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Cytochrome P450 gene StCYP84A from eggplant, and acquisition method and application thereof

A cytochrome and gene technology, applied in the fields of molecular biology and agriculture, can solve the problem of large variation of CYP72A gene, and achieve the effect of enriching gene selection

Inactive Publication Date: 2017-10-24
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, the study by Prall et al. also found that the CYP72A gene has the greatest degree of variation in Solanaceae crops, speculating that the loss of CYP72A gene during domestication may also be related to the difference in resistance between wild species and cultivated species

Method used

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  • Cytochrome P450 gene StCYP84A from eggplant, and acquisition method and application thereof
  • Cytochrome P450 gene StCYP84A from eggplant, and acquisition method and application thereof
  • Cytochrome P450 gene StCYP84A from eggplant, and acquisition method and application thereof

Examples

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Embodiment 1

[0028] In Example 1, the polyphenol oxidase gene was cloned using the RACE method using wild eggplant eggplant cDNA and genomic DNA as templates. The specific steps are as follows:

[0029] 1) Preparation of spore suspension:

[0030] Verticillium dahliae of eggplant was cultured on PDA medium plate for 7 days, and a mycelium block with an area of ​​about 1cm*1cm was picked from the edge with a dissecting needle, transferred into PL culture medium, and cultured on a shaker at 120rmp / min at 25°C for 7 days. -9 days, at this time, the PDA culture fluid (potato dextrose agar medium) changed from a transparent liquid to a milky white liquid. Filter the milky white liquid with double-layer gauze, keep the filtrate, observe the number of spores with a hemocytometer, calculate the number of spores per milliliter, dilute the bacterial spores with sterilized distilled water, and finally prepare a concentration of 1x10 7 spores / mL of the spore suspension, which was then used for inocul...

Embodiment 2

[0057] In this example, the eggplant gene obtained by cloning in Example 1 was analyzed and detected, and it was confirmed by cluster analysis and amino acid comparison that the gene belongs to the cytochrome oxidase gene family, which is the same as the cytochrome P450 gene in potato. Moreover, the use of real-time quantitative PCR analysis showed that its expression was related to the resistance of Verticillium dahliae in watermelon, further confirming its function. details as follows:

[0058] 1) Bioinformatics analysis of StCYP84A gene and its encoded protein in tomato

[0059] A cDNA sequence (SEQ ID NO.1) with a length of 2035 bp was cloned by RACE technology, and it was named StCYP84A. The 5'UTR of the gene contains 129bp, and the 3'UTR contains 331bp. Through ORF Finder combined with BLAST X for sequence comparison, it is speculated that StCYP84A contains an open reading frame (open reading frame, ORF) of 1575bp, encoding 324 amino acids, its isoelectric point is pre...

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Abstract

The invention provides a cytochrome P450 gene StCYP84A from eggplant, and an acquisition method and application thereof. The cytochrome P450 gene StCYP84A has a nucleotide sequence as shown in SEQ ID. No. 1. The acquisition method for the gene comprises the following steps: 1) extracting the total RNAs and genomic DNAs of a Solanum torvum sample; 2) with cDNAs obtained after inverse transcription of the RNAs extracted in the step 1) as a template, designing specific primers and carrying out RACE-PCR amplification; and 3) recovering a PCR product obtained in the step 2) and carrying out sequencing and sequence analysis. The cytochrome P450 gene StCYP84A from eggplant and coded related proteins can be used for resisting verticillium wilt and can be further applied to improvement of inherited characteristics in the fields of plant molecular marker-aided breeding and genetic engineering. The cytochrome P450 gene StCYP84A provided by the invention is of great significance to cultivation of plant stress resistance varieties, especially anti-verticillium wilt eggplant varieties, and to improvement of crop output and quality.

Description

technical field [0001] The invention relates to the fields of molecular biology and agriculture, in particular to a method for obtaining eggplant verticillium wilt resistance cytochrome P450 gene and its application. Background technique [0002] Eggplant (Solanum melongena L.) is one of the important vegetables in my country. It is rich in nutrition and has high economic benefits. However, most cultivated eggplants are susceptible to Verticillium wilt. Verticillium wilt of eggplant is a soil-borne vascular disease caused by Verticillium fungus. Its symptoms usually manifest as yellowing and wilting of half of the plant or half of the leaves. When the disease is severe, the stems are split and the vascular bundles turn brown. The disease has a long incubation period, strong transmission ability, and a wide range of damage. When the disease is severe, the output loss can reach more than 60%. The United States, Japan and India have all had outbreaks of eggplant Verticillium w...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/10C12N15/82A01H5/00C12Q1/68
CPCC12N9/0071C12N15/1003C12N15/8282C12Q1/6895C12Q2600/13C12Q2600/158C12Q2531/113
Inventor 杨旭张宇王露成玉富
Owner YANGZHOU UNIV
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