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水稻条斑病菌和白叶枯病菌致病相关基因的敲除

DOI: 10.7685/j.issn.1000-2030.2006.02.011, PP. 50-56

Keywords: 水稻条斑病菌,水稻白叶枯病菌,基因敲除,LPS合成基因,无毒基因

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Abstract:

wxocA、wxocB、wxocD、wxocE和wzt是水稻条斑病菌(Xanthomonasoryzaepv.oryzicola,Xooc)的脂多糖(lipopo-lysaccharide,LPS)合成基因,avrXa3是水稻白叶枯病菌(Xanthomonasoryzaepv.oryzae,Xoo)的一个无毒基因。利用pBCSK(-)和pKNG101两个质粒的复制起点不被水稻黄单胞菌识别的特点,把wxocA、wxocD、wxocE和wzt的中心区域克隆在pBCSK(-)上,获得突变转化单元pBCSK∷ΔwxocA、pBCSK∷ΔwxocD、pBCSK∷ΔwxocE和pBCSK∷Δwzt。把wxocB和avrXa3基因的旁侧序列和一个氯霉素抗性基因cm构建在pKNG101上,获得突变转化单元pKNG101∷ΔwxocB和pKNG101∷Δavr。以电转化方式把这些LPS合成基因突变转化单元DNA转入Xooc菌株RS105,把无毒基因突变转化单元转入Xoo菌株PXO99,通过同源重组分别获得了RS105中5个lps相应基因突变体MwxocA、MwxocB、MwxocD、MwxocE、Mwzt和一个无毒基因的突变体PXO99Δavr。SDS-PAGE分析发现,lps突变体的O抗原或核心寡糖的合成被破坏。致病性分析结果显示,基因wxocB、wxocE和wzt与致病性有关,前两基因的突变导致细菌完全失去毒性,而后一基因的突变导致细菌部分丧失毒性。与PXO99相比无毒基因突变体PXO99Δavr改变了原有的毒性表型,在IRBB50(Xa4/xa5)和Asominori(Xa17)上由较感转变为较抗表型。因此,本试验证明,以pBCSK(-)和pKNG101为载体敲除水稻条斑病菌和白叶枯病菌致病相关基因是可行的。

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