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Persimmon proanthocyanidins precursor transmembrane transport gene DkMATE5 and application thereof

A technology for transmembrane transport and proanthocyanidins, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of less research on transmembrane transport, incomplete clarity, and differences in types of astringent persimmon varieties

Active Publication Date: 2021-05-07
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the structural genes and regulatory genes of proanthocyanidin biosynthesis have been cloned and identified, but the transmembrane transport of proanthocyanidin precursors is less studied
In particular, whether the preferential transport of persimmon proanthocyanidin precursors causes the differences in the type of astringency of persimmon varieties is not yet fully clear.

Method used

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  • Persimmon proanthocyanidins precursor transmembrane transport gene DkMATE5 and application thereof
  • Persimmon proanthocyanidins precursor transmembrane transport gene DkMATE5 and application thereof
  • Persimmon proanthocyanidins precursor transmembrane transport gene DkMATE5 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Acquisition and Analysis of DkMATE5 and DkMATE4 Gene Sequences

[0020] 1. Acquisition of DkMATE5 and DkMATE4 gene sequences in the fruit of 'E Shi No. 1'

[0021] (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) to obtain candidate MATE protein sequences, compare the candidate protein sequences to the Pfam database, and retain the MATE genes with conserved domains as DkMATE family gene members, use the transcriptome database to search for DkMATE differentially expressed genes, and determine which Differential expression of DkMATE4 and DkMATE5 genes;

[0022] (2) Utilize the Primer Premier 5.0 software to design RACE primers therebetween, wherein the RACE primers used to amplify the DkMATE4 gene are shown in SEQ ID NO.13-14, and the RACE primers used to amplify the DkMATE5 gene are...

Embodiment 2

[0034] Example 2 Analysis of transient transformation of DkMATE4 and DkMATE5 in persimmon leaves

[0035] (1) According to the DkMATE5 gene sequence shown in SEQ ID NO.1 obtained in Example 1 and the DkMATE4 gene sequence shown in SEQ ID NO.12, respectively design the MATE5-OEF shown in SEQ ID NO.8-9 and MATE5-OER and MATE4-OEF and MATE4-OER as shown in SEQ ID NO.19-20, and according to the instructions, use the Gateway method to introduce the DkMATE5 gene and DkMATE4 gene into the entry vector pDONR207, and then introduce the overexpression through the LR reaction In the vector pK2GW7, overexpression recombinant plasmids were respectively constructed, and the overexpression recombinant plasmids were transformed into Agrobacterium (GV3101) by conventional methods.

[0036] (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 ...

Embodiment 3

[0038] Example 3 Escherichia coli functional complementation experiment to determine the transport preference of DkMATE5

[0039] 1. Construction of PGEX-6P-1-DkMATE5 expression vector

[0040] (1) Use PrimeSTAR Max Premix (2x) high-fidelity enzyme (TaKaRa, Japan), use the full-length plasmid of the DkMATE5 gene as a template, and use primers containing BamH I and Not I restriction sites (such as SEQ ID NO.10 -11), amplified and cloned into the prokaryotic expression vector PGEX-6P-1 to construct the prokaryotic expression plasmid PGEX-6P-1-DkMATE5.

[0041] 2. Escherichia coli functional complementation experiment

[0042] The prokaryotic expression plasmid PGEX-6P-1-DkMATE5 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 li...

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Abstract

The invention discloses a persimmon proanthocyanidins precursor transmembrane transport gene DkMATE5 and application thereof, and belongs to the technical field of plant genetic engineering. DkMATE4 and DkMATE5 genes are obtained through first screening of a transcriptome database, and DkMATE5 protein has a proanthocyanidins transport function. In-vivo complementary experimental analysis of escherichia coli proves that the DkMATE5 gene promotes preferential transmembrane transport of persimmon proanthocyanidins precursor catechin, epicatechin and epicatechin gallic acid. The persimmon proanthocyanidins precursor transmembrane transport gene DkMATE5 provides a scientific basis and a new gene resource for analysis of difference of proanthocyanidins components and contents of different deastringency types of persimmons and genetic improvement of sweet persimmons.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to persimmon proanthocyanidin precursor transmembrane transport gene DkMATE5 and application thereof. Background technique [0002] Persimmon fruit contains a large amount of Proanthocyanidins (PAs; commonly known as "persimmon tannin"), which is an important indicator of fruit maturity and commodity quality. The biosynthesis of proanthocyanidins involves shikimic acid, public phenylpropanoid, core flavonoid-anthocyanidins and proanthocyanidin-specific pathways. Proanthocyanidin precursors are synthesized in the cytoplasm, recognized by transport proteins located on the tonoplast membrane and transported to the vacuole for polymerization, thereby forming macromolecular proanthocyanidins. At present, the structural genes and regulatory genes of proanthocyanidin biosynthesis have been cloned and identified, but the transmembrane transport of proanthocyani...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C12N15/11C12Q1/6851A01H5/00A01H6/00
CPCC07K14/415C12Q1/6851C12N15/8243C12Q2531/113C12Q2563/107
Inventor 徐莉清刘颖罗正荣张青林陈文兴郭大勇
Owner HUAZHONG AGRI UNIV
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