家蚕Rab1基因的克隆及在裸蛹(Nd)突变品系幼虫丝腺中的表达分析  

Cloning of Bombyx mori Rab1 Gene and Expressional Analysis in Silk Gland of Naked Pupa (Nd) Mutant Strain Larvae

作　　者：胡文波[1] 刘春[1] 魏丽婉[1] 赵小明[1] 陈志伟[1] 李琼艳[1] 夏庆友[1] 

机构地区：[1]家蚕基因组生物学国家重点实验室,西南大学,重庆400716

出　　处：《蚕业科学》2013年第2期266-274,共9页ACTA SERICOLOGICA SINICA

基　　金：国家重点基础研究发展计划"973"项目(No.2012CB-114600);教育部"新世纪优秀人才支持计划"(No.NCET-11-0699);国家自然科学基金青年基金项目(No.31000545)

摘　　要：Rab1基因是大鼠肉瘤鸟苷三磷酸酶(Ras)基因家族的一员,调控囊泡从内质网到高尔基体的运输等重要细胞生理过程。通过生物信息学分析发现,家蚕Rab1(BmRab1)基因CDS长609 bp,含有5个外显子,编码202个氨基酸,位于第25染色体nscaf2823:1286217..1289996(+strand),与丝素重链基因(fib-H)相距200 kb左右。对正常结茧家蚕品系大造和无丝素分泌的裸蛹(Nd)突变品系的BmRab1的序列结构分析显示:大造和Nd品系的BmRab1 cDNA序列第543个碱基处存在单碱基突变,但翻译的氨基酸序列完全相同;在Nd品系中BmRab1的3'端非翻译区缺失8 bp。组织表达分析表明,BmRab1基因在Nd品系4眠和整个5龄期幼虫的后部丝腺异常高量表达,荧光定量PCR显示BmRab1在Nd品系5龄第3天幼虫后部丝腺的表达量是大造的3倍左右。进一步对Nd品系与大造的BmRab1上游启动子序列(-2 200 bp)及其内含子序列进行比较分析发现,Nd品系BmRab1启动子区域出现了2处缺失,并在第3内含子有81 bp插入,第4内含子453 bp替换了大造的263 bp。初步推测BmRab1可能参与了Nd品系幼虫后部丝腺退化萎缩的生理变化,但该基因是否是导致Nd品系后部丝腺退化萎缩的突变基因,还有待进一步证实。Rab1 is a member of rat sarcoma guanosine triphosphatase(Ras) gene family which plays an essential role in regulating important cellular processes such as vesicle transportation from endoplasmic reticulum to Golgi.Bioinformatic analysis showed that Bombyx mori Rab1 gene(BmRab1) has five exons and locates on nscaf2823:1286217..1289996(+strand) of chromosome 25,having a distance of about 200 kb with the fibroin heavy-chain gene(fib-H).Sequence structure analysis of BmRab1 in normal silkworm strain Dazao and naked pupa(Nd) mutant strain without fibroin secretion revealed that both of them have a single base mutation at the 543th base of cDNA sequence but encode the same amino acid sequence.An 8 bp deletion occurred to 3′ untranslated region of BmRab1 in Nd strain.Tissue expression analysis showed that BmRab1 gene was highly expressed in posterior silk gland of the 4th molting larvae and all larvae of the 5th instar of Nd strain.Fluorescent quantitative PCR indicated that the expression level of BmRab1 in posterior silk gland of day 3 Nd larvae of the 5th instar was about 3 times as much as of that of the normal strain Dazao.Comparison on upstream sequence(-2 200 bp) of promoter and introns of BmRab1 in Nd strain and Dazao strain indicated that there are two deletions in the promoter region and one insertion of 81 bp in the third intron.A segment of 453 bp in the fourth intron of Nd strain has replaced the 263 bp sequence in the fourth intron of Dazao strain.It was preliminarily inferred that BmRab1 participates in the atrophy of larval posterior silk gland in Nd mutant strain.Yet,whether this gene is the mutation gene that leads to the atrophy of larval posterior silk gland in Nd mutant strain needs further verifications.

关 键 词：家蚕 裸蛹 Rab1基因 丝腺 序列特征 组织表达 

分 类 号：S881.2[农业科学—特种经济动物饲养] Q781[农业科学—畜牧兽医]