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为揭示来源于番茄细菌性斑点病菌(Xanthomonas campestris pv.vesicatoria,Xcv)菌株XV18鞭毛素(FliCxcv)作为一种病原相关分子模式(PAMP)诱导水稻免疫反应的功能,本研究对FliCxcv编码基因flicxcv进行了基因克隆、序列分析、原核表达、蛋白纯化和诱导活性测定。结果表明,通过PCR特异性扩增,从Xcv菌株XV18中克隆了1 200bp的fliCxcv基因片段,其序列与GenBank中己测序菌株的完全一致。在大肠杆菌中对该基因全长、N端和C端截短序列进行了原核表达,并获得了纯化的FliCxcv全长及其截短蛋白。将纯化蛋白浸润接种到水稻品种日本晴叶片组织,发现FliCxcv全长及其截短蛋白均能诱导水稻叶片细胞死亡、H2O2产生以及防卫基因(OsPAL和OsPR1b)表达等免疫反应,但诱导活性存在差异。因此,本研究验证了FliCxcv具有激发水稻细胞免疫反应的PAMP功能,为水稻免疫诱导制剂的研发提供了材料。
In order to elucidate the function of FliCxcv (X18) from Xanthomonas campestris pv. Vesicatoria (Xcv) as a pathogen-related molecular model (PAMP) to induce rice immune response, we investigated the flicxcv gene encoding FliCxcv Gene cloning, sequence analysis, prokaryotic expression, protein purification and induction activity determination. The results showed that a fragment of fliCxcv of 1 200 bp was cloned from Xcv strain XV18 by PCR-specific amplification. The sequence of the fliCxcv gene was identical with that of the sequenced strain in GenBank. The full-length, N-terminal and C-terminal truncated sequences of the gene were prokaryotic in Escherichia coli, and the full-length FliCxcv and its truncated protein were obtained. The purified protein was inoculated into Nipponbare leaves of rice variety Nipponbare. The results showed that FliCxcv full-length and its truncated protein could induce the cell death, the production of H2O2 and the expression of defense genes (OsPAL and OsPR1b) in rice leaves, but the induction activity was different. Therefore, this study demonstrated that FliCxcv has the function of PAMP that stimulates the cellular immune response of rice, and provided the material for the research and development of rice immune-inducing agents.