An isolated csCARME protein and uses thereof

Abstract

This invention relates to the field of genetic engineering technology, and more particularly to an isolated CsCARME protein and its applications. The invention provides an isolated CsCARME protein, the amino acid sequence of which is shown in SEQ ID NO.2. Through multi-level experimental verification including genome-wide association analysis, expression association analysis, transient overexpression, virus-induced gene silencing, heterologous transgenesis, and Arabidopsis mutant complementation, this invention fully demonstrates that increasing CsCARME gene expression significantly promotes the accumulation of free amino acids, while decreasing its expression reduces amino acid content. This invention provides a novel molecular target for the targeted regulation of free amino acid content in tea plants, filling a gap in the field of amino acid metabolism regulation in tea plants and possessing significant theoretical innovation value.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to an isolated CsCARME protein and its applications. Background Technology

[0002] Tea, processed from the leaves of the tea plant, is a widely consumed health beverage globally. Its quality characteristics, especially its fresh, sweet, and mellow flavor, are primarily determined by the secondary metabolites accumulated in the leaves. Free amino acids are a key class of non-volatile quality components in tea. Among them, L-theanine, a characteristic amino acid unique to tea, not only imparts a significant umami flavor to the tea infusion but also possesses various health benefits such as stress relief, sleep improvement, and immune regulation. Therefore, increasing the accumulation of free amino acids in tea leaves has become one of the core objectives of tea quality improvement and functional component breeding.

[0003] However, traditional tea breeding methods mainly rely on hybridization and phenotypic selection, which suffer from problems such as long cycles, low efficiency, and low precision in trait selection. As a perennial woody plant, the tea tree has a complex genetic background, high heterozygosity, and long generation cycle, which limits the practical application of traditional phenotypic-based breeding strategies. In recent years, with the rapid development of modern molecular biology and genetic engineering technologies, identifying key functional genes that regulate important quality traits in tea trees and using techniques such as gene overexpression, gene silencing, and gene editing for targeted improvement has become an effective technical approach for cultivating high-quality new tea varieties.

[0004] Although some studies have reported genes related to amino acid metabolism in tea plants, such as CsGS and CsTS, the molecular regulatory mechanisms of free amino acid accumulation in tea plants remain incompletely understood, especially the limited resources of key genes involved in amino acid transport, methylation modification, or upstream regulation of metabolic pathways. Carnosine N-methyltransferase has been reported to participate in dipeptide methylation modification in animals, but the function of its homologous gene in plants, particularly tea plants, has not been reported. The lack of research on this functional gene limits the in-depth analysis of the amino acid metabolism regulatory network in tea plants.

[0005] Genome-wide association analysis (GWAS) is an effective tool for elucidating the genetic basis of complex traits. It can rapidly locate genetic loci significantly associated with target traits at the whole genome level, providing important clues for the discovery of candidate genes. This invention utilizes the GWAS strategy, combined with transcriptional expression analysis and functional validation, to identify for the first time a gene significantly associated with free amino acid content—CsCARME—in tea plants, and systematically validates its positive function in regulating the accumulation of free amino acids in tea leaves. This gene provides a new molecular target for tea quality improvement and also provides a theoretical basis and genetic resource for marker-assisted breeding and genetic engineering breeding of tea plants. Summary of the Invention

[0006] The purpose of this invention is to provide an isolated CsCARME protein and its applications.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an isolated CsCARME protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] The present invention provides an isolated nucleic acid molecule that encodes the CsCARME protein.

[0009] Preferably, its nucleotide sequence is shown in SEQ ID NO.1.

[0010] The present invention provides a recombinant expression vector containing the nucleic acid molecule.

[0011] The present invention provides that the recombinant expression vector is an overexpression vector pSH737-35S-CsCARME or a silencing vector pTRV2-CsCARME.

[0012] The present invention provides a recombinant host cell containing the recombinant expression vector.

[0013] This invention provides the application of CsCARME protein or the nucleic acid molecule encoding it in regulating the content of free amino acids in tea plants or improving the quality of tea. The amino acid sequence of the CsCARME protein is shown in SEQ ID NO.2, and this is achieved by regulating the expression level or activity of the protein.

[0014] This invention provides a method for regulating the content of free amino acids in tea leaves. The method regulates the accumulation of free amino acids in tea leaves by controlling the expression level or activity of CsCARME protein. The amino acid sequence of CsCARME protein is shown in SEQ ID NO.2.

[0015] This invention provides the application of the protein, or the nucleic acid molecule, or the recombinant expression vector, or the recombinant host cell in the quality improvement or breeding of tea plants.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first to isolate and identify the CsCARME gene from tea plants and systematically reveal its key biological function in positively regulating the accumulation of free amino acids in tea leaves. Through multi-level experimental verification including genome-wide association analysis, expression association analysis, transient overexpression, virus-induced gene silencing, heterologous transgenesis, and Arabidopsis mutant complementation, it is fully demonstrated that increasing CsCARME gene expression significantly promotes free amino acid accumulation, while decreasing its expression reduces amino acid content. This invention provides a novel molecular target for the targeted regulation of free amino acid content in tea plants, filling a gap in the field of amino acid metabolism regulation in tea plants and possessing significant theoretical innovation value.

[0017] This invention provides a practical technical solution based on the CsCARME gene, including various regulatory methods such as gene overexpression, gene silencing, or gene editing, as well as specific recombinant expression vectors and recombinant host cells. Through Agrobacterium-mediated transient transformation or VIGS technology, positive or negative regulation of free amino acid content can be efficiently achieved in tea plants, thus providing a direct and feasible genetic engineering strategy for improving the freshness and flavor quality of tea. Simultaneously, haplotype analysis results of the CsCARME gene region lay the genetic foundation for developing marker-assisted selection for high-amino acid traits.

[0018] Furthermore, this invention, through stable transformation in tobacco and replacement experiments with Arabidopsis homologous gene mutants, confirms the cross-species functional conservation of the CsCARME gene in regulating amino acid accumulation, further broadening its application prospects. The gene, protein, nucleic acid molecules, recombinant vector, and host cell described can be widely applied to tea quality improvement or breeding, providing important gene resources and tools for cultivating new tea varieties with high free amino acid content, demonstrating significant industrial application value and promising commercial prospects. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 This is a normal distribution diagram of the free amino acid content of 108 strains.

[0021] Figure 2 Manhattan plot (left) of CsCARME gene localization for genome-wide association analysis (GWAS) and QQ plot of GWAS results (right).

[0022] Figure 3 The correlation analysis of the relative expression level of the CsCARME gene in tea varieties with different free amino acid contents (left figure) and free amino acid content (right figure).

[0023] Figure 4 This is a subcellular localization map of the CsCARME protein in tobacco leaf cells.

[0024] Figure 5 The graph shows the changes in relative gene expression (left) and free amino acid content (right) after transient overexpression of CsCARME in tea leaves.

[0025] Figure 6 The graph shows the changes in relative gene expression levels (left) and free amino acid content in tea leaves after virus-induced gene silencing (VIGS) CsCARME (right).

[0026] Figure 7 The left figure shows the relative gene expression level (left figure) and free amino acid content of CsCARME transgenic tobacco plants (right figure).

[0027] Figure 8 The results of the complementation experiment for transforming Arabidopsis homologous gene mutants with the CsCARME gene include phenotype (left), relative gene expression level (middle), and free amino acid content (right). Detailed Implementation

[0028] Preparation of related culture media in this invention: (1) LB solid medium: 10 g / L tryptone + 10 g / L sodium chloride + 5 g / L yeast extract + 7.5 g / L agar powder (2) LB liquid medium: 10 g / L tryptone + 10 g / L sodium chloride + 5 g / L yeast extract (3) YEP solid medium: 10 g / L peptone + 10 g / L yeast extract + 5 g / L sodium chloride + 7.5 g / L agar powder (4) YEP liquid medium: 10 g / L yeast extract + 10 g / L peptone + 5 g / L sodium chloride All biological materials used in the following experiments are commercially available.

[0029] The sequences used in the following examples: The nucleotide sequence of the CsCARME gene, SEQ ID NO.1 (CSS0043590) ATGTTTTGTAAACTGAGGCGGAAGGAGAGAAAAAGAGTCGTGTTTGGCTGGGAGGAAAAA GGAAAAAGAAAATGTCGAAGAGCGACGTGGAGGAGGAGCAGCATGAGGAACTGCGACGCC GAAAGCTTGAAGAAAGCCCTTGAAGTCAAATCTCTTCGACGTATTATCAGCGCATACCTC AATTATCCAGATGCTGCAGAAGATGATGTAAAAAGATATGAAAGATCTTTTGCAAGGCTT CCACCTGCCCACAAGGGTCTGCTGTCCCACCTCCCAGTAAAATTTCAAAGACTGAGATGG TGTATTTCAAAAAATTCATTTTTCATATTTAACATGCTTCAGGCATTTGATCCCCCAATT GATATGAGCCAGGAAATTGATATTTGTCAACACCAAAATCTGGAAAATTTCCCGGACTAT AATATTCTTGCTGGTGAAAGAAATGCTTGCTCTTGCCAATCTGCCTCAACAAGTGGAAGA GTAGCTTTATTGAAATCTGATGAAGCTTGCTGTGGAGAAGGAAGCAATATAACATGCAGG TCACCTGATGGGCATTTCTCTTTATTCTTTTCTGATCCCATTGGCAATATATTTTGTGTT ATTTTATTCAACCATGATTACGGATTCCTAACTGATGTATCCTCATCACCTCCTGATTGG TTAGATCCATCATTTCAGTTACATGTCCCCTTAGTTGATGTGGATAAGGTTCGCTGCATT ATCAGGAACATTGTAAGAGATTGGGCAGCTGAGGGGCAGAGAGAACGTGATCAGTGCTAC AAGCCCATTCTTGAAGAACTTGAAATTCAATTCTCTGTTCGCCAGAAGGATTGCCCTCCT GCCTGTTTAGTTCCTGGTGCTGGACTTGGTAGGCTGGCTTTGGAAATTTCATGTCTAGGT TTTGTAAGCCAGGGGAATGAATTTTCATACTATATGATGATTTGTTCAAGTTTTATTCTT AACCAGACTCAGACTGTTGGGGAGTGGACAATATATCCTTGGATTCATAGCAATTGCAAT TCACTTTCTGACAGTGATCAGCTTCGTCCAGTTTCAATTCCAGATATTTTTCCAGCTAGT GCAGGGATTACTGATGGCTTTTCTATGTGTGGAGGTGACTTTGTTGAAGTGTACAGTGAT CCAAGCCAAGTAGGAGTTTGGGATGCAGTTGTGACATGCTTCTTTATTGATACAGCACAC AACATCGTAGAATATATTGAAATCATTTCAAGAATCCTCAAAGATGGGGGAGTATGGATA AACTTGGGTCCTCTCCTTTATCACTTTGCAGACGTGTATGGTCAGGAAGATGAGATGTCC ATTGAACTAAGTTTAGAAGATGTAAGAAGGGTCGCTTTCCATTATGGTTTTCAGTTGGAG AAAGAGAAGACCATTGAGACAACCTATACTACAAACCCTAGGTCAATGATGCAATTTTGG AGTATGGGAGTTTGTAATGATTTGATATTTTGTTGTAGTTGA Amino acid sequence of CsCARME gene SEQ ID NO.2 (CSS0043590) MFCKLRRKERKRVVFGWEEKGKRKCRRATWRRSSMRNCDAESLKKALEVKSLRRIISAYL NYPDAAAEDDVKRYERSFARLPPAHKGLLSHLPVKFQRLRWCISKNSFFIFNMLQAFDPPI DMSQEIDICQHQNLENFPDYNILAGERNACSCQSASTSGRVALLKSDEASCGEGSNITCR SPDGHFSLFFSDPIGNIFCVILFNHDYGFLTDVSSSPPDWLDPSFQLHVVPLVDVDKVRCI IRNIVRDWAAEGQRERDQCYKPILEELEIQFSVRQKDCPPACLVPGAGLGRLALEISCLG FVSQGNEFSYYMMICSSFILNQTQTVGEWTIYPWIHSNCNSLSDSDQLRPVSIPDIFPAS AGITDGFSMCGGDFVEVYSDPSQVGVWDAVVTCFIDTAHNIVEYIEIISRILKDGGVWI NLGPLLYHFADVYGQEDEMSIELSLEDVRRVAFHYGFQLEKEKTIETTYTTNPRSMMQFW SMGVCNDLIFCCS The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1: GWAS Localization and Haplotype Analysis

[0031] (I) Experimental Methods

[0032] 1. Test Materials and Determination of Free Amino Acid Content: The test materials were 108 varieties of Yunwu Gongcha tea, collected from tender leaves. The determination of free amino acid content was carried out in accordance with the People's Republic of China National Standard GB / T 8314-2013 "Determination of Total Free Amino Acids in Tea". The specific steps are as follows: Accurately weigh 1.0 g of ground tea sample (accurate to 0.001 g), place it in a 250 mL Erlenmeyer flask, add 100 mL of boiling distilled water, and immediately extract in a boiling water bath for 30 min (shaking once every 10 min). After extraction, immediately filter under reduced pressure, cool the filtrate, and dilute to 100 mL with distilled water, then mix well. Accurately pipette 2 mL of the test solution into a 25 mL volumetric flask, add 2 mL of pH 5.6 phosphate buffer and 2 mL of 2% ninhydrin solution, mix well, heat in a boiling water bath for 15 min, cool to room temperature, dilute to 25 mL with water, and let stand for 10 min. Using a blank solution as a reference, the absorbance value was measured at a wavelength of 570 nm, and the total amount of free amino acids in the sample was calculated (expressed as dry mass fraction) based on the glutamic acid standard curve.

[0033] 2. Genome-wide association analysis: 108 strains were resequencing, and GWAS analysis was performed using the obtained SNP data and the free amino acid content mentioned above. Association analysis was conducted using TASSEL software with a mixed linear model (MLM).

[0034] 3. Candidate Gene Screening and Haplotype Analysis: Based on the GWAS mapping results, candidate genes were annotated in the Tea Tree Genome Information Database (TPIA), resulting in a gene with the ID CSS0043590 (from the TPIA database), named CsCARME. Haplotype analysis was performed on the SNPs in this gene region to identify different haplotypes and their association with free amino acid content.

[0035] (II) Experimental Results

[0036] like Figure 1 As shown, the content of free amino acids in the Yunwu Gongcha tea group exhibits a continuous normal distribution with rich variation. For example... Figure 2 As shown in the left and right figures, GWAS analysis identified a locus on chromosome 5 that was highly significantly associated with the content of free amino acids (-log). 10(P) ≈ 5.8). Analysis revealed that Hap001 was the dominant haplotype, accounting for 87.9% (29 / 33) of the total sample, with the allele combination GAATG. Hap002 (AATAG) and Hap003 (GAATA) each accounted for 6.1% (2 / 33 each), and Hap004 (AGTAG) accounted for 3.0% (1 / 33). E represents the locus of the haplotype. The p-value for Hap003 was 0.001, reaching a highly significant level; the p-value for Hap002 was 0.059, close to but not reaching the significance threshold. The candidate gene in this region was CsCARME. Haplotype analysis showed that this gene has four haplotypes, among which Hap003 was highly significantly associated with high amino acid content (p=0.001). These results indicate that CsCARME is a key candidate gene closely related to the variation in free amino acid content in tea plants.

[0037] Example 2: Association Analysis of CsCARME Gene Expression and Free Amino Acid Content

[0038] (I) Experimental Methods

[0039] 1. Sample selection: Seven tea tree strains with significant differences in the content of free amino acids in the Guiding Yunwu 108 Bird King variety were selected, namely NW9, NW81, NW42, NW64, NW70, NW73 and NW25.

[0040] 2. Total RNA Extraction: Total RNA was extracted from leaves of each strain using the CTAB method. 0.1 g of fresh leaves were ground into powder using liquid nitrogen, and 1 mL of preheated RNA extraction buffer (containing 2% β-mercaptoethanol) was added. The mixture was incubated at 65°C for 10 min. An equal volume of chloroform:isoamyl alcohol (24:1) mixture was added, and the mixture was vigorously shaken and centrifuged. The supernatant was collected, and an equal volume of 5M NaCl and an equal volume of isopropanol pre-cooled at -20°C were added. The mixture was incubated at -20°C for 2-3 hours to precipitate. The precipitate was collected by centrifugation, washed twice with 75% ethanol, dried, and then dissolved in RNase-free water.

[0041] 3. cDNA synthesis: Using the extracted total RNA as a template, the first strand of cDNA was synthesized according to the instructions of the reverse transcription kit.

[0042] 4. Real-time quantitative PCR: The expression level of the CsCARME gene was detected by qRT-PCR. The tea plant Actin gene was used as an internal control. The quantitative primer sequences for the CsCARME gene were: forward primer 5'-GTTCTGTCTTCTCCCATGCAGCC-3' (SEQ ID NO.3), and reverse primer 5'-GGAGGAGGAGCAGCATGAGGAA-3' (SEQ ID NO.4). The PCR reaction system consisted of 20 μL: 2 μL cDNA, 10 μL 2×SYBR Green Master Mix, 0.4 μL each of the forward and reverse primers, and 7.2 μL ddH2O. The reaction program was: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 10 sec, 60℃ annealing for 20 sec, and 72℃ extension for 30 sec, for a total of 40 cycles. 2 -ΔΔCt The method calculates the relative expression level of genes.

[0043] 5. Correlation analysis: The expression level of CsCARME gene in each strain was correlated with the free amino acid content determined according to GB / T 8314-2013.

[0044] (II) Experimental Results

[0045] like Figure 3 As shown, the expression trend of the CsCARME gene is highly consistent with the changing trends of free amino acid content in each strain, and the two are significantly positively correlated. This result indicates that the expression level of CsCARME directly affects the accumulation of free amino acids in tea leaves.

[0046] Example 3 Subcellular localization of CsCARME protein

[0047] (I) Experimental Methods

[0048] 1. Vector construction: The CDS sequence of CsCARME was cloned into the pCAMBIA2301-35S-EGFP vector by homologous recombination or double enzyme digestion ligation method to construct the pCAMBIA2301-35S-CsCARME-EGFP fusion expression vector, and the sequence was verified to be correct.

[0049] 2. Agrobacterium transformation: The above recombinant plasmid and empty vector control were transformed into Agrobacterium tumefaciens GV3101 competent cells by freeze-thaw method, respectively, and plated on YEP solid plates containing kanamycin (100 mg / L) and rifampin (20 mg / L), and cultured at 28°C for 48 h.

[0050] 3. Transient expression in tobacco: Single colonies of positive Agrobacterium were picked and inoculated into YEP liquid medium (containing the corresponding antibiotics) and cultured at 28°C with shaking at 220 rpm until OD500.600 The concentration was 0.6-0.8. The bacterial cells were collected by centrifugation, resuspended in a resuspension buffer (containing 10 mM MES, 10 mM MgCl2, 150 μM acetylsalicylic acid, pH 5.6), and the OD was adjusted. 600 To a concentration of 0.6. After standing at room temperature for 3 hours, inject the bacterial solution into the lower epidermis of *Tobacco Benedict* leaves using a syringe without a needle. Incubate in the dark overnight after injection, then resume normal light incubation for 2-3 days.

[0051] 4. Fluorescence observation: Cut off the leaf from the injection area, peel off the lower epidermis, prepare a temporary slide, and observe the green fluorescence signal under a laser confocal scanning microscope.

[0052] (II) Experimental Results

[0053] like Figure 4 As shown, the green fluorescence signal of the CsCARME-EGFP fusion protein is specifically located on the cell membrane, while the fluorescence of the empty control EGFP is diffuse throughout the cell. This result indicates that the CsCARME protein is localized to the cell membrane, suggesting its potential involvement in membrane-associated transport or signal transduction.

[0054] Example 4: Transient overexpression of CsCARME in tea leaves

[0055] (I) Experimental Methods

[0056] 1. Vector construction: The CDS sequence of CsCARME was cloned into the pSH737 vector to obtain the pSH737-35S-CsCARME overexpression vector, and the sequence was verified to be correct.

[0057] 2. Preparation of Agrobacterium infection solution: Agrobacterium LBA4404 strain containing the overexpression vector and empty vector pSH737 was cultured and the cells were collected according to the method in Example 3. The cells were resuspended in infection buffer (containing 4.74 g / L MS, 30 g / L sucrose, 2 mg / L 6-BA, 150 μmol / L AS, 0.1 mg / L NAA, pH 5.8) and the OD was adjusted. 600 Up to 0.8.

[0058] 3. Leaf Injection: Using mature branches of the "Wuniuzao" tea tree as material, the 3rd and 4th unfolded leaves were selected. Overexpressing bacterial suspension and empty bacterial suspension were injected into different areas of the same leaf using a syringe without a needle. Each treatment was repeated three times, with three leaves per replicate. Leaves from the injected areas were collected 3-5 days after injection.

[0059] 4. Expression and Content Detection: The collected leaf samples were divided into two parts. One part was flash-frozen in liquid nitrogen and RNA was extracted. The expression level of CsCARME was detected by qRT-PCR according to the method in Example 2. The other part was immediately blanched, dried, and ground. The free amino acid content was determined according to the national standard GB / T 8314-2013.

[0060] (II) Experimental Results

[0061] like Figure 5 As shown, compared with the empty control, the expression level of the CsCARME gene was upregulated by up to 2.5 times in leaves overexpressing CsCARME, and the content of free amino acids increased by up to 25%. The results indicate that transient overexpression of CsCARME can significantly promote the accumulation of free amino acids in tea leaves.

[0062] Example 5: VIGS Silencing CsCARME

[0063] (I) Experimental Methods

[0064] 1. Construction of silencing vector: A specific fragment of the CsCARME gene (approximately 300-500 bp) was cloned into the pTRV2 vector to obtain the pTRV2-CsCARME recombinant plasmid, which was verified to be correct by sequencing.

[0065] 2. Preparation of Agrobacterium culture: pTRV1, pTRV2 empty vector, and pTRV2-CsCARME plasmid were transformed into Agrobacterium GV3101, respectively. The cells were cultured and collected according to the method in Example 3, and resuspended in infection buffer (containing 2 mg / L 6-BA, 0.1 mg / L NAA, 150 μmol / L AS, 10 mmol / L MES, 10 mmol / L MgCl2, pH 5.6) to adjust OD. 600 1.2. Mix pTRV1 bacterial culture with pTRV2 empty vector or pTRV2-CsCARME bacterial culture at a volume ratio of 1:1.

[0066] 3. Vacuum Infiltration of Tea Trees: Using "Fuding Dabai" tea tree cuttings as material, the cuttings are pruned to 15-20 cm, retaining only one mature leaf. The cuttings are immersed in a mixed bacterial solution and placed in a Buchner bottle for vacuum infiltration treatment. After treatment, the surface bacterial solution is absorbed with absorbent paper, and the cuttings are placed in a polyethylene basin, covered with plastic wrap, and incubated in the dark at 25℃ for 3 days. Subsequently, they are transferred to a greenhouse (16 hours of light / 8 hours of darkness) for continued cultivation for 30-40 days.

[0067] 4. Silencing effect detection: New leaves were collected, RNA was extracted according to the method in Example 2, the silencing efficiency of CsCARME was detected by qRT-PCR, and the free amino acid content was determined according to the national standard GB / T 8314-2013.

[0068] (II) Experimental Results

[0069] like Figure 6 As shown, compared with the empty vector control, the expression level of this gene was reduced by up to 77.8% in the CsCARME-silenced lines, while the free amino acid content was reduced by up to 31%. This result confirms the key positive regulatory role of CsCARME in the accumulation of free amino acids from the perspective of reverse genetics.

[0070] Example 6: CsCARME gene heterologous overexpression in tobacco

[0071] (I) Experimental Methods

[0072] 1. Genetic transformation: Agrobacterium-mediated leaf disc method was used. Agrobacterium LBA4404 bacterial culture containing the pSH737-35S-CsCARME overexpression vector (OD) was transformed... 600 =0.6) infected sterile tobacco (Nicotiana tabacum 'Xanthi') leaves (1 cm × 1 cm) and cultured for 2 days. Then transferred to selection medium (MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 100 mg / L Kan + 100 mg / L Tim) to induce adventitious shoots. When the shoots reached 1-2 cm in length, they were cut off and transferred to rooting medium (1 / 2 MS + 100 mg / L Kan + 100 mg / L Tim) to induce rooting.

[0073] 2. Transgenic identification: Young leaves from regenerated plants were subjected to GUS histochemical staining, with untransformed wild-type plants serving as a negative control. Simultaneously, leaf DNA was extracted and subjected to PCR identification. Positive transgenic lines (TP1, TP2, TP3) were screened.

[0074] 3. Phenotypic analysis: Mature leaves of transgenic tobacco and wild-type tobacco were taken. The expression level of CsCARME in one part was detected by qRT-PCR according to the method in Example 2, and the content of free amino acids in the other part was determined according to the national standard GB / T 8314-2013.

[0075] (II) Experimental Results

[0076] like Figure 7 As shown, multiple transgenic tobacco lines were successfully obtained. qRT-PCR showed that CsCARME was highly expressed in the transgenic lines. Compared with wild-type (WT), the free amino acid content in transgenic tobacco leaves was significantly increased by 20%-33%, indicating that CsCARME can also promote the accumulation of free amino acids in heterologous species.

[0077] Example 7: CsCARME gene replacement Arabidopsis mutant

[0078] (I) Experimental Methods

[0079] 1. Material preparation: Arabidopsis thaliana At2g32170 mutant (SALK-062342C) was purchased from AraShare. This mutant is a T-DNA insertion homozygous mutant of the CsCARME homolog, and its free amino acid content is significantly lower than that of the wild type.

[0080] 2. Genetic transformation: The inflorescence immersion method was used. Agrobacterium GV3101 bacterial suspension containing the pSH737-35S-CsCARME overexpression vector was resuspended and used to immerse Arabidopsis mutant inflorescences for 3 min. After dark incubation for 24 h, normal culture was performed. T1 generation seeds were collected, and resistant plants were screened on 1 / 2 MS medium containing kanamycin.

[0081] 3. Homozygous screening: Resistant plants were transplanted, and positive lines were identified by PCR. Seeds of the T2 generation were harvested from individual plants, and screening continued until the T3 generation to obtain homozygous supplementary lines.

[0082] 4. Verification of Complementation: Rosette leaves of wild-type (WT), mutant, and homozygous complemented line (35S-homozygous complemented line) were taken, and the expression level of CsCARME was detected by qRT-PCR according to the method in Example 2. The content of free amino acids was determined according to the national standard GB / T 8314-2013.

[0083] (II) Experimental Results

[0084] like Figure 8 As shown, compared with the mutant, the complemented line restored the normal growth phenotype, the CsCARME gene was successfully expressed, and its free amino acid content recovered from the low level of the mutant to near the wild type (0.38 mg / g). This complementation experiment further demonstrates that the CsCARME gene plays a key and conserved role in regulating amino acid synthesis.

[0085] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An isolated CsCARME protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

2. An isolated nucleic acid molecule, characterized in that, It encodes the CsCARME protein as described in claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

4. A recombinant expression vector containing the nucleic acid molecule of claim 2 or 3.

5. The recombinant expression vector according to claim 4, characterized in that, The recombinant expression vector is either the overexpression vector pSH737-35S-CsCARME or the silencing vector pTRV2-CsCARME.

6. A recombinant host cell containing the recombinant expression vector of claim 4 or 5.

7. The application of CsCARME protein or its encoding nucleic acid molecule in regulating the content of free amino acids in tea plants or improving tea quality, characterized in that... The amino acid sequence of the CsCARME protein is shown in SEQ ID NO.2, and this is achieved by regulating the expression level or activity of the protein.

8. A method for regulating the content of free amino acids in tea plants, characterized in that, The accumulation of free amino acids in tea leaves is regulated by modulating the expression level or activity of the CsCARME protein, the amino acid sequence of which is shown in SEQ ID NO.

2.

9. The application of the protein of claim 1, or the nucleic acid molecule of claim 2 or 3, or the recombinant expression vector of claim 4 or 5, or the recombinant host cell of claim 6 in the quality improvement or breeding of tea trees.

Images