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利用一对抗瘟性近等基因系(Near-isogenic lines, NILs)H7R(抗病)和H7S(感病),经稻瘟病菌小种早期诱导,提取RNA,进行mRNA差异显示(Differential display,DD)分析.在获得多个DD片段的基础上,经PCR重扩增、Sourthern杂交粗筛及Northern blotting进一步鉴定.阳性片段克隆于PGEM-T载体,经序列测定,国际联网查询,已获得两个受稻瘟病菌诱导的水稻新基因cDNA片段克隆. 在我国,Magnaporthe grisea是水稻稻瘟病的主要病害菌,由于生理小种的复杂性,抗病品种的抗性丧失已成为传统育种的一大难题.国际上对采用基因工程改良水稻的抗瘟性越来越重视并已成了各国竞争的热点.然而期望通过RFLP基因定位,并借助图谱克隆抗瘟性基因尚需多年的工作和大量的投入.此外,对于水稻与稻瘟病菌之间“基因对基因”的互作在分子水平上尚缺乏了解.本研究采用了本实验室完善的mRNA差异显示技术体系,在国际上首次用DD方法,鉴定和克隆受稻瘟病菌诱导的新的水稻抗瘟性和防卫反应候选基因.
Using a pair of near-isogenic lines (NILs) H7R (resistant to disease) and H7S (susceptible), RNA was extracted and differentially expressed by differential display (DD ) .According to multiple DD fragments obtained by PCR re-amplification, Sourthern hybridization screened and Northern blotting further identified positive fragment cloned in the PGEM-T vector, the determination of the sequence, the international network query, have been two Magnaporthe grisea is the main pathogen of rice blast in our country, and the loss of resistance of resistant varieties has become a major challenge in traditional breeding due to the complexity of physiological races .In the world, more and more attention has been paid to the anti-rice blast resistance using genetic engineering and it has become a hot issue in all countries.However, it is expected that the RFLP gene localization and the cloning of the blast resistance gene will still require years of work and substantial investment In addition, there is still a lack of understanding on the molecular level of the “gene-to-gene” interaction between rice and Magnaporthe grisea.In this study, we used our laboratory’s perfect mRNA differential display technology system, Using the DD method, the new rice blast resistance and defense response candidate genes induced by M. grisea were identified and cloned.