Persimmon proanthocyanidin precursor transmembrane transport gene dkmate7 and its application

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

Active Publication Date: 2022-02-11
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Recent studies speculate that PAs monomers may be combined with glutathione-S-transferase (AtGST26 / AtGSTF12 in Arabidopsis) and transported to the tonoplast membrane, and then MATE family proteins (Arabidopsis thaliana) located on the tonoplast membrane In AtDTX41) and H + -ATPase (AHA10 in Arabidopsis) transports it across the membrane into the vacuole, and finally condenses it into PA polymers with different polymerization degrees by oxidases such as laccase (LAC15 in Arabidopsis), but there is still a lack of Systematic Research Report

Method used

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  • Persimmon proanthocyanidin precursor transmembrane transport gene dkmate7 and its application
  • Persimmon proanthocyanidin precursor transmembrane transport gene dkmate7 and its application
  • Persimmon proanthocyanidin precursor transmembrane transport gene dkmate7 and its application

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Experimental program
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Effect test

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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Abstract

The invention discloses a persimmon proanthocyanidin precursor transmembrane transport gene DkMATE7 and its application, which belongs to the technical field of plant genetic engineering. The present invention screens and obtains the differentially expressed gene DkMATE7 gene for the first time through a transcriptome database, and at the same time discovers that the DkMATE7 protein has proanthocyanidin for the first time. Transport function. The analysis of in vivo complementation experiments in Escherichia coli further proved that the DkMATE7 gene promotes the preferential transmembrane transport of persimmon proanthocyanidin precursors catechin, epicatechin and epicatechin gallate. It provides a scientific basis for in-depth research on the transport and accumulation of proanthocyanidins in sweet persimmon, and also provides a new gene resource for genetic improvement.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to a persimmon proanthocyanidin precursor transmembrane transport gene DkMATE7 and an application thereof. Background technique [0002] Originally produced in China, persimmon is delicious in taste, produces body fluids and quenches thirst, and is good for human health. It is one of the most popular fruits in East Asia (China, Japan, Korea, etc.). The unique feature of persimmon is that a large amount of Proanthocyanidins (PAs, also known as "tannins") can be accumulated in the pulp tannin cells during the fruit development period, which becomes the main reason for the astringent feeling. In persimmons, the synthesis of proanthocyanidin precursors and the polymerization of PAs are located in the cytoplasm and vacuoles, respectively, so the initial unit and extension unit of PAs may be transmembrane transported into the vacuole by transporters and polyme...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12Q1/6895C12N15/70C12N1/21C12N15/11C12R1/19
CPCC07K14/415C12Q1/6895C12Q2600/158C12Q2600/13
Inventor 徐莉清吴鑫罗正荣张青林陈文兴郭大勇
Owner HUAZHONG AGRI UNIV
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