Tea tree-derived transmembrane transporter gene CsMATE40, recombinant expression vector, genetically engineered bacteria and applications

Abstract

The present invention provides a tea plant-derived transmembrane transporter gene, CsMATE40, a recombinant expression vector, a genetically engineered bacterium, and applications thereof, belonging to the field of plant genetic engineering technology. The nucleotide sequence of the tea plant-derived transmembrane transporter gene, CsMATE40, is shown in SEQ ID No. 1. Experimental results show that the tea plant-derived transmembrane transporter gene, CsMATE40, can increase the accumulation of epicatechin and the expression of genes related to epicatechin synthesis in host plants. These results demonstrate that the present invention provides an excellent candidate gene for breeding new high-epicatechin plant varieties, possessing potential for application and significant value prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and in particular relates to a tea tree-derived transmembrane transporter gene CsMATE40, a recombinant expression vector, a genetically engineered bacterium and applications. Background Art

[0002] The tea plant (Camellia sinensis) is a commercial crop, with tea being its primary product. Depending on processing methods and the degree of fermentation, tea can be divided into green tea, black tea, oolong tea, white tea, yellow tea, and dark tea. Tea plants are rich in a variety of bioactive secondary metabolites, such as flavonoids, caffeine, amino acids, and vitamins. Flavonoids are particularly abundant, comprising approximately 18% to 36% of the dry weight of fresh leaves. These include catechins, flavonols, and anthocyanins. Catechins are synthesized in large quantities in the endoplasmic reticulum and subsequently transported to the vacuole for storage, reducing cytotoxicity and ensuring the plant's resistance to external stresses. This process involves a catechin transport mechanism. Specifically, catechins participate in the regulation of tea plant growth and development, as well as in the regulation of various biotic and abiotic stresses, such as chelating heavy metal ions and protecting against ultraviolet light, heat, drought, and salt stress. Furthermore, catechins possess numerous physiological activities, including antioxidant, anti-inflammatory, antibacterial, and cardiovascular disease prevention.

[0003] In plants, secondary metabolites are generally synthesized in the cytoplasm and accumulated in specific tissues and organs, with spatiotemporal specificity. For example, artemisinin, a sesquiterpene lactone used to treat malaria, is synthesized and accumulated at high levels only in specific glandular trichomes in inflorescences, leaves, and stems. Within cells, secondary metabolites are generally stored in vacuoles. When plants need to utilize these secondary metabolites, the vacuole fuses with the plasma membrane, releasing the stored substances into the cytoplasm for the corresponding metabolic activities. The transfer of these substances requires an efficient transport mechanism, and transport proteins play an important role in this process.

[0004] MATE transporters (Multidrug And Toxic Compound Extrusion) are widely distributed in plants. The MATE transporter family has been reported to have a wide range of biological functions, playing key roles in plant growth and development and in response to environmental stresses, including regulating ion homeostasis, drug absorption and transport, and excretion of pathogenic toxins. They are also closely related to plant growth and development and responses to environmental stress. Overexpression of AtTT12 in Arabidopsis thaliana promotes the vacuolar accumulation of anthocyanidin protosomes synthesized from glycosylated epicatechin in the seed coat. MtMATE2 is specifically expressed in alfalfa and participates in pigment deposition. MATE transporters are also present on vesicle membranes. After flavonoids are synthesized in the endoplasmic reticulum, they are transported by transporters to vesicles derived from the endoplasmic reticulum. Vesicles carrying flavonoids are transported to the vacuole, where they are then imported into the vacuole through membrane fusion.

[0005] However, current research on plant secondary metabolite transporters has only conducted preliminary functional identification and has not studied the transport activity of tea plant-derived MATE transporter genes. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a tea tree-derived transmembrane transporter gene CsMATE40, a recombinant expression vector, a genetically engineered bacterium and its application. The tea tree-derived transmembrane transporter gene CsMATE40 can increase the content of epicatechin in plant leaves and the expression of genes related to epicatechin synthesis.

[0007] To achieve the above objectives, the present invention provides the following technical solutions:

[0008] The present invention provides a tea tree-derived transmembrane transporter gene CsMATE40, and the nucleotide sequence of the tea tree-derived transmembrane transporter gene CsMATE40 is shown in SEQ ID No. 1.

[0009] The present invention also provides the use of the tea tree-derived transmembrane transporter gene CsMATE40 in increasing the epicatechin content in plant leaves, and overexpressing the tea tree-derived transmembrane transporter gene CsMATE40 in plants.

[0010] The present invention also provides the use of the tea tree-derived transmembrane transporter gene CsMATE40 in increasing the expression of genes related to epicatechin synthesis in plant leaves, and overexpressing the tea tree-derived transmembrane transporter gene CsMATE40 in plants.

[0011] Preferably, the genes related to epicatechin synthesis include NtDFR, NtLAR, NtANS, and NtANR.

[0012] The present invention also provides a primer pair for amplifying the tea plant-derived transmembrane transporter gene CsMATE40, comprising the primer pair described in SEQ ID No. 2 and SEQ ID No. 3;

[0013] Or the primer pair shown as SEQ ID No.4 and SEQ ID No.5.

[0014] The present invention also provides a recombinant expression vector for overexpressing the tea tree-derived transmembrane transporter gene CsMATE40, comprising the tea tree-derived transmembrane transporter gene CsMATE40 and an expression vector.

[0015] Preferably, the expression vector is pBI121 or pDR196.

[0016] The present invention also provides a genetically engineered bacterium that overexpresses the tea tree-derived transmembrane transporter gene CsMATE40, which is obtained by transferring the recombinant expression vector into the genetically engineered bacterium.

[0017] Preferably, the genetically engineered bacteria include Agrobacterium GV3101 or yeast AD1-8.

[0018] The present invention also provides the use of the recombinant expression vector and the genetically engineered bacteria in increasing the epicatechin content in plant leaves and / or the expression level of genes related to epicatechin synthesis.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] Through transcriptome data analysis, this study isolated and cloned the differentially expressed gene CsMATE40 for the first time. Agrobacterium-mediated transient transformation of tobacco leaves demonstrated for the first time that CsMATE40 can increase epicatechin accumulation in host plants. In vivo complementation experiments in yeast further demonstrated that CsMATE40 functions as a transmembrane transporter of epicatechin. This invention provides a gene resource for regulating epicatechin accumulation in plants and an excellent candidate gene for the breeding of new high-epicatechin plant varieties, possessing potential applications and significant value prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of gene expression of CsMATE40 in different tissues of the tea variety "Longjing 43" are shown;

[0022] Figure 2 is a map of vector pBI121;

[0023] Figure 3 is a map of the recombinant vector pBI121-CsMATE40;

[0024] Figure 4 The results show the determination of epicatechin (EC) content after transient transformation of CsMATE40 in tobacco leaves;

[0025] Figure 5 The expression levels of genes related to epicatechin (EC) synthesis were detected after transient transformation of CsMATE40 in tobacco leaves;

[0026] Figure 6 is a map of vector pDR196;

[0027] Figure 7 is a map of the recombinant vector pDR196-CsMATE40;

[0028] Figure 8These are the results of a functional complementation experiment in yeast. DETAILED DESCRIPTION

[0029] The present invention provides a tea tree-derived transmembrane transporter gene CsMATE40, and the nucleotide sequence of the tea tree-derived transmembrane transporter gene CsMATE40 is shown in SEQ ID No. 1.

[0030] In the present invention, the nucleotide sequence of the tea plant-derived transmembrane transporter gene CsMATE40 is shown in Table 1.

[0031] Table 1 Nucleotide sequence of tea plant-derived transmembrane transporter gene CsMATE40 and its amplification primers

[0032]

[0033]

[0034]

[0035] The present invention also provides the use of the tea tree-derived transmembrane transporter gene CsMATE40 in increasing the epicatechin content in plant leaves, and overexpressing the tea tree-derived transmembrane transporter gene CsMATE40 in plants.

[0036] The present invention also provides the use of the tea tree-derived transmembrane transporter gene CsMATE40 in increasing the expression of genes related to epicatechin synthesis in plant leaves, and overexpressing the tea tree-derived transmembrane transporter gene CsMATE40 in plants.

[0037] In the present invention, the genes related to epicatechin synthesis preferably include NtDFR, NtLAR, NtANS, and NtANR.

[0038] The present invention also provides a primer pair for amplifying the tea plant-derived transmembrane transporter gene CsMATE40, comprising the primer pair described in SEQ ID No. 2 and SEQ ID No. 3;

[0039] Or the primer pair shown as SEQ ID No.4 and SEQ ID No.5.

[0040] In the present invention, the nucleotide sequences of the primer pairs are shown in Table 1.

[0041] The present invention also provides a recombinant expression vector for overexpressing the tea tree-derived transmembrane transporter gene CsMATE40, comprising the tea tree-derived transmembrane transporter gene CsMATE40 and an expression vector.

[0042] In the present invention, the expression vector is preferably pBI121 or pDR196.

[0043] The present invention also provides a genetically engineered bacterium that overexpresses the tea tree-derived transmembrane transporter gene CsMATE40, which is obtained by transferring the recombinant expression vector into the genetically engineered bacterium.

[0044] In the present invention, the genetically engineered bacteria preferably include Agrobacterium GV3101 or yeast AD1-8.

[0045] The present invention also provides the use of the recombinant expression vector and the genetically engineered bacteria in increasing the epicatechin content in plant leaves and / or the expression level of genes related to epicatechin synthesis.

[0046] The technical solutions provided by the present invention are 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.

[0047] Example 1: Acquisition of the tea tree-derived gene CsMATE40 sequence

[0048] 1.1 Total RNA was extracted from leaves of 'Longjing 43' (two-year-old potted seedlings, obtained by cuttings and propagation in the laboratory) using the RNAPlant Plus Kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). The extracted total RNA concentration and quality were measured using a Nanodrop instrument. RNA was considered qualified if the OD260 / 280 ratio was >2.0 and the OD260 / 230 ratio was >1.8. RNA that qualified was synthesized into cDNA using the PrimeScript™ RT reagent kit (purchased from TaKaRa, Japan). Detailed procedures were followed according to the manufacturer's instructions.

[0049] 1.2 Primers capable of amplifying the full length of the CsMATE40 gene were designed using the NCBI Primer Blast website, resulting in the upstream primer shown in SEQ ID No. 2 and the downstream primer shown in SEQ ID No. 3. Using the cDNA obtained in 1.1 as a template, PCR amplification was performed using KOD FX high-fidelity enzyme (purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.) to obtain a purified PCR product containing the complete CsMATE40 gene coding region sequence.

[0050] The purified PCR product was ligated into the pEASYblunt zero vector (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) to generate the pEASY-CsMATE40 plasmid. Plasmid identification was performed by PCR using universal primers M13 (upstream primer: 5'-GTAAAACGACGGCCAGT-3' (SEQ ID No. 6), downstream primer: 5'-CAGGAAACAGCTATGAC-3' (SEQ ID No. 7)). Specific ligation and identification methods were performed according to the manufacturer's instructions. The positive plasmids identified were sequenced by Hangzhou Youkang Biotechnology Co., Ltd., and the CsMATE40 gene sequence was obtained as shown in SEQ ID No. 1.

[0051] Example 2 Gene expression patterns of CsMATE40 in different tissues of Longjing 43

[0052] Total RNA was extracted from different tissue samples (terminal buds, young leaves, mature leaves, old leaves, stems, and roots) of "Longjing 43" (two-year-old potted seedlings, obtained by cutting propagation in the laboratory) using the RNAPlant Plus Kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). TM The total RNA was reverse transcribed and cDNA was synthesized using the RT reagent Kit (purchased from TaKaRa, Japan). The detailed operation method was carried out according to the manufacturer's instructions.

[0053] Fluorescent quantitative PCR primers for quantitative detection of CsMATE40 gene were designed using the NCBI PrimerBlast website. The primers consisted of an upstream primer as shown in SEQ ID NO.4 and a downstream primer as shown in SEQ ID NO.5.

[0054] Six replicates were set for each biological sample, and the tea plant CsGAPDH gene (NCBI, XM_028229956.1) was used as an internal reference. CsMATE40 was quantitatively analyzed using Light Cycler 480 SYBR Green I Master fluorescent dye (purchased from Roche, Germany) and a Light Cycler 480 II real-time fluorescence quantitative PCR instrument. Figure 1 shown.

[0055] The fluorescent quantitative PCR reaction system was as follows: per 10 μL volume, add 2 μL of cDNA template, 0.8 μL of 10 pmol / μL upstream primer, 0.8 μL of 10 pmol / μL downstream primer, 5 μL of Light Cycler 480 SYBR Green I Master fluorescent dye, and 1.4 μL of water. Amplification conditions were: 95°C × 30 sec, (95°C × 15 sec, 57°C × 30 sec, 72°C × 20 sec) × 40 cycles, then 95°C × 15 sec, then 40°C × 30 sec.

[0056] The results showed that the expression level of CsMATE40 was higher in tea leaves and roots, and in the aboveground parts, the expression level in mature leaves and old leaves was higher than that in other tissues.

[0057] Example 3 Transient transformation of CsMATE40 in tobacco leaves

[0058] The upstream primer: 5'-ACGGGGGACTCTAGAGGATCCATGGAAGAGGGATTGTTGTTA-3' (SEQ ID No. 8) and the downstream primer: 5'-TCAACAGATGATGGATTAAAGCTTTAATCCATCATC-3' (SEQ ID No. 9) were designed using Snap Gene 4.1.8 software. The pEASY-CsMATE40 plasmid obtained in Example 1 was used as a template to perform PCR amplification to obtain the amplified product rDNA.

[0059] The PCR amplification system consisted of 0.4 μL of amplified rDNA, 5 μL of 2× Buffer, 2 μL of dNTPs, 0.3 μL of 10 pmol / μL upstream primer, 0.5 μL of 10 pmol / μL downstream primer, 0.2 μL of KOD high-fidelity enzyme, and 1.6 μL of water per 10 μL aliquot. Amplification conditions were: 98°C for 4 min, 35 cycles of (98°C for 20 sec, 60°C for 20 sec, 68°C for 60 sec), and 68°C for 3 min.

[0060] The amplified rDNA was purified using Gene JET PCR purification kit (purchased from Thermo Fisher Scientific), and the purified product and expression vector pBI121 (stored in the laboratory, vector map as shown in the figure) were cloned. Figure 2 As shown) were subjected to homologous recombination ligation reaction to obtain the recombinant vector pBI121-CsMATE40 (the map of the recombinant vector is shown Figure 3 shown).

[0061] The recombinant vector pBI121-CsMATE40 was transformed into Agrobacterium GV3101 (purchased from Shanghai Weidi Biotechnology Co., Ltd.) using a chemical transformation method to obtain an overexpression genetically engineered bacterium.

[0062] The pBI121 empty vector was transformed into Agrobacterium GV3101 (purchased from Shanghai Weidi Biotechnology Co., Ltd.) to obtain a control genetically engineered bacterium.

[0063] The chemical transformation method involves adding 2 μL of the recombinant vector pBI121-CsMATE40 plasmid to 100 μL of Agrobacterium tumefaciens GV3101 competent cells. After a 5-minute ice bath, 5-minute liquid nitrogen freezing, 5-minute 37°C water bath, and 5-minute ice bath, the cells are added to 700 μL of antibiotic-free LB liquid medium (purchased from Beijing Coolbo Technology Co., Ltd.) and incubated at 28°C for 2 hours. 100 μL of the bacterial solution is then spread onto LB solid medium containing 50 mg / L kanamycin (purchased from Beijing Coolbo Technology Co., Ltd.). After incubation at 28°C for 2 days, a single colony is selected to obtain the overexpressing genetically engineered bacterium.

[0064] The genetically engineered bacteria expressing pBI121-CsMATE40 and the control genetically engineered bacteria expressing an empty pBI121 vector were injected into tobacco leaves using syringes. After 48 hours of transient expression of the recombinant vector in the tobacco leaves, the leaves were harvested and quickly frozen in liquid nitrogen.

[0065] Example 4 Analysis of epicatechin content in transiently transformed tobacco leaves

[0066] The tobacco leaf sample after instantaneous transformation for 48 h in Example 3 was ground into powder in a mortar, 0.1 g of the sample was weighed and placed in a 2 ml centrifuge tube (purchased from Axygen, USA), 70% methanol solution was added, and after thorough mixing, the centrifuge tube was placed in a 70°C water bath. After extraction for 20 min, it was taken out and cooled to room temperature. The sample was centrifuged at 4000 r / min for 10 min in a Centrifuge 5424R centrifuge (purchased from Eppendorf, Germany). The supernatant was passed through a 0.22 μm organic needle filter (purchased from Tianjin Jinteng Experimental Equipment Co., Ltd.) and placed in a glass transparent sample bottle (purchased from Tianjin Jinteng Experimental Equipment Co., Ltd.).

[0067] According to GB / T 8313-2018, the content of epicatechin in tobacco transiently transformed leaves was tested. The test results are as follows: Figure 4 shown.

[0068] Depend on Figure 4It can be seen that compared with the control, the epicatechin content in tobacco leaves transiently expressing the recombinant plasmid pBI121-CsMATE40 was extremely significantly higher than that in the empty control (P<0.001), indicating that CsMATE40 can increase the epicatechin content in tobacco leaves.

[0069] Example 5 Detection of the expression of epicatechin synthesis-related genes in transiently transformed tobacco leaves

[0070] The total RNA from the tobacco leaves after 48 h of transient transformation in Example 3 was extracted using the RNAPlantPlus Kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). The concentration and quality of the extracted total RNA were detected using a Nanodrop instrument. If the test results met the requirements of OD260 / 280>2.0 and OD260 / 230>1.8, the RNA sample was judged to be qualified. The above qualified biological samples were analyzed by PrimeScript TM cDNA was synthesized using RTreagentKit reverse transcription kit (purchased from TaKaRa, Japan), and the detailed operation method was carried out according to the manufacturer's instructions.

[0071] Using tobacco NtACTIN gene (NCBI, NM_001425946.1) as internal reference, Light Cycler 480SYBR Green I Master fluorescent dye (purchased from Roche, Germany) and Light Cycler 480Ⅱ real-time fluorescence quantitative PCR instrument, the genes related to epicatechin synthesis in tobacco leaves were quantitatively analyzed. The primers for the fluorescence quantitative detection are shown in Table 2. The quantitative detection results are shown in Table 2. Figure 5 shown.

[0072] Table 2 Primers for fluorescence quantitative detection of genes related to epicatechin synthesis

[0073]

[0074] Depend on Figure 5 It can be seen that the expression levels of genes related to epicatechin synthesis in tobacco leaves transiently transformed with the recombinant plasmid pBI121-CsMATE40 were significantly upregulated, further proving that the expression of the tea tree-derived transporter gene CsMATE40 has the effect of increasing the epicatechin content in plant leaves.

[0075] Example 6 In vivo yeast complementation experiment to determine the transport function of CsMATE40

[0076] 6.1 Construction of the recombinant vector pDR196-CsMATE40

[0077] Using the pEASY-CsMATE40 plasmid in Example 1 as a template, the upstream primer shown in SEQ ID No. 18: 5'-TCCCCCGGGCTGCAGGAATTCATGGAAGAGGGATTGTTGTTA-3' and the downstream primer shown in SEQ ID No. 19: 5'-ACAGATGATGGATTAAAGTGATCACTTTAATCCATC-3' were designed using Snap Gene 4.1.8 software. PCR amplification reaction was performed to obtain the amplified product rDNA.

[0078] The PCR amplification system consisted of 0.4 μL of amplified rDNA, 5 μL of 2× Buffer, 2 μL of dNTPs, 0.3 μL of 10 pmol / μL upstream primer, 0.5 μL of 10 pmol / μL downstream primer, 0.2 μL of KOD high-fidelity enzyme, and 1.6 μL of water per 10 μL aliquot. Amplification conditions were: 98°C for 4 min, 35 cycles of (98°C for 20 sec, 60°C for 20 sec, 68°C for 60 sec), and 68°C for 3 min.

[0079] The amplified rDNA was purified using Gene JET PCR purification kit (purchased from Thermo Fisher Scientific), and the purified product was combined with the expression vector pDR196 (vector map shown in Figure 6 As shown) for homologous recombination ligation reaction, the recombinant vector pDR196-CsMATE40 plasmid was obtained (the recombinant vector map is shown in Figure 7 shown).

[0080] The recombinant vector plasmid pDR196-CsMATE40 was transformed into a mutant yeast strain AD1-8 (gifted from Professor Mohan Gupta of the University of Chicago) lacking the ability to efflux multiple drugs and toxic compounds using a yeast classic transformation kit (purchased from Beijing Coolaibo Technology Co., Ltd.) to obtain genetically engineered bacteria overexpressing the recombinant vector pDR196-CsMATE40 plasmid. The detailed operation method was carried out according to the manufacturer's instructions.

[0081] The pDR196 empty vector was transformed into the mutant yeast strain AD1-8 to obtain a control genetically engineered bacterium.

[0082] 6.2 In vivo yeast complementation experiments

[0083] The yeast strain containing the overexpression recombinant vector pDR196-CsMATE40 plasmid obtained in 6.1 was picked up and placed in 3 mL of uracil-deficient liquid culture medium Minimal SD Base (purchased from Beijing Coolbo Technology Co., Ltd.), and cultured at 30°C and 200 rpm for 12 h to obtain a bacterial solution.

[0084] 1 mL of bacterial culture was added to 150 mL of uracil-deficient liquid medium, Minimal SD Base, and cultured with shaking at 30°C and 200 rpm for 24 h. The OD600 value of the bacterial culture was measured using a NanoDrop spectrophotometer. If the OD600 value was 0.8-1, 50 mL of uracil-deficient liquid medium, Minimal SD Base, containing 2 mM epicatechin was added and cultured with shaking at 30°C and 200 rpm for 24 h. The culture was centrifuged at 4000 rpm for 10 min in a Centrifuge 5810R centrifuge (Eppendorf, Germany). The culture was washed twice with 0.9% saline and centrifuged again at 4000 rpm for 10 min. The cells were collected in 2 mL centrifuge tubes (Axygen, USA).

[0085] Add 1 ml of 70% methanol solution to the centrifuge tube, mix thoroughly, and place the centrifuge tube in a 70°C water bath. After extraction for 20 minutes, take it out and cool to room temperature. Centrifuge it at 4000 r / min for 10 minutes in a Centrifuge 5424R centrifuge (purchased from Eppendorf, Germany). Take the supernatant and pass it through a 0.22 μm organic needle filter (purchased from Tianjin Jinteng Experimental Equipment Co., Ltd.) and put it into a glass transparent sample bottle (purchased from Tianjin Jinteng Experimental Equipment Co., Ltd.).

[0086] The epicatechin content in the above yeast was detected according to the "GB / T 8313-2018 Method for Determination of Tea Polyphenols and Catechins in Tea", and the mutant yeast AD1-8 transformed with the pDR196 empty plasmid was used as a control. The results are as follows: Figure 8 shown.

[0087] Depend on Figure 8 After 24 hours of epicatechin incubation, the epicatechin content in the mutant yeast expressing the recombinant plasmid pDR196-CsMATE40 was significantly higher than that in the control yeast. Because the mutant yeast AD1-8 lacks multidrug and efflux transport functions, complementation of the tea plant-derived transmembrane transporter CsMATE40 restored the transport function, demonstrating that CsMATE40 has epicatechin transport function.

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Tea tree-derived transmembrane transporter gene CsMATE40 The application of the method for increasing the epicatechin content in plant leaves is characterized in that: Overexpression of tea tree-derived transmembrane transporter genes in plants CsMATE40 The plant is tobacco, and the tea tree-derived transmembrane transporter gene CsMATE40 The nucleotide sequence is shown in SEQ ID No.

1.

2. Overexpression of tea tree-derived transmembrane transporter genes CsMATE40 Recombinant expression vector, overexpression of tea tree-derived transmembrane transporter gene CsMATE40 The use of a genetically engineered bacterium in increasing the epicatechin content in plant leaves is characterized in that: The plant is tobacco, and the tea tree-derived transmembrane transporter gene CsMATE40 The nucleotide sequence is shown in SEQ ID No.1.

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