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Rho族蛋白是重要的分子开关,受GTP酶激活蛋白(GAP)调控。MGG_09303在稻瘟病菌中编码一个假定的Rho GTP酶激活蛋白。为探讨其与Rho族蛋白的互作关系,本文首先将基因MGG_09303与酵母中RhoGAPs蛋白序列进行多重比对,结果表明MGG_09303与酿酒酵母中的RhoGAP蛋白的氨基酸同源性较高的有BEM3(29%)、BEM2(28%)、LRG1(27%),而相似性较高的有RGD2(53%)、LRG1(52%),还发现MGG_09303与酿酒酵母中的RhoGAP具有共同的保守的精氨酸。据此初步预测它有可能与Rho1和Rho3存在互作,本研究构建了pGADT7-MGG_09303捕获表达载体,利用酵母双杂交技术将捕获载体pGADT7-MGG_09303分别与Rho族蛋白持续激活态(CA)和失活态(DN)诱饵载体共转AH109酵母细胞,进行双杂交实验。结果表明,MGG_09303蛋白与Rho1和Rho3的持续激活态(CA)互作,而不与其负显性失活态(DN)互作,研究结果验证了预测的结果,为进一步研究RhoGAP蛋白对Rho族蛋白负调控的分子机制奠定了重要的基础。
The Rho family of proteins is an important molecular switch regulated by GTPase (GAP). MGG_09303 encodes a putative Rho GTPase activating protein in Magnaporthe grisea. In order to explore its interaction with Rho family proteins, we firstly compared the MGG_09303 gene with the RhoGAPs in yeast. The results showed that the amino acid sequence of MGH_09303 and RhoGAP in Saccharomyces cerevisiae were highly homologous with BEM3 (29 %), BEM2 (28%) and LRG1 (27%), whereas RGD2 (53%) and LRG1 (52%) were highly similar. MGG_09303 was also found to have conserved arginine in common with RhoGAP in Saccharomyces cerevisiae acid. According to this, it is predicted that it may interact with Rho1 and Rho3. In this study, a pGADT7-MGG_09303 capture expression vector was constructed. The pGADT7-MGG_09303 and the Rho family protein, respectively, Live (DN) bait vectors co-transferred AH109 yeast cells for two-hybrid experiments. The results showed that MGG_09303 interacts with the continuous activation state (CA) of Rho1 and Rho3, but not with its negative dominant negative (DN). The results validated the predicted results. To further investigate the effect of RhoGAP on Rho Protein negative regulation of molecular mechanisms laid an important foundation.