Application of Mitochondrial Carrier CsTHS1 in Regulating Theanine Accumulation in New Shoots of Late Spring Tea Plant

Abstract

The present application relates to the technical field of theanine content regulation in tea leaves, and particularly relates to the application of mitochondrial carrier CsTHS1 in regulating the intracellular transport of theanine in tea shoots and further regulating the accumulation of theanine, and the mechanism of regulating the reduction of theanine content in late spring shoots is studied. The results show that CsTHS1 is significantly induced in late spring, can promote the transport of theanine in the intracellular of the shoots to mitochondria, promote the degradation of theanine in mitochondria, and negatively regulate the accumulation of theanine in tea shoots. Silencing CsTHS1 in tea plants can significantly increase the content of theanine, and overexpression of CsTHS1 in tea plants can significantly reduce the content of theanine. Therefore, by reducing the expression level of CsTHS1 in tea plants, the transport and degradation of theanine to mitochondria are reduced, the content of theanine in tea shoots is improved, and the quality of tea leaves is improved.

Description

Technical Field

[0001] This invention relates to the field of theanine content regulation technology in tea, specifically to the application of mitochondrial carrier CsTHS1 in regulating the accumulation of theanine in the new shoots of late spring tea trees. Background Technology

[0002] Tea is an important economic crop, widely cultivated in most countries worldwide. Its shoots are used to produce various types of tea, one of the world's most widely consumed non-alcoholic beverages. The popularity of tea beverages stems primarily from their rich content of secondary metabolites, such as catechins, caffeine, and theanine, which contribute to their rich flavor and health benefits. Theanine, in particular, is one of the most important components contributing to the unique sensory qualities and health benefits of green tea. It imparts a refreshing sensation and alleviates bitterness. Theanine is the most abundant non-protein amino acid in tea plants, accounting for 1-2% of the dry weight of tea leaves and 40%-70% of the total free amino acids in tea shoots. Therefore, the theanine content determines the quality of green tea.

[0003] For tea plants, theanine is the most important form of nitrogen storage and transport. Theanine metabolism mainly consists of two parts: synthesis and hydrolysis. In winter, theanine is synthesized from glutamate and ethylamine by theanine synthase (CsTSI) and stored in the roots. Alanine decarboxylase (CsAlaDC) can catalyze the production of ethylamine from alanine, and its synergistic effect with CsTSI promotes high accumulation of theanine in tea plant roots. In addition, glutamine synthase (CsGSs) also has theanine synthase activity. In early spring, theanine is transported from roots to new shoots via CsAAPs transporters, causing theanine content in new shoots to gradually increase, reaching its peak in early April. In late spring, theanine is degraded in new shoots, possibly by CsPDX2.1 into glutamate and ethylamine. Recent studies have found that γ-glutamyl transpeptidase (CsGGT2) has theanine hydrolase activity. However, how theanine is transported from roots to new shoots and how it is transported and degraded in new shoot cells remains largely unknown.

[0004] Amino acids play crucial roles in plant growth and development, carbon and nitrogen balance, energy metabolism, and resistance to biotic and abiotic stresses. Amino acid degradation is the main pathway for dynamically regulating the content of free amino acids in plants, with degradation reactions primarily occurring in mitochondria and cytoplasm, and more frequently in mitochondria. Glutamine, a structural analogue of theanine, can be degraded in plant mitochondria, cytoplasm, and chloroplasts, but only in animal cell mitochondria. The enzyme most closely associated with the degradation of branched-chain amino acids (leucine, isoleucine, and valine) is the mitochondrial subtype BCAT2. In tea plants, glutamate can be oxidized and deaminated in mitochondria to form α-ketoglutarate. In animal cells, theanine is degraded to glutamate in mitochondria, thereby increasing glutathione synthesis and promoting apoptosis in animal cancer cells. Therefore, it is hypothesized that the degradation of theanine in tea plants occurs partially or entirely in mitochondria.

[0005] Mitochondria are organelles composed of two membranes: an outer membrane and an inner membrane. The outer membrane allows the entry of ions and uncharged molecules, while the inner membrane requires specific mitochondrial carriers to transport metabolites; therefore, amino acids require mitochondrial carriers to enter the mitochondria. Most mitochondrial carriers (MCs) have a molecular weight between 30-35 kDa and contain a transmembrane domain consisting of six α-helices. Adjacent transmembrane domains are linked by a characteristic sequence PX[D / E]XX[K / R]X[K / R](20-30 residues)[D / E]GXXXX[W / Y / F][K / R]G (PFAM PF00153). Currently, Arabidopsis mitochondrial carriers are the most studied in plants. BOU promotes the degradation of glutamate in mitochondria by transporting it into the mitochondria. Additionally, it promotes the synthesis of tetrahydrofolate from glutamate in mitochondria to regulate photorespiration and carbon and nitrogen metabolism. BAC1 and BAC2 transport arginine into the mitochondria, promoting arginine degradation to provide nitrogen nutrition for seedling development. AtUCP1 and AtUCP2 regulate the metabolism of aspartate, glycine, serine, and other amino acids, as well as photorespiration, by mediating aspartate / glutamate transport. However, mitochondrial carriers in tea plants have not been reported, and the regulatory mechanism of theanine accumulation in late spring tea shoots remains unclear.

[0006] In view of the above-mentioned defects, the inventors of this invention have finally obtained this invention after a long period of research and practice. Summary of the Invention

[0007] The purpose of this invention is to solve the problem of how to apply CsTHS1 to regulate the theanine content in late spring tea leaves, and to provide the application of the mitochondrial carrier CsTHS1 in regulating the accumulation of theanine in the new shoots of late spring tea trees.

[0008] To achieve the above objectives, this invention discloses the application of the mitochondrial vector CsTHS1 in regulating theanine accumulation in late spring tea shoots. By reducing CsTHS1 expression, the theanine content in spring tea leaves is increased. The base sequence of CsTHS1 is shown in SEQ ID NO.1. The expression of CsTHS1 promotes the transport of theanine into the mitochondria, where theanine is degraded, thus reducing the theanine content in the shoots.

[0009] Late spring is the period when the new shoots of tea trees grow rapidly.

[0010] The expression of CsTHS1 promotes the transport of theanine into mitochondria in new shoot cells.

[0011] The expression of CsTHS1 promotes the transport of theanine into mitochondria in new shoot cells and synergistically promotes the degradation of theanine with CsGGT2 and CsGDH2.1.

[0012] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention studies the regulatory mechanism of theanine accumulation in late spring tea shoots and discovers that the mitochondrial carrier CsTHS1 transports theanine into the mitochondria and synergistically promotes theanine degradation with CsGGT2 and CsGDH2.1, thereby reducing the theanine content in late spring tea shoots. Therefore, the mitochondrial carrier CsTHS1 negatively regulates theanine accumulation in tea shoots. Overexpression of CsTHS1 can significantly reduce theanine content in tea shoots, while reducing CsTHS1 expression can significantly increase theanine content in shoots. Therefore, this invention provides a technique to increase theanine content in late spring tea shoots by reducing the expression of the mitochondrial carrier CsTHS1. Attached Figure Description

[0013] Figure 1 A model for the accumulation of theanine in spring tea shoots regulated by CsTHS1, OG: α-ketoglutarate;

[0014] Figure 2 To assess the theanine sensitivity and accumulation of wild-type BY4743 and ths1 mutants, (A) the spotting experiment of BY4743 and ths1 on YNB solid medium containing 40 mM theanine, and the yeast strains were cultured on YNB solid medium containing 0 mM or 40 mM theanine for 3 days; (B) the theanine content in yeast after 6 h, 12 h and 18 h of treatment with 40 mM theanine.

[0015] Figure 3This is a phylogenetic tree of mitochondrial carriers with amino acid transport activity in yeast, humans, and Arabidopsis thaliana. Different colored circles represent different species: red for humans; green for Arabidopsis thaliana; and blue for Saccharomyces cerevisiae. The amino acids on the right side of the phylogenetic tree are the main substrates of the mitochondrial carriers. The numbers in the phylogenetic tree nodes are the branching values, indicating the confidence level of that branch.

[0016] Figure 4 Phylogenetic and structural analysis of THS1 homologs in tea plants: (A) Phylogenetic analysis of SLC25A44 homologs in different species. Different colored circles represent different species: red, tea plant; green, Arabidopsis thaliana; blue, human. The numbers of the phylogenetic tree nodes are the expansion values, indicating the confidence level of that branch. (B) These data were downloaded from the Tea Plant Genomes website (TPIA, https: / / tpia.teaplants.cn). (C) Sequence alignment and structural analysis of CSS0042480, CSS0016491, and CSS0021371. Characteristic motifs I, II, and III are mitochondrial vector characteristic sequences PX[D / E]XX[K / R]X[K / R] (20-30 amino acid residues)[D / E]GXXXX[W / Y / F][K / R]G. Blue indicates 100% sequence identity, magenta indicates 100%-80% sequence identity, and light blue indicates 80%-60% sequence identity. (D) By modeling the homology of SLC25A44, the stereo structures of CSS0042480, CSS0016491 and CSS0021371 were predicted. H1-H6 are transmembrane α-helical structures.

[0017] Figure 5 For yeast complementation experiments of CSS0042480 / ths1, CSS0016491 / ths1, and CSS0021371 / ths1, (A) wild-type BY4743, mutant ths1, pDR196 / ths1, CSS0042480 / ths1, CSS0016491 / ths1, and CSS0021371 / ths1 were dotted on YNB solid medium containing 40 mM theanine, and (B) yeast strains were cultured in YNB liquid medium containing 0 mM or 40 mM theanine, and the optical density (OD) at different time points was measured. 600 () indicates the growth status of yeast, (C) the theanine content in yeast after treatment with 10mM theanine for 1h, 3h and 6h;

[0018] Figure 6To analyze the theanine transport activity in yeast mitochondria, (A) subcellular localization of CsTHS1 in yeast cells. CsTHS1-EGFP is expressed in the ths1 mutant. Mito-Tracker is a mitochondrial-specific dye (MitoTrackerRed CMXRos). Scale bar: 3.1 μm. (B) Flowchart of yeast mitochondrial isolation and theanine uptake. Yeast mitochondria were isolated by differential centrifugation. After incubation with theanine for a period of time, the mitochondria were separated from the solution using a silicone oil separation method. (C) The transport rate of theanine in yeast mitochondria under different concentrations of theanine (50-500 μM). (D) The theanine content in mitochondria at different incubation times in the presence of 100 μM exogenous theanine.

[0019] Figure 7 To analyze the binding capacity of CsTHS1 to theanine and the theanine transport activity, (A) the response values ​​(RU) of CsTHS1 protein to different concentrations of theanine, glutamate, and glutamine. The KD value indicates the strength of CsTHS1's binding capacity to amino acids; the smaller the KD value, the stronger the binding capacity. (B) The recombinant and theanine uptake experimental procedure of CsTHS1 protein liposomes. (C) SDS-PAGE electrophoresis and Coomassie brilliant blue staining of CsTHS1 protein liposomes and blank liposomes. (D) Theanine content in protein liposomes after different incubation times with theanine. (E) The theanine uptake rate of protein liposomes after incubation with 4 mM theanine for 2 h.

[0020] Figure 8 Subcellular localization of CsTHS1 and CsGGT2 in tobacco, scale bar: 20 μm;

[0021] Figure 9 The expression patterns of CsTHS1 in tea plants (AB) and CsGGT2 in different tissues of tea plants, including buds, first leaf, second leaf, third leaf, and fifth leaf. (CE) The expression levels of CsTHS1 and CsGGT2 and theanine content in the first leaf of three tea varieties on April 6 and 19. (FI) The expression levels of CsTHS1, CsGGT2, and CsGDH2.1 and theanine content in the first leaf of tea plants treated at 22℃ and 33℃.

[0022] Figure 10To investigate the effects of transient silencing and overexpression of CsTHS1 on theanine accumulation, (A) new shoots of tea plants were treated with sODN-CsTHS1 and asODN-CsTHS1, respectively, with sODN-CsTHS1 treatment serving as a control. Scale bar: 2 cm. (B) The expression level of CsTHS1 and theanine content of new shoots treated with asODN-CsTHS1 and sODN-CsTHS1. (C) Transient overexpression of CsTHS1 in the first leaf of tea plants, with an empty vector EV serving as a control. Scale bar: 2 cm. (D) The expression level of CsTHS1 and theanine content of the first leaf of tea plants with transient overexpression of CsTHS1. Detailed Implementation

[0023] The above-mentioned and other technical features and advantages of the present invention will be described in more detail below with reference to the accompanying drawings.

[0024] I. Sensitivity of yeast ths1 mutant to high concentrations of theanine

[0025] 1. Yeast strains and culture methods

[0026] The yeast culture medium was prepared as follows: 1.7 g / L YNB (excluding ammonium sulfate and amino acids), 20 mg / L uracil; 30 mg / L leucine; 10 mg / L histidine; 5 g / L (NH4)2SO4; 20 g / L glucose; 20 g / L agar powder (solid medium); pH 5.5-6.0, autoclaved at 121℃ for 20 min. Theanine used in the theanine treatment was filtered through a 0.22 μm sterile filter and then added to the culture medium in the specified proportions. Wild-type BY4743 and the yeast mutant ths1 were cultured to OD200. 600 The absorbance was 0.6-0.8, and then diluted 10-fold, 100-fold, and 1000-fold. 2 μL of the diluted yeast solution was spotted onto YNB solid medium containing 0 mM or 40 mM theanine and incubated at 30°C for 3 days.

[0027] 2. Determination of theanine content in yeast

[0028] Add 1 mL of yeast to 99 mL of YNB liquid medium containing 0 mM or 40 mM theanine, and incubate at 30 °C for 6 h, 12 h, and 18 h. Collect 3 mL of yeast cells and measure OD. 600 The absorbance and theanine content were determined by HPLC. Yeast cells were collected by centrifuging 3 mL of yeast at 12000 g for 2 min. The yeast cells were washed twice with buffer and then once with sterile water. The yeast was resuspended in 1 mL of sterile water and heated in a 100℃ water bath for 30 min, vortexing every 10 min. After the water bath, the cells were centrifuged at 12000 g for 30 min. The supernatant was filtered through a 0.22 μm filter, and the theanine content was determined by HPLC.

[0029] The results showed that the mutant ths1 exhibited a hypersensitive phenotype to 40 mM theanine compared to the wild-type BY4743. Figure 2 A), and the theanine content in mutant ths1 was significantly higher than that in BY4743, indicating that ths1 accumulated more theanine in yeast cells than BY4743. Figure 2 B). These results indicate that THS1 is involved in regulating theanine accumulation in yeast, suggesting that its homolog in tea plants may be involved in regulating theanine accumulation in tea plant shoots.

[0030] II. Identification and Structural Characterization of THS1 Homologous Sequences in Tea Plants

[0031] 1. Construction of a phylogenetic tree of mitochondrial amino acid carrier proteins in Arabidopsis thaliana, yeast, and humans.

[0032] Nineteen mitochondrial amino acid carrier proteins from Arabidopsis thaliana, yeast, and humans were identified. A phylogenetic tree was constructed using MEGA7 software with THS1 and the 19 amino acid carrier proteins, employing the neighbor-joining method, and the Bootstrap value was set to 1000.

[0033] 2. Screening of THS1 homologous proteins in tea plants

[0034] Using the SLC25A44 sequence, THS1 homologs in tea and Arabidopsis were identified using the BLAST tool in the Tea Genome Database (TPIA) and the Arabidopsis Genome Database (TAIR), respectively. A phylogenetic tree was then constructed using MEGA7 software. The expression of THS1 homologs in different tissues of tea was searched on the Tea Genome Database to further screen for THS1 functional homologs.

[0035] 3. Sequence structure characteristics analysis of THS1 homologous proteins in tea plants

[0036] Sequence alignment of Group I members with SLC25A44 was performed using the online software Clustalw (https: / / www.genome.jp / tools-bin / clustalw), and visualization was performed using Genedoc to analyze conserved sequences and their characteristic sequences. The tertiary structure of THS1 homologous proteins in tea plants was predicted using the online tool Swissmodel.

[0037] Phylogenetic analysis of mitochondrial amino acid carriers in Arabidopsis thaliana, yeast, and humans revealed that THS1 is in the same branch as neutral amino acid carriers, and is closely related to SLC25A44. Figure 3SLC25A44 can regulate the catabolism of BCAAs (leucine, isoleucine, and valine) by transporting them into mitochondria; therefore, SLC25A44 should be a functional homolog of THS1, which is a neutral amino acid carrier. The SLC25A44 sequence was used to identify THS1 homologs in the tea plant genome (TPIA). Six possible THS1 functional homologs were identified in tea (CSS0042480, CSS0016491, CSS0021371, CSS0004119, CSS0031303, and CSS0023341). Figure 4 A).

[0038] Given that theanine degradation in tea trees mainly occurs in new shoots in late spring, we further screened THS1 functional homologs by analyzing the expression of CSS0042480, CSS0016491, CSS0021371, CSS0004119, CSS0031303, and CSS0023341 in different tissues of tea trees. CSS0042480, CSS0016491, and CSS0021371 showed higher expression in the aboveground parts and lower expression in the roots, especially CSS0042480, which was mainly expressed in young leaves. Figure 4 B). CSS0004119 and CSS0023341 have low expression levels in different organizations, while CSS0031303 is mainly expressed in the root. Figure 4 B). The tissue expression specificity of CSS0042480, CSS0016491, and CSS0021371 matches that of theanine-degrading tissues, suggesting that CSS0042480, CSS0016491, and CSS0021371 may be involved in regulating the accumulation of theanine in tea shoots. Sequence alignment revealed that CSS0042480, CSS0016491, and CSS0021371 possess a typical conserved sequence of mitochondrial vectors (PX[D / E]XX[K / R]X[K / R](20-30 amino acid residues)[D / E]GXXXX[W / Y / F][K / R]G)( Figure 4 C). Tertiary structure predictions indicate that they possess the typical tertiary structure of mitochondrial carriers. Figure 4 Therefore, these three MCs are considered as candidate MCs for transporting theanine and regulating the accumulation of theanine.

[0039] III. Effects of CSS0042480, CSS0016491, and CSS0021371 expression on theanine in yeast ths1 mutants.

[0040] 1. Gene cloning of CSS0042480, CSS0016491, and CSS0021371

[0041] Open reading frames CSS0042480, CSS0016491, and CSS0021371 were retrieved from the Tea Genome Database (TPIA) and amplified by polymerase chain reaction (PCR). The PCR products were then inserted into the pDR196 plasmid.

[0042] 2. Yeast Functional Complementarity Experiment

[0043] Plasmids CSS0042480, CSS0016491, and CSS0021371, cloned from the pDR196 vector, were introduced into the ths1 mutant. The ths1 mutant transformed from pDR196 served as a negative control. The cells were cultured until OD650. 600 The absorbance values ​​were 0.6-0.8, and then diluted 10-fold, 100-fold, and 1000-fold. 2 μL of the diluted yeast solution was spotted onto YNB solid medium containing 0 mM or 40 mM theanine and incubated at 30°C for 3 days. Yeast was also cultured in YNB liquid medium containing 40 mM theanine, and its OD values ​​were measured at different time points. 600 The absorbance value indicates the growth status. The yeast was treated with 10 mM theanine at different time points, and the theanine content in the yeast was measured using the method described in section one for the determination of theanine content in yeast.

[0044] like Figure 5 As shown in Figure A, the growth of all strains was inhibited on the medium containing 40 mM theanine, while CSS0042480 / ths1 recovered to wild-type levels. The absorbance of yeast in the medium containing 40 mM theanine was lower than that in the medium without theanine, with the yeast mutant ths1 being severely inhibited, while CSS0042480 / ths1 recovered to wild-type levels. Figure 5 B). Similarly, the theanine content in CSS0042480 / ths1 also decreased to the wild-type level. Figure 5 C), therefore, CSS0042480 can regulate the accumulation of theanine, and CSS0042480 is named CsTHS1.

[0045] IV. Subcellular localization of CsTHS1 and CsGGT2

[0046] Because mitochondrial vectors are not all localized within mitochondria, to determine the cellular localization of CsTHS1 and the degradation site of theanine, CsTHS1 and CsGGT2 were introduced into pCAMBIA1305 and transformed into Agrobacterium tumefaciens GV3101. Then, 35s::CsTHS1-GFP and 35s::CsGGT2-GFP were transiently expressed in tobacco epidermal cells along with the mitochondrial marker mt-rK-CD3-991. The subcellular localization of CsTHS1 and CsGGT2 was observed using a laser confocal microscope. CsTHS1-EGFP was also inserted into pDR196 and transformed into the yeast mutant ths1. The yeast cells were cultured at 30°C for 20 h. 4 ml of yeast solution was centrifuged at 12000 g for 5 min to collect the yeast cells. The cells were washed twice with HBSS and then incubated at 30°C for 30 min in HBSS solution containing the mitochondrial red fluorescent probe (MitoTracker Red CMXRos). After centrifugation at 12000g for 5 min, the sample was washed once with HBSS, then resuspended in 1 ml of HBSS and observed using a laser confocal microscope.

[0047] The results showed that the green fluorescence of CsTHS1-GFP completely overlapped with the red fluorescence of mtrK-CD-3991. Figure 8 The same result was observed with CsGGT2-GFP, which can co-localize with mt-rK-CD3-991. Therefore, CsTHS1 and CsGGT2 are located in mitochondria. Since CsGGT2 can catalyze the hydrolysis of theanine to glutamate and ethylamine, the degradation of theanine occurs in the mitochondria. Figure 8 Simultaneously, subcellular localization in yeast revealed that CsTHS1 is also located in mitochondria within yeast cells. Figure 6 A) Further analysis can be conducted by isolating yeast mitochondria to determine whether the mitochondrial carrier CsTHS1 may transport theanine.

[0048] V. Expression of CsTHS1 in yeast can promote theanine entry into mitochondria.

[0049] Yeast pDR196 / ths1 and CsTHS1 / ths1 were cultured in YPDA medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 6.5) until OD500. 600The absorbance was 1.5. Yeast cells were collected by centrifugation at 6000g for 5 min, washed once with sterile water, and then resuspended in buffer A (15 mM DTT, 0.1 M Tris-SO4, pH 9.4). The cells were incubated at 220g and 30°C for 20 min. Then, the cells were collected by centrifugation at 2000g for 5 min, washed once with 40 mL of buffer B (1.2 M sorbitol, 20 mM K3PO4, pH 7.4), and then resuspended in buffer B containing lysozyme. The cells were incubated at 30°C for 1 h.

[0050] The following procedures were performed on ice, with centrifugation at 4°C. Cell particles were washed twice with pre-chilled buffer B and centrifuged at 3500g for 5 min to collect globules. The globules were resuspended in 50 mL of buffer C (0.6 M sorbitol, 20 mM MES, pH 6.0) containing 1 mM PMSF. The mixture was then transferred to a glass homogenizer and homogenized 15 times. After homogenization, the mixture was centrifuged at 1700g for 5 min, and the supernatant was collected. The supernatant was centrifuged at 13500g for 10 min. Mitochondrial particles were washed with 35 mL of buffer C (PMSF-free), centrifuged at 1700g for 5 min, and the supernatant was collected. The supernatant was centrifuged at 13500g for 10 min. Mitochondrial particles were washed once with approximately 30 mL of buffer D (0.6 M sorbitol, 20 mM HEPES, pH 7.4) and transferred to a 50 mL centrifuge tube. The final mitochondrial particles were collected by centrifugation at 13500g for 10 min.

[0051] Freshly prepared yeast mitochondria (100 μg) were placed in 200 μL of theanine transport buffer (250 mM sucrose, 10 mM MTES, 0.5 mM ATP), and theanine was added to a final concentration of 100 μM. The mixture was incubated at 25 °C for different times. Alternatively, it was incubated for 60 min in transport buffers containing different concentrations of theanine. The mitochondria were then separated using a silicone oil layer separation method, and the theanine content of the supernatant and mitochondria was determined.

[0052] like Figure 6 As shown in Figure C, after incubation in 50-500 μM theanine for 60 min, the theanine content in the mitochondria of both yeast strains gradually increased. The theanine content in the mitochondria of CsTHS1 / ths1 was significantly higher than that of pDR196 / ths1. This indicates that CsTHS1 transports theanine into yeast mitochondria in a concentration-dependent manner. After incubation in 100 μM theanine for different times, the theanine content in these mitochondria showed a trend of first increasing and then decreasing. Figure 6(D) The theanine content in the mitochondria of pDR196 / ths1 yeast was highest at 10 min, while the theanine content in the mitochondria of CsTHS1 / ths1 yeast was highest at 60 min. Afterward, the theanine content gradually decreased with increasing culture time. The greatest difference in theanine content between the two yeast mitochondria occurred at 60 min, at which point the theanine content in the mitochondria of CsTHS1 / ths1 was almost twice that of the pDR196 / ths1 mitochondria. These results further support the theory that CsTHS1 transports theanine into the mitochondria.

[0053] VI. CsTHS1 has a high affinity for theanine.

[0054] CsTHS1 was introduced into the pGEX4T-1 vector and expressed as inclusion bodies in *E. coli*. Inclusion bodies were purified using an inclusion body protein lysis and refolding kit. Following the protein and small molecule binding guidelines, the protein was coupled to a CM7 chip using an amino-coupling kit. Theanine, glutamine, and glutamate were diluted to different concentrations with 1.05xPBS-P and then injected into the CM7 chip. The refractive index of the SPR sensor surface increased when amino acids bound to the protein. This change was expressed in resonance or response units (RUs). The affinities of CsTHS1 for glutamate, glutamine, and glutamate KD were calculated using a kinetic model.

[0055] The equilibrium dissociation constants (KD) of CsTHS1 for theanine, glutamine, and glutamate were 2.706 μM, 106.2 μM, and 291.4 μM, respectively. Figure 7 A). These results indicate that CsTHS1 can directly bind to amino acids, exhibiting a high affinity for theanine, a moderate affinity for glutamine, and a weak affinity for glutamate.

[0056] VII. CsTHS1 can directly transport theanine.

[0057] Total yeast lipids (25 mg / mL) were dissolved in a chloroform-methanol (3:1) solution. The lipids were dried in a rotary evaporator at 30 °C, followed by further drying in a vacuum desiccator for 30 min to remove methanol and trace amounts of chloroform. The dried lipids were then resuspended in buffer E (20 mM HEPES, 100 mM potassium chloride, 15 mM sodium chloride, 0.5 mM ATP, pH 7.0) at room temperature to a final concentration of 10 mg / mL. The lipid suspension was then sonicated to produce monovesicular (SUV) suspensions. The SUV suspensions were rapidly frozen in liquid nitrogen and then slowly thawed at room temperature for three cycles. 0.5% (w / v) of octyl-β-D-glucopyranoside was then added to destabilize the liposomes, and the mixture was incubated with stirring at room temperature for 3 h. Purified CsTHS1 was added to a mixture containing 0.5% (w / v) octyl-β-D-glucopyranoside at a liposome / protein ratio of 40:1 (w / w), and then incubated at 4°C with stirring for 2 h. Detergent was removed using Bio-Beads SM-2 (Bio-Rad) (wet weight) / detergent ratio of 60:1 (w / w), and the mixture was stirred at 4°C for 1–2 h. Bio-Beads SM-2 were then removed from the solution, and a second aliquot of Bio-Beads SM-2 was added. The sample was stirred at 4°C for 2–3 h, after which SM-2 was removed. Protein liposomes were separated by centrifugation at 210,000 g for 30 min at 4°C. The protein liposome microspheres were resuspended in buffer F (20 mM HEPES, 100 mM sodium chloride; pH = 6.0).

[0058] Protein liposomes were resuspended in transport buffer (20 mM HEPES, 100 mM sodium chloride, 4 mM theanine, pH 6.0) and incubated at 30 °C for different times. The protein liposomes were collected and centrifuged at 210,000 g for 30 min at 4 °C. The protein liposomes were washed three times with buffer F (20 mM HEPES, 100 mM sodium chloride, pH 6.0). The theanine content in the protein liposomes was determined.

[0059] like Figure 7 As shown in Figure C, the SDS-PAGE electrophoresis results of CsTHS1 recombinant protein liposomes and empty liposomes showed that the CsTHS1 protein liposomes contained a CsTHS1-GST band, while the empty liposomes did not, indicating that CsTHS1 had been successfully recombined into CsTHS1 protein liposomes. Then, the CsTHS1 protein liposomes were incubated with theanine for different times (…). Figure 7 (D) After 90 min of incubation, the theanine content in CsTHS1 protein liposomes was significantly higher than that in empty liposomes. Simultaneously, the theanine uptake rate of CsTHS1 protein liposomes was significantly higher than that of empty liposomes. Figure 7 E). These results confirm that CsTHS1 can directly transport theanine.

[0060] 8. CsTHS1 is highly correlated with the theanine content in the new shoots of late spring tea trees.

[0061] The samples for CsTHS1 tissue-specific expression analysis were from the cultivar “Shucha Zao” (SCZ) cultivated at the Nongcui Garden of Anhui Agricultural University. Total RNA was extracted from six tissues, including buds, stems, first leaf, second leaf, third leaf, and fifth leaf. Samples for CsTHS1 expression analysis in the first leaf of different tea varieties were from tea gardens in Xuancheng, Anhui. First leaf samples from three varieties were collected on April 9th ​​and April 16th. These varieties included “Zhongcha 108” (ZC108), “Zhenong 113” (ZN113), and “Suyu Huang” (SYH). Tea seedlings treated at different temperatures were from tea gardens in Chaohu. They were cultured at 22℃, light / dark for 16h / 8h, and humidity at 75% until new shoots emerged. Half of the seedlings were then transferred to 33℃, light / dark for 16h / 8h, and humidity at 75% for three consecutive days. The first leaves of the seedlings cultured at 22℃ and 33℃ were collected.

[0062] Total RNA was extracted using the FastPure Universal Plant Total RNA Kit. RNA was reverse transcribed into cDNA using the HiScriptIII All-in-one RT SuperMix Perfect for qPCR kit. qRT-PCR experiments were performed using the AceQ Universal SYBR qPCR Master Mix reagent on a QuantStudio 6Flex Real-Time System. CsGAPDH was used as an internal control gene. Data are presented as mean ± standard deviation (SD) of three independent replicates. 2 -ΔCt The relative expression level of genes can be calculated.

[0063] Given that theanine degradation mainly occurs in the young leaves of tea plants, the expression patterns of CsTHS1 and CsGGT2 in the aboveground parts of tea plants were then examined, including the buds, first leaf, second leaf, third leaf, fifth leaf, and stem of late spring shoots. The results showed that CsTHS1 and CsGGT2 were highly expressed in the first and second leaves, and their expression levels gradually decreased with increasing leaf maturity. Figure 9 A, B) correspond to the degradation sites of theanine in the leaves.

[0064] Given the significant decrease in theanine content in new shoots in late spring, the expression of CsTHS1 and CsGGT2 in tea shoots on April 6th and April 19th was further analyzed. The results showed that compared to leaves on April 6th, the expression of both CsTHS1 and CsGGT2 was significantly increased on April 19th, while the theanine content was significantly decreased. Figure 9CD). Considering the rapid rise in temperature in late spring, high temperatures reduce the accumulation of theanine in tea shoots. Tea seedlings were treated at 22℃ or 33℃ for 3 days, and the expression of CsTHS1, CsGGT2, and CsGDH2.1 and the theanine content in the first leaf were detected. Compared with 22℃, the theanine content at 33℃ was significantly reduced, while the 33℃ treatment significantly induced the expression of CsTHS1, CsGGT2, and CsGDH2.1 (CD). Figure 9 CsGDH2.1 has been reported to regulate theanine accumulation by modulating glutamate degradation in mitochondria, and is significantly induced in late spring. These results indicate that the expression levels of CsTHS1 and CsGGT2 increase in late spring, which may promote the transport of theanine to mitochondria for degradation. In this process, CsTHS1, CsGGT2, and CsGDH2.1 may work synergistically to promote theanine degradation in mitochondria.

[0065] 9. Transient silencing and overexpression of CsTHS1 altered the accumulation of theanine.

[0066] Using CsTHS1 as the input sequence, candidate asODN sequences were obtained using the Solido online software. New shoots containing buds and two leaves were immersed in 20 μM asODN solution at 22°C for 24 h. The sODN-treated shoots served as controls. The first leaf was collected for CsTHS1 expression and theanine content analysis.

[0067] CsTHS1 was inserted into the PK7WGF2.0 vector and introduced into Agrobacterium GV3101. Agrobacterium was cultured at 28°C, and the cells were then collected, resuspended in MMG buffer (10 mM magnesium chloride, 10 mM MES, 0.1 mM acetylsuccine), and the OD was adjusted. 600 The absorbance was adjusted to 1.0. Then, Agrobacterium was injected into the first leaf of the tea seedling using a syringe, with PK7WGF2.0 empty Agrobacterium as a control. After 24 h of dark culture and 3 days of normal light (light / dark, 16 h / 8 h) culture, the first leaf was collected for CsTHS1 expression analysis and theanine content detection.

[0068] To investigate the role of CsTHS1 in theanine accumulation, the expression of CsTHS1 in tea buds was transiently silenced using antisense oligonucleotides (asODN). Figure 10 A). The results showed that, compared with the control group (sODN-CsTHS1), the expression of CsTHS1 was significantly reduced after treatment with asODN-CsTHS1, while the content of theanine was significantly increased. Figure 10 B). Furthermore, CsTHS1 was transiently overexpressed in the first leaf of the tea plant. Figure 10 C). For example Figure 10As shown in Figure D, the expression level of CsTHS1 in the first leaf of the overexpressing tea plant was significantly increased, while the content of theanine was significantly decreased. These results indicate that CsTHS1 negatively regulates the accumulation of theanine in tea plants.

[0069] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, all of which will fall within the protection scope of the present invention.

Claims

1. The application of mitochondrial carrier CsTHS1 in regulating the accumulation of theanine in late spring tea shoots, characterized in that, The theanine content in spring tea can be increased by reducing the expression level of CsTHS1, the base sequence of which is shown in SEQ ID NO.

1.

2. The application of the mitochondrial carrier CsTHS1 as described in claim 1 in regulating the accumulation of theanine in late spring tea shoots, characterized in that, Late spring is the period when the new shoots of tea trees grow rapidly.

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