Tea tree epicatechin galloyl transferase gene CsECGT as well as coded protein and application thereof
A technology of galloyl group and epicatechin, applied in the field of genetic engineering, can solve the problems of high technical difficulty, unknown amino acid sequence and high cost, and achieve the effect of reducing cost
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Embodiment 1
[0036] Embodiment 1, the acquisition of CsECGT gene sequence
[0037] Using tea tree (Camellia sinensis) young and fresh leaves as materials, RNA was extracted and reverse-transcribed into cDNA as a template. Primers were designed according to the published conserved sequence of the acyltransferase gene (the primer sequence is as follows), and the sequence of the coding region was amplified by PCR.
[0038] Upstream primer: 5'-TGAGTATAGACCCAGCAA-3', as shown in SEQ ID No.3;
[0039] Downstream primer: 5'-CATTTCACAGTACCCACA-3', as shown in SEQ ID No.4.
[0040] Perform agarose gel electrophoresis detection on the PCR amplification product, cut out the target band, purify and recover, connect the recovered DNA fragment to the pGEM-T easy vector, transform Escherichia coli (Esherichia coli) DH5α competent cells, and pass blue-white spot Screening, extraction of positive clone plasmids and restriction analysis, and then sequence the monoclonal, the insert fragment is 488bp. Usin...
Embodiment 2
[0048] Example 2 Heterologous expression of epicatechin galloyltransferase gene CsECGT
[0049] 1. Construction of a recombinant expression vector carrying the tea tree epicatechin galloyltransferase gene CsECGT
[0050] The tea tree cDNA is used as a template, primers are designed according to the sequence SEQ ID No.1, and the deoxyribonucleotide sequence of the tea tree epicatechin galloacyltransferase gene coding region is amplified by PCR. BamHI and Xho I restriction sites were introduced at both ends of the primers respectively, and the primer sequences are as follows:
[0051] Upstream: 5'-ATGTTTCCACCAAAGTCATACAG-3' (as shown in SEQ ID No.7), introducing a BamHI restriction site,
[0052] Downstream: 5'-CTAAAGAGGATAGTAATGAATCCATC-3' (as shown in SEQ ID No.8), introducing the Xho I restriction site;
[0053] Perform agarose gel electrophoresis on the PCR amplification product, cut out the target band, purify and recover, connect the recovered DNA fragment to the pGEM-T ...
example 3
[0061] Example 3 Inhibition of expression of epicatechin galloyltransferase gene CsECGT
[0062] 1. Construction of epicatechin galloacyltransferase gene silencing vector
[0063] Take one 0.5mL centrifuge tube, add 5μL 10×PCR Buffer, 3μL 25mmol / L MgC12, 0.4μL 25mmol / L dNTP mix, 1μL 10μmol / L upstream primer ECGTRNAiF (as shown in SEQ ID No.9), 1μL 10μmol / L downstream primer ECGTRNAiR (as shown in SEQ ID No.10), 1 μL 2U / μL Taq enzyme, 2 μL cDNA obtained in Example 1, add double distilled water to a total volume of 20 μL. PCR was performed using the following program: 95°C for 4 min, 94°C for 1.5 min, 62°C for 1 min, 72°C for 2 min, 35 cycles, and 72°C for 10 min. PCR products were detected by 1.5% agarose gel electrophoresis, such as Figure 7A fragment of about 300bp was obtained as shown. Add 150 μL TE pH8.0 buffer solution and 50 μL attB-PCR product, then add 100 μL 30% PEG8000 / 30mM MgCl2 solution, mix well, centrifuge at 10,000×g for 15 min at room temperature, remove th...
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