【作者】 张冬梅；

【导师】 王宗华； 鲁国东；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 Rab族蛋白是小G蛋白Ras家族最大的亚家族之一。研究表明它主要参与细胞内的囊泡运输,调节生物体内各种蛋白在细胞内外的正确运输和分配。为明确Rab族蛋白在稻瘟病菌等丝状真菌生长发育和侵染过程的作用,对稻瘟病菌Rab蛋白进行了生物信息学、基因表达和功能分析。生物信息学分析表明,在稻瘟病菌基因组中有11个Rab蛋白家族成员,这些Rab家族成员都具有其保守的结构域并且与其它物种间的Rab族蛋白存在着高度的同源性。荧光实时定量PCR分析表明,稻瘟病菌11个假定的Rab蛋白MoRab1、MoRab4、MoRab52、MoRab53和MGG₀7191相对于其它发育阶段,均是孢子发育阶段表达最高;MoRab6、MoRab7和MoSec4在菌丝发育阶段表达最高;MoRab51和MoRab8在芽管发育阶段表达最高;MGG₀1079附着胞阶段的表达量最高。本研究利用同源重组方法,获得一个MoRab51（Mgg01185.6）和MoRab7（Mgg08144.6）的基因敲除突变体。表型分析发现两个基因敲除突变体的气生菌丝较野生菌株明显减少,菌丝生长速度明显减慢,致病力丧失。Rab51敲除突变体分生孢子形成减少,而MoRab7的敲除突变体不形成分生孢子。MoRab7的敲除突变体菌丝块接种洋葱表皮可以形成附着胞,但无法成功侵入洋葱表皮细胞。以孢子悬浮液和菌丝块分别接种水稻叶片结果表明,MoRab51和MoRab7敲除突变体均失去了对水稻的致病能力。透射电子显微镜观察发现,MoRab51和MoRab7敲除突变体相对对照菌株WT细胞内部囊泡数目增多,变小。综上所述,稻瘟病菌MoRab51和MoRab7蛋白可能参与了稻瘟病菌极性生长相关蛋白的转运,该蛋白的缺失阻碍细胞的极性生长,表现为菌丝生长速度、产孢能力丧失,进一步导致其致病力丧失。更多还原

【Abstract】 Rab family proteins (also called Rab GTPases) belong to small GTPases Ras superfamily. It has been shown that Rab-GTPase plays a critical role in mediating vesicular transport between different cytoplasmic membranes, so it also plays a key role in fungal development and is involved in regulation of cell polarity growth.To characterize the function of Rab GTPases in Magnaporthe oryzae, we conducted functional analysis of Rab GTPases of the fungus at whole genome level. There are 11 putative members of Rab family in Magnaporthe grisea genome database, all of which have the conserved domains and display high homology with Rab members in other organisms. Then, we designed primers according to gene sequences of the putatitve Rab genes, and conducted Real-time PCR to obtain the expression profile during the different stages including hypha, spore, germ tube and appressorium. In order to identify the gene function, we constructed a series of gene knock-out vectors for gene deletion through homologous recombination approach. TheΔMorab51 andΔMorab7 mutants were confirmed by PCR and Real-time PCR. Further, we analyzed the phenotype ofΔMorab51 andΔMorab7 mutants. It showed that growth rate of the mycelia reduced dramatically.ΔMorab7 mutants,as well asΔMorab51 failed to cause lesions on infected rice leaves. The conidiation of theΔMorab51 mutant decreased remarkably, while theΔMorab7 mutant failed to produce conidia. The mycelia still formed normal appressoira and didn’t penetrate the onion epidermics. TheΔMorab51 andΔMorab7 mutants lost pathogenicity to rice. TEM observation showed that the vacuole maturation was blocked either inΔMorab51 or inΔMorab7 mutant.Our results suggested that MoRab51 and MoRab7 participates in the transportation of proteins related to cell polarity growth and pathogenicity, which were involved in regulation of vegetative growth and conidiation in M.oryzae.更多还原