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The Proteomics Analysis of Resistance to Downy Mildew and Isolation of Resistance Gene Analogs in Radish(Raphanus Sativus L.)

Author: WangXianLi
Tutor: GongYiQin;LiuLiWang
School: Nanjing Agricultural College
Course: Olericulture
Keywords: Radish (Raphanus sativus L.) Downy mildew Proteomics DDRT-PCR Resistance gene analogs (RGAs)
CLC: S436.31
Type: Master's thesis
Year: 2013
Downloads: 5
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Abstract

Radish (Raphanus sativus L.) belongs to the Brassicaceae family and is an important worldwide vegetable crop with high nutrition and medical value. Downy mildew (DM) is one of the main diseases of Brassicaceae vegetables induced by the (Oomycete Peronospora parasitica). In recent years, radish downy mildew occurred with increasing severity, coupled with mixed infection of viral disease, resulted in the radish production and quality seriously declining, while the previous researches about radish downy mildew have not attracted the attention of breeders. When the plants infected by pathogen, series of chemical changes will occur in vivo. For a better understanding of the mechanisms of plant resistance to disease, in this study,2-DE technology and mRNA differential display technical were used to analysis proteome changes and the expression of the differentially expressed genes before and after the radish leaves affected by downy mildew. In addition, we have cloned a variety of/?-gene like sequences, and developed them into marks to understand the resistance of different radish varieties. These results would help to analyze Disease-Resistance Mechanism from different levels when radish affected by pathogen and provide some theoretical basis for the genetic improvement of radish quality and yield. The main findings are as follows:1. A proteomic approach was employed to study the mechanism of disease resistance when radish affected by downy mildew with the high-generation inbred line’NAU-BJQ’as material. Proteins were extracted from leaves at7days post-inoculation by using Tris-Hcl/TCA-acetone extraction method and profiled by two-dimensional gel electrophoresis. The differentially expressed proteins were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). A total of24protein spots showed significant difference and22were successfully identified. The identified proteins had functions related to protein synthesis and metabolism, sucrose and energy metabolism, signal transduction, resistance related proteins and regulation proteins. qRT-PCR approach based on peptide sequences was used to compare transcript and protein accumulation patterns for two up-regulated and two down-regulated proteins.2. Differential-display reverse transcription-PCR (DDRT-PCR) was used to investigate the difference of gene expression during the taproot development of radish. A total of56differential fragments had been obtained in total of80selective amplifications.53fragments showed high sequence similarity to the genes of known or putative function, which were involved in transcriptional regulation, signal transduction, metabolism, transport channel and stress responding as well as protein metabolism. The results indicated that a variety of metabolic pathways changed when radish subject to pathogens. qRT-PCR was used to characterize the expression feature of six genes in different stages post-inoculation. All of the6induced transcript accumulation was consistent with DDRT-PCR.3. In this study, two prevalent approaches, PCR amplification with degenerate primers and data-mining of radish EST databases, were used to isolate radish resistance gene candidate sequences. A total of26RsRGAs with high homology to known R-genes or RGAs were obtained by PCR amplification. Further analysis indicated that20sequences encoding a single open reading frame (ORF). Using data-mining method,257R-genes-like unigenes/ESTs were identified. For RGA-based marker development, totally124specific primers were designed according to RGA sequences and71primers could be amplified polymorphic bands in32radish accessions. Based on the data from RGA marker analysis,32radish accessions were divided into four main groups at the similarity index of0.68, which was in high accordance with disease index from downy mildew evaluation.

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