具有还原型烟酰胺腺嘌呤二核苷酸脱氢酶功能的家蚕l(3)neo18基因的克隆与表达特征  

Molecular Cloning and Expression Profile of Bm-l(3) neo18 Gene With NADH Dehydrogenase Activity in the Silkworm,Bombyx mori

作　　者：王更先[1] 鲁延军[1] 司马杨虎[1] 张升祥[1,2] 徐世清[1] 

机构地区：[1]现代丝绸国家工程实验室,苏州大学基础医学与生物科学学院,江苏苏州215123 [2]山东农业大学林学院,山东泰安271018

出　　处：《蚕业科学》2009年第1期58-65,共8页ACTA SERICOLOGICA SINICA

基　　金：国家重点基础研究发展计划“973”项目(编号2005CB12-1005)

摘　　要：果蝇l(3)neo18基因可以通过P转座子诱发致死突变,编码的蛋白质具有还原型烟酰胺腺嘌呤二核苷酸(NADH)脱氢酶(ND)的功能。利用生物信息学、cDNA末端快速扩增(RACE)和反转录聚合酶链式反应(RT-PCR)等方法,克隆了家蚕l(3)neo18同源基因,分析了基因结构与表达谱。Bm-l(3)neo18基因(EU826676)的cDNA全长为868 bp,由573 bp的完整开放读码框(ORF)序列、25 bp的5′端非翻译区序列(5′-UTR)和251 bp的3′端非编码区序列(3′-UTR)组成,编码蛋白质为190个氨基酸残基,分子质量22.7 kD,pI 9.60。Bm-l(3)neo18基因由3个外显子和2个内含子组成,定位于家蚕第3染色体,位于nscaf2930的1 203.8-1 205.6 knt。Bm-l(3)neo18蛋白质在1-185氨基酸残基位置为ND的SGDH亚基保守区,在61-83氨基酸残基位置具有一个保守的跨膜区域。采用Clustal W进行多序列比对发现,Bm-l(3)neo18与埃及伊蚊等昆虫ND具有50%以上的蛋白质同源性,ND的保守区域高度一致,NJ法分子进化分析也显示Bm-l(3)neo18与昆虫ND进化上同源。该基因除在家蚕卵期的表达量较低外,在幼虫、蛹和蛾期均有较高表达,且存在组织差异性。1（3）neo18 gene can cause lethal mutation through P element transposition. Its protein has NADH dehydrogenase （ND） activity. By using the methods of bioinformatics, rapid amplication of cDNA ends （RACE） and reverse transcription-polymerase chain reaction （RT-PCR）, a full-length cDNA was obtained from silkworm and was named Bm-1（3）neo18 （GenBank accession No. EU826676）. Sequence analysis revealed that the cDNA of Bm-1（3）neo18 is 868 bp long with 573 bp open reading frame （ORF）, 25 bp 5＇-UTR （untranslated region） and 251 bp 3＇-UTR. The ORF encodes 190 amino acids with predicted molecular mass of 22.7 kD and isoelectric point of 9.60. Bm-1（3） neo18 contains 3 exons and 2 introns and maps within 1 203.8 knt to 1 205.6 knt region of nscaf 2 930 on chromosome 3. The deduced amino acid sequence has an SGDH domain of ND from amino acids 1 to 185 and a transmembrane region from amino acids 61 to 83. Multiple sequence alignment using ClustalW showed that it has over 50% identity with other insect such as Aedes aegypti. The ND conservative regions were very similar with each other. Phylogenetic analysis by Neighbor Joining method indicated that Bm-1（3） neo18 was homologous with ND of other insects. Expression of Bm-1（3）neo18 was down-regulated at egg stage, and up-regulated at larval, pupal and moth stages. Besides, a tissue-specific differential expression was also observed.

关 键 词：家蚕 l(3)neo18基因 还原型烟酰胺腺嘌呤二核苷酸脱氢酶 基因表达 

分 类 号：S881.2[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]