关键词: 稻瘟病, 水稻地方品种, 抗病相关基因, 遗传转化, 实时荧光定量PCR

Abstract:

【Objective】 Acuce, currently the predominant cultivating rice landrace at Yuanyang Hani terrace, Yunnan Province, has been continuously planted for more than 100 years. Interestingly, there has never been a major outbreak of rice blast during the past decades. Clarifying the types, quantities, expression characteristics, and phenotypic effects of functional genes involved in the resistance response process so as to exploring the molecular mechanism of Acuce resistance to Magnaporthe oryzae will optimize facilitate the conservation and utilization of rice landrace. 【Method】 The present research focuses on the biological effect of disease resistance-related candidate gene BGIOSGA023826 which screened from Acuce -M. oryzae interaction transcriptome. Initially, biological function of this gene was analyzed by using bioinformatics software and conducted gene cloning. Subsequently, overexpression vector was constructed through Agrobacterium-mediated genetic transformation, then carry out positive identification and disease resistance phenotype analysis of overexpressed plants. Finally, Real-time fluorescence quantitative PCR was used to analyze the expression of PR genes in overexpressed plants, and enzyme-linked immunosorbent assay was used to determine changes in endogenous hormone content. 【Result】 The candidate gene BGIOSGA023826 encodes 354 amino acids figuring unstable hydrophobic characteristic without transmembrane domains, coding a non-membrane and non-secretory protein, classified as a hydrolase gene. The overexpression of BGIOSGA023826 in transgenic plants significantly enhances resistance to M. oryzae compared to Nipponbare. The results of real-time fluorescent quantitative PCR indicate that the relative expression levels of the PR genes PR1b, OsNPR1, and OsAOS2 as well as transcription factors WRKY45 significantly upregulated. Both of the PR1b gene and the key gene OsAOS2 functioning in the JA synthesis pathway continuously upregulated from 12 hpi. Meanwhile, the expression level of the transcription factor gene WRKY45 drastically increased at 24 hpi and reached its peak at 36 hpi, which were nearly two folds higher than that of Nipponbare. As to the results of endogenous hormone content measurement, the JA increased to 263.88 pmol·L^-1 at 12 hpi, reaching peak 304 pmol·L^-1 at 36 hpi; Similarly, the SA increased to 186.5 pmol·L^-1 at 24 hpi, reaching peak 198.88 pmol·L^-1 at 36 hpi; At the same time, the content of ROS also rises continually and reached its peak at 36 hpi, MDA also increased from 40.43 nmol·g^-1 to 53.94 nmol·g^-1; However, SOD did not exhibit significant differences at individual time points in transgenic plant, but showed an upward trend in Nipponbare. 【Conclusion】 Preliminary elucidating of the temporal expression relationship between the hydrolase gene BGIOSGA023826 and the PR genes as well as endogenous hormone levels, demonstrating it involved in the landrace Acuce resistance to M. oryzae infection process.

Key words: Magnaporthe oryzae, rice landraces, disease resistance related genes, genetic transformation, real-time fluorescent quantitative PCR