Application of key active site of mitochondrial vector CsTHS1

Abstract

The invention relates to the technical field of molecular biology, in particular to application of a mitochondrial vector CsTHS1 key active site, sequence evolution analysis is carried out on an amino acid sequence of CsTHS1, and it is found that an amino acid residue at the 307th position of the mitochondrial vector CsTHS1 sequence is evolved into arginine (R) from conservative glycine (G) of homologous species of the CsTHS1 sequence to regulate and control theanine transport activity of CsTHS1. R at the 307 position of the CsTHS1 sequence is mutated into G, so that the transport activity of theanine is reduced, and G at the corresponding site of homologous CSS0016491 is mutated into R, so that the transport activity of theanine is remarkably enhanced. Therefore, R307 in the CsTHS1 sequence is a key amino acid residue influencing the theanine transport activity, and the CsTHS1 theanine transport activity can be regulated and controlled by editing the site, so that theanine metabolism in the tea tree is regulated and controlled.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly relates to the application of key active sites of a mitochondrial carrier CsTHS1. Background Art

[0002] Camellia sinensis, a shrub or small tree of the genus Camellia in the family Theaceae, is an important cash crop and is widely cultivated in more than 50 countries around the world. Its leaves are processed into various types of tea, which is one of the most widely consumed non-alcoholic beverages globally. The wide popularity of tea is attributed to its pleasant taste and the various health benefits conferred by a large number of high-level secondary metabolites. The polyphenols, amino acids, catechins, caffeine, and aromatic compounds contained in tea affect the function and quality of tea. Among them, amino acids not only affect the health benefits of tea but also are important indicators determining the quality of tea leaves, and determine the overall quality of tea leaves by affecting the freshness and mellowness of tea.

[0003] Various amino acids in Camellia sinensis have different effects on the quality of tea leaves. Among them, theanine is one of the most important components in the unique sensory quality of green tea, which can endow green tea with a fresh taste and can offset the bitterness and astringency brought by caffeine, catechins, and catechin derivatives in green tea. Some studies have shown that theanine contributes to the production of volatiles, which may be the main sources of rice fragrance and chestnut fragrance. It is the most abundant non-protein amino acid in Camellia sinensis, with its content accounting for 1-2% of the dry weight of tea leaves and 40%-70% of the total free amino acids in the new shoots of Camellia sinensis, being the amino acid with the highest content in Camellia sinensis. Followed by glutamic acid (9%), arginine (7%), serine (5%), and aspartic acid (4%). Therefore, the content of theanine determines the quality of green tea.

[0004] Previous studies found that the yeast mitochondrial carrier YFR045Wp / THS1 may act as a neutral amino acid carrier in yeast cells and participate in regulating the accumulation of theanine in yeast under the condition of exogenous addition of theanine. In addition, its mutant ths1 can also be used as a genetic complementation system to study the role of neutral amino acids MCs in other species (including plants). Based on the analysis in yeast, a mitochondrial carrier CsTHS1 was found in Camellia sinensis, which can mediate the transport of theanine into mitochondria and promote the degradation of theanine in mitochondria, thereby reducing the content of theanine in the new shoots of Camellia sinensis in late spring. Currently, it is found that the expression of CsTHS1 increases significantly in late spring and is also significantly induced by high temperature, which is consistent with the increase in environmental temperature in late spring. However, theanine is a non-protein amino acid unique to Camellia sinensis. As a mitochondrial carrier for transporting theanine, whether there is evolution in the amino acid sequence of CsTHS1 and whether there are certain key amino acid residues affecting the theanine transport activity of CsTHS1 are currently unknown.

[0005] In view of the above defects, the inventors of the present invention have finally obtained the present invention through long-term research and practice. Summary of the Invention

[0006] The object of the present invention is to solve the problem of how to regulate the activity of tea amino acid transport through the mitochondrial carrier CsTHS1 so as to regulate the accumulation of theanine in the new shoots of tea trees, and provide an application of the key active site of the mitochondrial carrier CsTHS1.

[0007] To achieve the above object, the present invention discloses an application of the key active site of the mitochondrial carrier CsTHS1. The amino acid sequence of the mitochondrial carrier CsTHS1 is shown in SEQ ID NO.1. The key active site of the mitochondrial carrier CsTHS1 is arginine at position 307. After mutating the arginine at the key active site of the mitochondrial carrier CsTHS1 to glycine, it is used to increase the accumulation of theanine in the new shoots of tea trees.

[0008] After mutating the arginine at position 307 of the mitochondrial carrier CsTHS1 to glycine, it becomes CsTHS1 R307G , and the CsTHS1 R307G has an amino acid sequence shown in SEQ ID NO.2.

[0009] The homologous sequence of the mitochondrial carrier CsTHS1 is CSS0016491. After mutating the glycine corresponding to position 307 of the CsTHS1 sequence in the sequence CSS0016491 to arginine, the theanine transport activity is significantly enhanced. After mutating the glycine corresponding to position 307 of the CsTHS1 sequence in the homologous sequence CSS0016491 to arginine, it becomes CSS0019491 G297R .

[0010] The amino acid sequence of the sequence CSS0016491 is shown in SEQ ID NO.3, and the amino acid sequence of the sequence CSS0019491 G297R is shown in SEQ ID NO.4.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: Through the evolutionary analysis of the CsTHS1 sequence, the present invention identified that the arginine at position 307 in CsTHS1 is the key amino acid residue affecting its theanine transport activity. The increase in theanine transport activity after mutating the corresponding glycine at the same position in CSS0016491 to arginine further supports the above conclusion. Therefore, the present invention provides a key amino acid residue R307 affecting the theanine transport activity of CsTHS1, providing a target for gene editing. By reducing the theanine transport activity of CsTHS1, the accumulation of theanine in the new shoots of tea trees is increased, and the tea quality is improved. Brief Description of the Drawings

[0012] Figure 1 For the yeast functional complementation analysis of CsTHS1 / ths1 and CSS0016491 / ths1, BY4743 is the yeast wild type; ths1 is the yeast mutant with the YFR045W gene mutated and hypersensitive to 40 mM theanine; pDR196 / ths1 is the yeast mutant ths1 transformed with the empty vector pDR196 as the negative control; YFR045W / ths1, CsTHS1 / ths1, and CSS0016491 / ths1 are the strains that complement yeast ths1 with YFR045W, CsTHS1, and CSS0016491 respectively, and among them, YFR045W / ths1 is the positive control;

[0013] Figure 2 For the sequence alignment of the mitochondrial carrier CsTHS1 and its homolog CSS0016491 in tea plants, the sequence identity is 83.0%;

[0014] Figure 3 For the phylogenetic analysis of the homologs of CsTHS1 in related species, different colors represent different species; red represents tea plant (Camellia sinensis); blue represents kiwifruit (Actinidia chinensis); yellow represents grape (Vitis vinifera); purple represents mulberry (Morus notabilis); gray represents cacao (Theobroma cacao), and the numbers at the nodes of the phylogenetic tree are bootstrap values, indicating the confidence of the branch;

[0015] Figure 4 For the identification and site-directed mutagenesis of the specific amino acid sites of CsTHS1, among them, (A) is the multiple sequence alignment of CsTHS1 and its homologs, the asterisks represent the specific amino acid residues in the CsTHS1 sequence, and the numbers represent the positions of the corresponding amino acid residues in the CsTHS1 sequence; (B) is the changes of the amino acid residues and the corresponding bases in the site-directed mutagenesis of CsTHS1. The gray shadows represent the variations of the amino acid residues and their corresponding codons. The red arrows represent the variations of the corresponding bases;

[0016] Figure 5 For the yeast functional complementation analysis of CsTHS1 with different amino acid residue variations, among them, (A) is the theanine sensitivity analysis of the yeast complemented with different amino acid residue variation sequences of CsTHS1; (B) is the growth phenotypes of CsTHS1 / ths1 and CsTHS1 R307G / ths1 yeast in the medium of YNB ± 40 mM theanine; CsTHS1 R307GThe arginine at position 307 in the CsTHS1 sequence was mutated to glycine; the absorbance value of OD600 was used to represent the growth of yeast.

[0017] Figure 6 For the yeast functional complementation analysis of site-directed mutagenesis of amino acid residues in CSS0016491, where (A) is CSS0016491 G297R The changes in amino acid residues and corresponding bases in it. CSS0016491 G297R The glycine at position 297 in the CSS0016491 sequence was mutated to arginine; (B) CSS0016491 / ths1 and CSS0016491 G297R / ths1 yeast theanine sensitivity analysis; (C) is CSS0016491 / ths1 and CSS0016491 G297R / ths1 yeast growth phenotypes in the medium of YNB ± 40 mM theanine; the absorbance value of OD600 was used to represent the growth of yeast.

[0018] Figure 7 For CsTHS1 R307G and CSS0016491 G297R Analysis of theanine transport in proteoliposomes of proteins. Detailed implementation manners

[0019] The following combines the accompanying drawings to make a more detailed description of the above and other technical features and advantages of the present invention.

[0020] I. Yeast functional complementation analysis of CsTHS1 and its homolog CSS0016491 in tea plants

[0021] 1. Identification of homologs of CsTHS1 in tea plants and their multiple sequence alignments

[0022] Search for the amino acid sequences of CsTHS1 and CSS0016491 in the tea plant genome database (TPIA, Tea Plant Information Archive) (https: / / tpia.teaplants.cn / ). Then perform multiple sequence alignments through the Clustalw online software (https: / / www.genome.jp / tools-bin / clustalw), and then use GeneDoc for visualization to analyze their sequence identities.

[0023] 2. Gene cloning of CsTHS1 and CSS0016491

[0024] First, retrieve the DNA sequences of CsTHS1 (CSS0042480) and CSS0016491 from the tea plant genome database TPIA. In addition, retrieve YFR045W from the yeast genome database (SGD, Saccharomyces Genome Database) (https: / / www.yeastgenome.org / ), and amplify it by polymerase chain reaction (PCR). Then, insert the PCR product into the pDR196 plasmid.

[0025] 3. Yeast functional complementation analysis

[0026] Transform the recombinant plasmids of CsTHS1, CSS0016491, and YFR045W cloned on the pDR196 vector into the yeast mitochondrial carrier YFR045Wp / THS1 mutant yeast cells ths1 to obtain CsTHS1 / ths1, CSS0016491 / ths1, and YFR045W / ths1. At the same time, transform the empty vector pDR96 into the yeast cells of the ths1 mutant (pDR196 / ths1) as a negative control. Culture the yeast in YNB medium (1.7 g / L YNB (without 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, adjust the pH with potassium hydroxide) until the absorbance value at OD600 is 0.6 - 0.8. Then, centrifuge the yeast cells at 12000 x g for 2 minutes to collect them, and then resuspend them in sterile water to an OD600 of 0.6, and then dilute them 10-fold and 100-fold in sequence. Spot 2 μL of the diluted yeast solution on the YNB solid medium containing 0 mM or 40 mM theanine, and culture it at 30 °C for 3 days.

[0027] During the study of the function of CsTHS1, it was found that the yeast YFR045W mutant ths1 is hypersensitive to 40 mM theanine, and CsTHS1 can complement its hypersensitive phenotype to theanine. CsTHS1 can complement the hypersensitive phenotype of the yeast mutant ths1 to theanine by reducing the accumulation of theanine in yeast. Further, CsTHS1 can mediate the entry of theanine into mitochondria for degradation, thereby reducing the accumulation of theanine in the new shoots of tea plants. In addition, the homolog CSS0016491 of CsTHS1 cannot complement the hypersensitive phenotype of the yeast mutant ths1 to theanine, so CSS0016491 / ths1 is still hypersensitive to 40 mM theanine ( Figure 1 ). Through sequence alignment, it was found that the sequence identity between CSS0016491 and CsTHS1 is 83.0% ( Figure 2) CsTHS1 has a high sequence identity with CSS0016491, but CSS0016491 cannot restore the growth phenotype of the yeast mutant ths1 to the wild-type level, indicating that they have differences in mitochondrial carrier function. This functional difference may be caused by the differences in key amino acids in the sequences of CsTHS1 and CSS0016491.

[0028] II. Sequence Evolution Analysis and Site-Directed Mutagenesis of CsTHS1

[0029] 1. Sequence Evolution Analysis of CsTHS1

[0030] To conduct sequence evolution analysis of CsTHS1, the CsTHS1 sequence was used as the query object to search for homologous sequences in Actinidia chinensis, Vitis vinifera, Morus notabilis, and Theobroma cacao through the BLAST tool. Then, the MEGA7.0 software was used to construct a phylogenetic tree using the neighbor-joining method (bootstrap value of 1000). Next, the Clustalw online analysis software was used to perform multiple sequence alignments of CsTHS1 and its homologs to identify the specific amino acid residues of CsTHS1.

[0031] 2. Site-Directed Mutagenesis of CsTHS1

[0032] Based on the results of multiple sequence alignments, the specific amino acid residues in CsTHS1 were determined, and the specific amino acid residues were mutated to the conserved amino acid residues in the CsTHS1 homologs through a two-step cloning method according to the corresponding codons of the amino acids.

[0033] As Figure 3 shown, a phylogenetic tree was constructed for CsTHS1 and its homologs in tea plants, Actinidia chinensis, Vitis vinifera, Morus notabilis, and Theobroma cacao. The phylogenetic tree can be divided into three groups: Group I-A, Group I-B, and Group-II. CsTHS1 and its closest homolog CSS0016491 belong to Group I-A. This group includes the CsTHS1 homologs in Actinidia chinensis, Vitis vinifera, Morus notabilis, and Theobroma cacao ( Figure 3 ). Then, multiple sequence alignments were performed on CsTHS1, CSS0016491, PSR96178, PSR96933, EXB29679, WJZ94319, and XP017979429, which are closely related to CsTHS1 in Group I-A of the phylogenetic tree. The results showed that CsTHS1 contains 8 specific amino acid residues, namely C59, G74, F117, S120, G249, S251, F281, and R307 (Figure 4 (A)). To analyze the effects of the specific amino acid residues in the above eight CsTHS1s on the function of CsTHS1, according to the changes in the corresponding bases in the codon table, they were site-directed mutated into the conserved amino acid residues in other homologs, namely C59A, G74R, F117L, S120P, G249V, S251T, F281L, and R307G( Figure 4 (B)).

[0034] III. Yeast functional complementation and theanine transport analysis of CsTHS1 site-directed mutation

[0035] 1. Yeast functional complementation of CsTHS1 site-directed mutation

[0036] The coding sequences of the CsTHS1 site-directed amino acid mutations obtained in Part II were inserted into pDR196, and the recombinant plasmids were transformed into the yeast mutant ths1 to obtain CsTHS1 C59A / ths1, CsTHS1 G74R / ths1, CsTHS1 F117L / ths1, CsTHS1 S120P / ths1, CsTHS1 G249V / ths1, CsTHS1 S251T / ths1, CsTHS1 F281L / ths1 and CsTHS1 R307G / ths1 yeast strains. Then, according to the yeast functional complementation analysis method in "Implementation Method 1", the complementation of the CsTHS1 site-directed mutation to the growth phenotype of the yeast mutant was analyzed.

[0037] 2. Theanine transport analysis of CsTHS1

[0038] Yeast total lipids were dissolved in a chloroform-methanol mixture (3:1) to make the final concentration of lipids 25 mg / mL. Then, the lipid solution was used to remove methanol and chloroform through a rotary evaporator, and then a small amount of residual methanol and chloroform was removed by vacuum drying for 2 hours. The dried lipids were suspended with buffer E (20 mM HEPES, 100 mM KCl, 0.5 mM ATP, adjusted to pH 7.0 with NaOH) at 10 mg / mL to form a lipid solution, and then unilamellar vesicles were generated by sonication. The unilamellar vesicle solution was rapidly frozen with liquid nitrogen and then slowly thawed at room temperature. Such freezing and thawing were carried out for 3 cycles to form liposomes. Then, 0.5% octyl β-D-glucopyranoside was added to the liposome solution and incubated at room temperature for 3 hours to make the liposomes unstable. Then, CsTHS1 was added to the above proteoliposome solution R307GProtein, with a protein-to-lipid ratio of 40:1 (w / w), was stirred at 4 °C for 2 hours. Subsequently, octyl β-D-glucopyranoside was removed using Bio-beads SM-2 beads, with a Bio-beads SM-2 beads / octyl β-D-glucopyranoside ratio of 60:1 (w / w), and this process was repeated twice. Then, the proteoliposomes were separated from the Bio-beads SM-2 by centrifugation at 210000x g for 30 minutes at 4 °C.

[0039] The resulting proteoliposomes were resuspended in buffer F (20 mM HEPES, 100 mM NaCl, 4 mM theanine, pH adjusted to 6.0 with NaOH) and incubated at 30 °C for 120 minutes respectively. The proteoliposomes were collected by centrifugation at 210000x g for 30 minutes at 4 °C, washed three times with buffer F without theanine, and the theanine content in the proteoliposomes was quantified by high performance liquid chromatography.

[0040] As Figure 5 (A) shows that the CsTHS1 / ths1, CsTHS1 C59A / ths1, CsTHS1 G74R / ths1, CsTHS1 F117L / ths1, CsTHS1 S120P / ths1, CsTHS1 G249V / ths1, CsTHS1 S251T / ths1, CsTHS1 F281L / ths1 and CsTHS1 R307G / ths1 yeast strains, after being cultured in YNB medium containing 40 mM theanine medium, it was found that the growth of CsTHS1 C59A / ths1, CsTHS1 G74R / ths1, CsTHS1 F117L / ths1, CsTHS1 S120P / ths1, CsTHS1 G249V / ths1, CsTHS1 S251T / ths1, CsTHS1 F281L / ths1 strains was similar, however, the growth of CsTHS1 R307G / ths1 yeast was significantly weaker than that of CsTHS1 / ths1, showing a phenotype sensitive to theanine. The results indicate that CsTHS1 R307G cannot complement the theanine hypersensitive phenotype of the yeast mutant ths1. Then, a shaking culture experiment was carried out in YNB liquid medium containing 0 mM and 40 mM theanine, and the absorbance value of OD600 was measured at different culture times to represent the growth phenotype of the yeast. The results showed that CsTHS1R307G The growth of / ths1 was significantly weaker than that of CsTHS1 / ths1 yeast ( Figure 5 (B)). These results suggest that the mutation of arginine (R) to glycine (G) at position 307 in CsTHS1 affects the function of the mitochondrial carrier.

[0041] To further analyze whether the R307G mutation in CsTHS1 affects the theanine transport activity of CsTHS1 and thus affects the sensitivity of CsTHS1 R307G / ths1 yeast to theanine. Through recombinant protein liposomes and theanine transport analysis, it was found that the theanine transport activity of CsTHS1 R307G protein liposomes was significantly lower than that of CsTHS1 protein liposomes ( Figure 7 ). These results indicate that the arginine at position 307 in CsTHS1 is a key amino acid residue affecting the theanine transport activity of CsTHS1.

[0042] IV. Yeast functional complementation and theanine transport analysis of CSS0016491 site-directed mutation

[0043] 1. Site-directed amino acid mutation of CSS0016491

[0044] According to the sequence alignment results, G297 was mutated to arginine (R) at the corresponding site of the mutation site in CsTHS1 in CSS0016491, and it was implemented according to the method in "II". R307G

[0045] 2. Yeast functional complementation of CSS0016491 site-directed mutation

[0046] Analyze the functional complementation of CSS0016491 site-directed mutation to the yeast mutant ths1 according to the method in "III".

[0047] 3. Theanine transport analysis of CSS0016491 site-directed mutation

[0048] Analyze the theanine transport of CSS0016491 site-directed mutation according to the analysis in "III".

[0049] Figure 6 To further determine the key role of arginine (R) at position 307 in the CsTHS1 sequence in theanine transport of CsTHS1, glycine (G) at the corresponding position of CSS0016491, which cannot complement the theanine sensitivity phenotype of the yeast mutant ths1, was mutated to arginine (R), that is, CSS0016491 G297R ( Figure 6 (A)). As Figure 6 (B) shows, CSS0016491 G297RThe growth of / ths1 yeast was significantly better than that of CSS0016491 / ths1 yeast, indicating that CSS0016491 G297R could complement the hypersensitive phenotype of the yeast mutant ths1 to L-theanine. Similar results were obtained in the yeast liquid shaking culture experiment ( Figure 6 (C)). Meanwhile, the results of the L-theanine transport experiment using proteoliposomes showed that CSS0016491 G297R accumulated significantly more L-theanine in proteoliposomes than CSS0016491 proteoliposomes, indicating that the mutation of glycine (G) to arginine (R) at position 297 in CSS0016491 significantly enhanced the L-theanine transport activity. These results further confirmed the important role of arginine (R) at this position in the L-theanine transport activities of CsTHS1 and CSS0016491.

[0050] The above are only the preferred embodiments of the present invention, which are illustrative rather than restrictive to the present invention. Those skilled in the art understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, but all of them will fall within the protection scope of the present invention.

Claims

1. Application of key active sites of mitochondrial vector CsTHS1, characterized in that, The amino acid sequence of the mitochondrial vector CsTHS1 is shown in SEQ ID NO.

1. The key active site of the mitochondrial vector CsTHS1 is the arginine at position 307. After mutating the arginine at the key active site of the mitochondrial vector CsTHS1 to glycine, it is used to increase the accumulation of theanine in the new shoots of tea plants.

2. Use of the key active site of mitochondrial vector CsTHS1 according to claim 1, characterized in that, After the arginine at position 307 of the mitochondrial vector CsTHS1 is mutated to glycine, it becomes CsTHS1 R307G , said CsTHS1 R307G has the amino acid sequence shown in SEQ ID NO.

2.

3. Use of the key active site of mitochondrial vector CsTHS1 according to claim 1, characterized in that The mitochondrial carrier CsTHS1 homologous sequence is CSS0016491. After the glycine corresponding to position 307 of the CsTHS1 sequence in the sequence CSS0016491 is mutated to arginine, the theanine transport activity is significantly enhanced. After the glycine corresponding to position 307 of the CsTHS1 sequence in the homologous sequence CSS0016491 is mutated to arginine, it becomes CSS0019491 G297R .

4. Use of a key active site of mitochondrial vector CsTHS1 according to claim 1, characterized in that, The amino acid sequence of the sequence CSS0016491 is shown in SEQ ID NO.3, and the sequence CSS0019491 G297R has an amino acid sequence shown in SEQ ID NO.4.

Images