased by 8%-35%.

6. The application of a method for synergistic regulation of γ-aminobutyric acid accumulation and quality by the CsBAT transporter protein and key genes in flavonoid metabolism in tea cultivation, characterized by: The method for regulating the accumulation and quality of γ-aminobutyric acid in tea plants as described in claim 1 is applied to tea cultivation.

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