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Grape hydrogen peroxide receptor gene as well as encoding protein and application thereof

A grape hydrogen peroxide and receptor gene technology, applied in the field of genetic engineering, can solve the problems of hydrogen peroxide receptor distribution and other information expression patterns and subcellular localization are not clear

Active Publication Date: 2021-09-21
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, until now, the distribution and other information (such as expression patterns and subcellular localization) of hydrogen peroxide receptors in the grape genome are still unclear.

Method used

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  • Grape hydrogen peroxide receptor gene as well as encoding protein and application thereof
  • Grape hydrogen peroxide receptor gene as well as encoding protein and application thereof
  • Grape hydrogen peroxide receptor gene as well as encoding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Identification and Cloning of Hydrogen Peroxide Receptor Members in Grape

[0047] Based on the characteristics of the hydrogen peroxide receptor, we used the grape genome (12X.v2) and the corresponding annotation information (VCost.v3) to perform a local BLAST search for HPCA within the grape genome (the E-value of the search result was less than 0.01), The hydrogen peroxide receptors of grapes were screened out. We identified four hydrogen peroxide receptors VvHPCA1, 2, 3 and 4 from grapevine genome. The MEGA software was used to construct the phylogenetic tree, and the characteristic sequence of HPCA in different species was analyzed by multiple sequence alignment ( figure 1 ). The above results indicate that the VvHPCAs identified in the present invention belong to the LRR-RK family and have a hydrogen peroxide characteristic domain.

[0048] Full-length cloning: high-fidelity DNA polymerase Max DNA Polymerase (Bao Biological Engineering Co., Ltd., Da...

Embodiment 2

[0054] Example 2 Motif and conserved domain analysis of VvHPCAs encoded protein

[0055] The above sequencing results (DNA sequences) were translated into amino acid sequences using Primer Premier v5 software. The amino acid sequence of the VvHPCA1 protein is shown in SEQ ID NO.1, the amino acid sequence of the VvHPCA2 protein is shown in SEQ ID NO.2, the amino acid sequence of the VvHPCA3 protein is shown in SEQ ID NO.3, and the amino acid sequence of the VvHPCA4 protein is shown in SEQ ID NO .4 shown.

[0056] Using the Pfam database (http: / / pfam.xfam.org / search#tabview=tab1), NCBI Conserved Domain Search (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) and SMART ( Simple Modular Architecture Research Tool) (http: / / smart.embl-heidelberg.de) for LRR-RK and HP domain analysis.

[0057] The results showed that VvHPCAs contained a conserved motif at the C-terminus ( figure 2 A), both contain serine threonine kinase catalytic domain (Catalytic domain of serine threonine...

Embodiment 3

[0062] Example 3H 2 o 2 and ROS inhibitors (hypotaurine, HT) to treat ‘Kyoho’ grapes

[0063] The 'Kyoho' grapes (Vitis vinifera cv. 'Kyoho') were collected during the commercial harvest period, and the grapes with the same ear size and fruit color were selected for processing. Referring to Guo et al. (Transcriptome analysis reveals mechanism of early ripening in Kyoho grape with hydrogen peroxide treatment. BMC Genomics. 21, 784. https: / / doi:10.1186 / s12864-020-07180-y .) method, configure 300mmol L - 1 h 2 o 2 , 150 mmol L -1 HT solution (containing 0.01% Silwet_L-77), with double distilled water as a control, soak the selected ears in the above solution for 15 minutes. Then put it into a fresh-keeping box, cover it with plastic wrap, and store it in a storage room at 25±1°C. The control, H 2 o 2 and HT-treated fruits ( image 3 ), fully frozen with liquid nitrogen and stored at -80°C for subsequent analysis and experiments.

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Abstract

The invention relates to a grape hydrogen peroxide receptor gene as well as an encoding protein and application thereof. According to the invention, the VvHPCAs gene of the grape hydrogen peroxide receptor is identified. Hydrogen peroxide and hypotaurine are adopted to treat Kyoho grapes, the exfoliation rate, the water loss rate, the hardness, the H2O2 content and the ROS metabolism related enzyme activity are measured, the postharvest quality and ROS level change are comprehensively monitored, VvHPCAs expression analysis is carried out on the sample, it is found that the VvHPCA1 expression quantity is in positive correlation with the H2O2 level and the aging degree mediated by the H2O2 level, and the expression modes of VvHPCA1 and VvHPCA3 are opposite; the expression level of the VvHPCA3 is in negative correlation with the H2O2 content and the post-harvest aging degree; the invention further provides application of the VvHPCA1 and VvHPCA3 genes, wherein overexpression of the VvHPCA1 genes or inhibition of the expression of the VvHPCA3 can enhance the sensitivity of grape fruits to H2O2, and then the ROS-mediated maturation and aging process is accelerated; by inhibiting the expression of the VvHPCA1 gene or overexpressing the VvHPCA3 gene, the sensitivity of grape fruits to H2O2 can be weakened, so that stress and senescence caused by H2O2 can be resisted.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a grape hydrogen peroxide receptor gene, its encoded protein and its application. Background technique [0002] Reactive oxygen species (ROS) are a class of molecules / ions with lively chemical properties and high oxidation activity, including hydrogen peroxide (H 2 o 2 ), hydroxyl radical (hydroxyl radical, OH . ) and other forms. It can be produced by cellular metabolic pathways (by-products) or actively synthesized by cells under special physiological conditions (Mittler 2017). ROS can regulate the physiological activities of cells in a certain concentration range, otherwise it will cause oxidative damage and even cell death. In recent years, the function of ROS in fruit ripening and senescence has been paid more and more attention. There are usually two accumulation peaks of ROS during fruit ripening and senescence. Generally, it rises sharply to ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/11C12N15/82C12Q1/6851C07K14/415A01H5/00A01H5/08A01H6/88
CPCC07K14/415C12N15/8271C12N15/8266C12Q1/6851C12Q2531/113C12Q2521/107
Inventor 郭大龙刘海楠裴茂松余义和韦同路
Owner HENAN UNIV OF SCI & TECH
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