摘要
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。
Rho GTPase is an important molecular switch, regulated by GTPase activation proteins （GAP）. MGG_ 09303 encodes a putative Rho GTPase activating protein （Rho GAP） in Magnaporthe oryzae. To explore the possible interaction between MGG_09303 and Rho proteins, we first analyzed sequence identity and similarity of MGG_9303 and RhoGAPs in Saccharornyces cerevisiae in this study. The results showed that it has a relatively high identity with BEM3 （29%）, BEM2 （28%）, LRG1 （27%） and a high similarity with RGD2 （53%）, LRG1 （52%） in S. cerevisiae. Therefore we predicted that MGG_09303 might interact with Rhol and Rho3 in M. oryzae. Then by yeast two-hybrid assay, we made pGADT7-MGG_09303 prey construct to co-transfrom into AH109 yeast cells with bait constructs of constitutively active （CA） and dominant negative （DN） forms of Rho proteins, respectively. The results showed that MGG 09303 could interact with CA forms of both Rhol and Rho3, but not their DN forms, which verified our prediction so that this study lays the foundation for further understanding in the negative regulatory mechanisms of Rho-family proteins by RhoGAPs.
出处
《分子植物育种》
CAS
CSCD
北大核心
2013年第6期719-724,共6页
Molecular Plant Breeding
基金
国家自然科学基金(31030004
31300132)资助
关键词
稻瘟病菌
酿酒酵母
RHO
GAP
序列比对
酵母双杂交
Rho族蛋白
相互作用
Magnaporthe oryzae, Saccharomyces cerevisiae, RhoGAP, Sequence alignment, Yeast two-hybrid, Rho- family protein, Interaction
