杆状病毒p35基因诱导烟草产生广谱抗病机理分析

Mechanism Analysis of Broad-spectrum Disease Resistance Induced by Expression of Anti-apoptotic p35 Gene in Tobacco

摘要:

来源于昆虫病毒和动物的抗细胞凋亡基因能够诱导植物对生物或者非生物胁迫产生抗性, 但其抗性机理有不同甚至相反的报道。本研究将来源于苜蓿银纹夜蛾核多角体病毒的p35基因转化烟草, T1代转化烟草Western blotting 检测P35蛋白的表达, 转化烟草接种烟草花叶病毒(Tobacco mosaic virus, TMV)抗病效果增强。进一步的抗病机理研究表明, 转化和野生型烟草感染TMV后诱导过氧化氢积累无明显区别, 野生型烟草感染24 h后出现DNA Laddering而转化烟草则没有; Western blotting 结果显示PR-1蛋白表达没有显著差异。但接种另外一种病原真菌核盘菌(Sclerotinia sclerotiorum)后的RT-PCR分析结果表明, 表达P35蛋白的烟草可增强感染核盘菌后PR-1基因的转录, 而且表达时间提前。以上结果说明p35基因介导的广谱抗病反应的机理与接种的不同病原有关, 对不同病原物的抗病机理存在差异, 除抑制细胞凋亡外, 还可能通过激活PR基因的表达提高对病原物的抗病能力。

Abstract:

Studies have shown that transgenic plants expressing antiapoptotic genes from baculovirus and animals increase resistance to biotic and abiotic stress. However, the mechanism under these resistances is conjectural, or in some cases even controversy. In the present study, the p35 gene from baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) was expressed in tobacco, and for the first time P35 protein was detected in transgenic plants by Western blotting. Inoculation of T1 transgenic tobacco leaves with tobacco mosaic virus (TMV) showed enhanced resistance, and DNA laddering was observed after TMV infection in control but not in transgenic plants. DAB staining showed that TMV infection did not affect peroxide induction of transgenic plants, Western blotting analysis of PR1 protein also showed no difference of control and transgenic plants. Inoculation of fungus (Sclerotinia sclerotiorum) using a detached leaf assay showed enhanced resistance of transgenic leave tissue. RT-PCR analysis demonstrated that p35 gene expression induced earlier expression of PR1 gene after S. sclerotiorum infection. Taken together, our results suggest that the mechanism under enhanced disease resistance by P35 protein is possibly related to the activation of PR-related proteins in addition to the inhibition of programmed cell death, depending on the pathogens challenged.