三角帆蚌Rab3 cDNA全长克隆及其与三角帆蚌瘟病相关性鉴定  

Full-length cDNA Cloning of Rab3 Gene from Hyriopsis cumingii and Related to Hyriopsis cumingii Plague

作　　者：许宝红[1] 肖调义[1] 刘巧林[1] 苏建明[1] 陈开健[1] 刘敏[1] 周伟[1] 

机构地区：[1]湖南农业大学动物科学技术学院,长沙410128

出　　处：《中国农学通报》2011年第29期64-71,共8页Chinese Agricultural Science Bulletin

基　　金：国家自然科学基金"三角帆蚌丝氨酸蛋白酶基因的克隆;表达调控与功能研究"(31040083)

摘　　要：根据三角帆蚌消减杂交cDNA文库获得的EST序列,运用cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术获得三角帆蚌Rab3基因的全长cDNA序列(GenBank登录号为HQ190954)。序列全长1707bp,开放阅读框666bp,编码221个氨基酸,5’端非编码区为158bp,3’端非编码区为883bp。NCBI Blastp进行氨基酸序列相似性分析表明,与居蟹皮海绵(Suberites domuncula)的Rab-3like(SdRab3)氨基酸相似性最高,为64%。以氨基酸序列构建进化树,首先与居蟹皮海绵的Rab3-like(SdRab3)聚为一支。原核表达结果表明,在30℃,5h,1mmol/LIPTG的诱导条件下,Rab3蛋白得到高效表达,重组菌体经裂解和SDS-PAGE电泳分析,发现一条26kD左右特异的蛋白表达条带,其表达量约占菌体总蛋白30%。用嗜水气单孢菌感染三角帆蚌后,Western-blot检测表明,在健康组和病变三角帆蚌肝脏中,Rab3蛋白与β-actin的表达量比值分别为0.438和0.580,说明Rab3基因在三角帆蚌经病原菌侵染后,表达量上调。初步推测三角帆蚌Rab3蛋白可能与三角帆蚌瘟病有关。According to the Hyriopsis cumingii （H.cumingii） expressed sequence tags （EST） obtained by constructing subtractive hybridization cDNA library,the gene full-length cDNA sequence of Rab3 from H.cumingii was cloned by RACE-PCR technique GenBank accession.Induced with 30℃,5 h,1 mmol/L IPTG,the HcRab3 was expressed at high levels,electrophoretic analysis by SDS-PAGE revealed that 26 kD protein expression band was obtained.After Western-blot in healthy and deseased Hyriopsis cumingii lever protein,the expression ratio of Rab3 protein and beta actin was 0.438 and 0.580.It was preliminarily presumed that the HcRab3 maybe related to Hyriopsis cumingii plague.

关 键 词：三角帆蚌 Rab3 原核表达 WESTERN-BLOT 

分 类 号：S917.4[农业科学—水产科学]