分子植物育种
分子植物育種
분자식물육충
MOLECULAR PLANT BREEDING
2013年
6期
719-724
,共6页
叶文雨%陈四妙%陈晓%林艺娟%汪洋%余文英%鲁国东%陈继圣%王宗华
葉文雨%陳四妙%陳曉%林藝娟%汪洋%餘文英%魯國東%陳繼聖%王宗華
협문우%진사묘%진효%림예연%왕양%여문영%로국동%진계골%왕종화
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
稻瘟病菌%釀酒酵母%Rho GAP%序列比對%酵母雙雜交%Rho族蛋白%相互作用
도온병균%양주효모%Rho GAP%서렬비대%효모쌍잡교%Rho족단백%상호작용
Magnaporthe oryzae%Saccharomyces cerevisiae%RhoGAP%Sequence alignment%Yeast two-hybrid%Rho-family protein%Interaction
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族蛋白是重要的分子開關,受GTP酶激活蛋白(GAP)調控。MGG_09303在稻瘟病菌中編碼一箇假定的Rho GTP酶激活蛋白。為探討其與Rho族蛋白的互作關繫,本文首先將基因MGG_09303與酵母中RhoGAPs蛋白序列進行多重比對,結果錶明MGG_09303與釀酒酵母中的RhoGAP蛋白的氨基痠同源性較高的有BEM3(29%)、BEM2(28%)、LRG1(27%),而相似性較高的有RGD2(53%)、LRG1(52%),還髮現MGG_09303與釀酒酵母中的RhoGAP具有共同的保守的精氨痠。據此初步預測它有可能與Rho1和Rho3存在互作,本研究構建瞭pGADT7-MGG_09303捕穫錶達載體,利用酵母雙雜交技術將捕穫載體pGADT7-MGG_09303分彆與Rho族蛋白持續激活態(CA)和失活態(DN)誘餌載體共轉AH109酵母細胞,進行雙雜交實驗。結果錶明,MGG_09303蛋白與Rho1和Rho3的持續激活態(CA)互作,而不與其負顯性失活態(DN)互作,研究結果驗證瞭預測的結果,為進一步研究RhoGAP蛋白對Rho族蛋白負調控的分子機製奠定瞭重要的基礎。
Rho족단백시중요적분자개관,수GTP매격활단백(GAP)조공。MGG_09303재도온병균중편마일개가정적Rho GTP매격활단백。위탐토기여Rho족단백적호작관계,본문수선장기인MGG_09303여효모중RhoGAPs단백서렬진행다중비대,결과표명MGG_09303여양주효모중적RhoGAP단백적안기산동원성교고적유BEM3(29%)、BEM2(28%)、LRG1(27%),이상사성교고적유RGD2(53%)、LRG1(52%),환발현MGG_09303여양주효모중적RhoGAP구유공동적보수적정안산。거차초보예측타유가능여Rho1화Rho3존재호작,본연구구건료pGADT7-MGG_09303포획표체재체,이용효모쌍잡교기술장포획재체pGADT7-MGG_09303분별여Rho족단백지속격활태(CA)화실활태(DN)유이재체공전AH109효모세포,진행쌍잡교실험。결과표명,MGG_09303단백여Rho1화Rho3적지속격활태(CA)호작,이불여기부현성실활태(DN)호작,연구결과험증료예측적결과,위진일보연구RhoGAP단백대Rho족단백부조공적분자궤제전정료중요적기출。
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 Saccharomyces 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 Rho1 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 Rho1 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.