【作者】 石振华；

【导师】 鲁国东；

【作者基本信息】 福建农林大学 ， 遗传学， 2011， 硕士

【摘要】 Rho1是小G蛋白Rho族的一个重要成员,它存在两种相互转化的状态:GDP结合的失活状态和GTP结合的活性状态。GDP/GTP交换因子（GEF1）的功能是促进Rho1从GDP结合态向GTP结合态的转换。本研究首先通过生物信息学的方法对MGG₁2644这个基因的理论等电点、分子量、结构域以及功能等进行预测分析,结果显示该基因是小G蛋白Rho1的GEF,同时该基因的变化与物种的进化相吻合。然后以稻瘟病菌为模式生物,通过同源重组的方法得到该基因的敲除突变体,发现该基因的缺失突变体虽然生长速度缓慢,然而隔膜间距与野生型相比却无明显差异,说明该基因可能参与了细胞分裂周期的调控。突变体在加有SDS的淀粉培养基中的生长速度受到抑制,说明对SDS的耐性能力降低。此外,突变体不能产生分生孢子,菌丝末端虽然可以分化生成附着胞,但产生的附着胞的数量也很少且附着胞丧失了侵染洋葱表皮及水稻叶片的能力,因而在造伤和非造伤的叶片上都不能形成病斑。这些表型都受到Rho1的调控,因而推测该基因与Rho1的功能有关。将带有启动子的MgRhoGEF1基因转入缺失敲除突变中,突变体的表型得以恢复,说明敲除突变体表型的出现是由于MgRhoGef1的缺失直接或间接导致。利用酵母双杂交的方法证实了MgRhoGef1及其结构域都能与Rho1互作,因而对MgRhoGef1的研究有利于了解Rho1的功能,完善对Rho1的认识。又由于MgRhoGef1的缺失导致稻瘟病菌丧失致病能力,因而有可能通过该基因的研究找到合适药物靶标,为稻瘟病的防治提供新的策略。更多还原

【Abstract】 Rho1 GTPase is one of the most important Rho family members belonging to small GTP-binding proteins. It has two convertible forms: GDP-bound inactive and GTP-bound active forms. Guanine nucleotide releasing factor （also called GEF） regulated by an upstream signal interacts with GDP-bound form and catalyze the exchange of GDP for GTP to generate the active state of Rho GTPase. Based on bioinformatics analysis, the gene MGG₁2644 has a putative RhoGEF domain in its amino acid sequence, homologous to MgRhoGef1 in Saccharomyces cerevisiae. Phylogenetic analysis showed that the RhoGEF domain in MGG₁2644 are conserved but also showed divergent while the species evolved. Magnaporthe oryzae as an ideal model organism is used for our study. Knock-out mutants by homologous recombination grow slower than wild-type strain （WT）, but the distance of two neighbor septa is nearly as normal, indicating that the MgRhoGef1 gene is related with cell division cycle. In addition, the mutants show lower ability to endure SDS than that of WT. The deletion mutants could not form conidia, but could develop appressoria at the hypha terminals, although there are fewer compared to the WT. These appressoria formed by deletion mutants lost the ability to infect the epidermal cell of onion as well as rice leaves, so we cannot see any lesions on the inoculated leaves. The complementary mutants recovered all these defects of deletion mutants indicating that the MgRhoGef1 is truly involved in these phenotypes. Based on the bioinformatics analysis, we infer that the MgRhoGef1 gene is the GEF of Rho1 protein. Yest two hybridization confirm that the interaction of MgRhoGef1 and its GEF domain with Rho1. Taking together, our study will be in favor of better understanding of Rho1 function, and further study would help to find an ideal target for controling the damage of Magnaporthe oryzae.更多还原