Persimmon procyanidine precursor transmembrane transport gene DkMATE7 and application thereof
A technology for transmembrane transport and proanthocyanidins, which can be used in applications, genetic engineering, plant genetic improvement, etc., and can solve problems such as lack of research reports
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Embodiment 1D
[0018] The acquisition and analysis of embodiment 1DkMATE7 gene sequence
[0019] 1. Obtaining the sequence of DkMATE7 gene in the fruit of 'E Shi No. 1'
[0020] (1) First, download the HMM information of the MATE gene family from the Pfam database (http: / / pfam.xfam.org), and use the HMMER software to compare the persimmon genome (http: / / www.kakiwi.zju.edu.cn / cgi- bin / persimmon / index.cgi), obtain the candidate MATE protein sequence, take the candidate protein sequence and compare it with the Pfam database, the MATE gene with the conserved domain is reserved as the DkMATE family gene member, use the transcriptome database to find the DkMATE differentially expressed genes, and determine the DkMATE7 Gene differential expression.
[0021] (2) Use the RNAplant Plus Kit (Tiangen Biotech Ltd., Beijing, China) to extract the RNA from the pulp of 'Eshi No. 1' persimmon, and use the PrimeScriptTM RT reagent Kit with gDNAEraser (TaKaRa, Japan) reverse transcription kit Synthesize cDNA...
Embodiment 2
[0031] Example 2 DkMATE7 transient transformation analysis in persimmon leaves
[0032] (1) According to the DkMATE7 gene sequence as shown in SEQ ID NO.1 obtained in Example 1, design MATE7-OEF and MATE7-OER as shown in SEQ ID NO.6-7 respectively, and according to the instructions, use the Gateway method to The DkMATE7 gene was introduced into the intermediate vector pDONR207, and then introduced into the overexpression vector pK2GW7 by LR reaction, and the overexpression recombinant plasmids were respectively constructed, and the overexpression recombinant plasmids were transferred into Agrobacterium (GV3101) by conventional methods.
[0033](2) The above-mentioned transfected Agrobacterium was instantly transformed into the leaves of 'Eshi 1' by conventional injection infiltration method, and the leaves infected by Agrobacterium were collected 10 days after the injection, and stored at -80°C after quick-freezing in liquid nitrogen use. According to the Folin-Ciocalteau met...
Embodiment 3
[0035] Example 3 Escherichia coli functional complementation experiment to determine the transport preference of DkMATE7
[0036] 1. Construction of PGEX-6P-1-DkMATE7 expression vector
[0037] Using PrimeSTAR Max Premix (2x) high-fidelity enzyme (TaKaRa, Japan), with the DkMATE7 gene full-length plasmid as a template, primers (as shown in SEQ ID NO.8-9) comprising BamH I and Not I restriction sites were used shown), amplified and cloned into the prokaryotic expression vector PGEX-6P-1 to construct the prokaryotic expression plasmid PGEX-6P-1-DkMATE7.
[0038] 2. Escherichia coli functional complementation experiment
[0039] The prokaryotic expression plasmid PGEX-6P-1-DkMATE7 constructed above was transformed into Escherichia coli mutant strain acrB lacking drug efflux ability according to conventional methods. Pick a successfully transformed single colony into 5mL LB liquid medium, shake at 200r / min at 37°C for about 12h. Take 500 μL of bacterial liquid into 50 mL of LB ...
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