α-tubulin基因的克隆、生物信息学分析及其在仿刺参肠道再生过程中的表达模式  

Molecular cloning, bioinformatics analysis and expression pattern detection ofα-tubulin gene during intestinal regeneration in the sea cucumber(Apostichopus japonicus)

作　　者：王怡[1] 高银雪 湛垚垚[1] 张向向[1] 杨立猛[1] 常亚青[1] 

机构地区：[1]大连海洋大学农业部北方海水增养殖重点实验室,辽宁大连116023

出　　处：《水产学报》2016年第4期547-557,共11页Journal of Fisheries of China

基　　金：国家"八六三"高技术研究发展计划(2012AA10A412);辽宁省农业攻关及成果产业化项目(2015203003)~~

摘　　要：为了明确仿刺参α-微管蛋白(α-tubulin)基因的序列及结构信息,初步研究该基因在仿刺参肠道再生过程中的生物学功能,本研究利用转录组数据挖掘和c DNA末端快速扩增技术(RACE)首次克隆得到仿刺参α-tubulin基因的全长c DNA序列。结果表明,仿刺参α-tubulin基因c DNA全长为1641 bp,共编码453个氨基酸,经生物信息学分析发现,该基因的5'端非编码区为153 bp,3'端非编码区为126 bp,推算该基因所编码的蛋白质分子量为50.33 ku,等电点为4.89,属于亲水性非跨膜蛋白质,且氨基酸序列中含有微管蛋白特有的信号序列GGGTGSG。通过与13种已公布物种的α-tubulin氨基酸序列进行多重序列比对及系统进化分析,发现仿刺参α-tubulin蛋白的氨基酸序列与其他真核生物的α-tubulin蛋白序列具有非常高的保守性,与南极岩斑鳕α-tubulin相似性高达90%。采用实时定量PCR技术对α-tubulin基因在仿刺参肠组织再生不同时期的表达情况进行检测,结果显示α-tubulin基因在仿刺参肠组织再生不同时期均有表达,其相对表达量在肠组织再生17 d时最高,5 d时最低。本研究在获得仿刺参α-tubulin基因结构信息的同时,进一步印证了真核生物α-tubulin基因的高度保守性,同时表明α-tubulin基因参与仿刺参肠道再生过程。In this study, full-length cDNA of a-tubulin in sea cucumber Apostichopusjaponicus was cloned for the first time by transcriptome data mining and rapid amplification of cDNA ends （RACE）, and qRT-PCR was used to preliminarily reveal the biology function of a-tubulin in the process of intestine regeneration. The results showed that full-length cDNA of a-tubulin was 1641 bp, containing a 1362 bp open reading frame encoding a putative polypeptide of 453 amino acids residues. Bioinformatics analysis showed that the sequence of a-tubulin contains a 5＇-untranslated region （UTR） of 153 bp, and a 3＇-UTR of 126 bp. The predicted molecular mass of the deduced amino acid ofa-tubulin was 50.33 ku, and the theoretical isoelectric point was 4.89. The amino acids inferred have a special sequence （GGGTGSG） which was a typical element in α-tubulin protein, belonging non-transmembrane and hydrophilic protein. Multiple sequence alignment analysis and phylogenetic analysis revealed that the α- tubulin in sea cucumber was very conservative with other eukaryotic organism and had 90% sequence identity with Notothenia coriiceps. Real-time quantitative PCR was carried out to measure the expression level ofa-tubulin gene in different stages of sea cucumber intestine regeneration. The expression of α-tubulin was detected at all stages of intestine regeneration. The maximum and the minimum expression levels were found on 17th and 5th day, respectively. This study first obtained the structure information of a-tubulin gene in sea cucumber, and further certified the high homology of α-tubulin in eukaryote. In addition, our results indicated that α-tubulin plays an important role during intestinal regeneration in the sea cucumber A. japonicus.

关 键 词：仿刺参 Α-微管蛋白 克隆 生物信息学分析 基因表达 

分 类 号：Q785[生物学—分子生物学] S917.1[农业科学—水产科学]