辣椒CaROPGEF1 cDNA的分离及其结构和表达的初步分析

Isolation of CaROPGEF1 cDNA of pepper and preliminay analysis for its structure and expression

ROP是植物特有的一类Rho相关GTPases,在植物体内通过GTP结合形式与GDP结合形式的切换而对生长发育和适应环境起调节作用.由GTP结合形式向GDP结合形式的转换主要依赖于鸟苷酸交换因子(guanine nucleotide exchangefactor,GEF).本文以锁定于GDP结合形式的辣椒DN-CaROP1突变体为诱饵,采用ProQuestTM酵母双杂交体系,从辣椒幼苗cDNA文库中分离了辣椒GEF阳性克隆,其推导的氨基酸序列与其他植物中发现的GEF具有较高的同源性,并含典型的植物ROPGEF保守域DUF315及N端与C端的高度变化域,将之命名为CaROPGEF1;RT-PCR分析表明,CaROPGEF1在辣椒多种组织中均有表达;进一步酵母双杂交分析表明,CaROPGEF1可与GDP结合态的ROP结合,而不能与GTP结合态的ROP结合.上述结果暗示CaROPGEF1调节的ROP从DN态向CA态的转化在辣椒植株中频繁发生,可能参与辣椒ROP介导的生长、发育和对环境适应的调控.

ROP,the sole family of a Rho-related GTPase of plants,play important roles in the regulation of plant growth,development and environment adaption by the conversion between GDP-bound and GTP-bound form,and transformation from GDP-bound form to GTP-bound form is catalyzed by guanine nucleotide exchange factor（GEF）.In this study,a positive clone of guanine nucleotide exchange factor of pepper was isolated with ProQuestTM yeast two-hybrid system from pepper seedlings using GDP-bound mutant of pepper ROP as a bait.The induced amino acid sequence was homologous highly with the GEF from other plants,and contained a conserved DUF315 domain and a variable N-and C-terminal regions,it was designated as CaROPGEF1;RT-PCR assay showed that CaROPGEF1 expressed in a variety of plant tissues and the result of yeast two-hybrid assay showed that CaROPGEF1 was interactive with GDP-bound CaROP,but not with GTP-bound CaROP.These results suggested the switch from GDP-bound CaROP to GTP-bound CaROP mediated by CaROPGEF1 occurs continually,which might play roles in the CaROP mediated regulation of plant growth,development and environment adaption in pepper.