三疣梭子蟹腺苷酸转移酶(ANT)基因的克隆与分析  

Cloning and Analysis of Gene for Adenine Nucleotide Translocase in Portunus trituberculatus

作　　者：李冰玥 朱冬发[1] 邱锡尔[1] 周彦琦 柳志业 谢熙[1] 

机构地区：[1]宁波大学海洋学院,宁波315211

出　　处：《生物技术通报》2016年第5期124-130,共7页Biotechnology Bulletin

基　　金：国家自然科学基金项目(41376152);浙江省自然科学基金项目(LY13C190006)

摘　　要：腺苷酸转移酶(Adenine nucleotide translocase,ANT)是线粒体内膜上负责能量分子传导的转运蛋白,在能量代谢中起着关键作用。为了研究ANT基因在甲壳动物蜕皮中的作用,采用RT-PCR和c DNA末端快速扩增技术(RACE技术)克隆得到三疣梭子蟹ANT基因的序列全长(Gen Bank登录号:KM921660),该序列全长1 414 bp,包括132 bp的5'端非编码区,352 bp的3'段非编码区,具有930 bp的完整开放阅读框(ORF),编码309个氨基酸。将该ANT基因序列推导的氨基酸序列与已公布的其他物种ANT序列进行系统进化树分析发现,三疣梭子蟹ANT基因与其他甲壳动物ANT基因聚为一支,其中与拟穴青蟹ANT一致性高达96%。通过氨基酸序列比对发现,三疣梭子蟹ANT序列具有3个保守的线粒体穿膜功能结构域,是形成能量分子传导的转运通道,催化细胞质中ADP和线粒体内ATP间进行跨膜交换。采用实时荧光定量PCR技术,分析三疣梭子蟹ANT基因的组织差异表达,结果表明ANT基因在三疣梭子蟹肌肉(Ms)中的表达量最高,在其他组织中表达量均较低,具显著差异(P<0.05);在三疣梭子蟹蜕皮周期中,肌肉中ANT基因的表达量A期最高(P<0.05),然后下降,至C期最低,随后又逐渐上升至D1期,在D1期出现第2个峰值后再逐渐下降。研究结果说明ANT基因与三疣梭子蟹肌肉活动密切相关,可能在蜕皮调控中发挥重要作用。Adenine nucleotide translocase（ANT）is a transport protein responsible for the conduction of energy molecules in the inner membrane of mitochondria,playing a vital role in the energy metabolism. In order to study the role of ANT gene in the molting of crustacean,a full-length c DNA of ANT gene（Gen Bank access number ：KM921660）was cloned from Portunus trituberculatus using RT-PCR and RACE. Sequence analysis showed that it was 1 414 bp long,including a 132 bp 5＇ non-coding region,a 352 bp 3＇ non-coding region,and a 930 bp coding region,and encoded 309 amino acids. The phylogenetic comparison of ANT gene sequence between P. trituberculatus and other species revealed that ANT gene from P. trituberculatus and other species clustered as one same branch,and the similarity with Scylla paramamosainwas up to 96%. Amino acid alignment proved that it had 3 conserved mitochondrial transmembrane domains,forming a channel for the conduction of energy molecules,and catalyzing transmembrane exchange between ATP in mitochondria and ADP in cytoplasm. Real-time PCR results indicated that ANT gene expressed in all 10 different tissues with the highest expression in muscle,while quite low in all other tissues,and there were significant differences among the expressions of the tissues（P0.05）. During the whole molting process,the level of ANT in muscle reached the maximum at postmolt（stage A）,then began to decline until the least during intermolt（stage C）,and then gradually rose until the second peak at stage D1,then declined again. The combined result indicated that ANT gene was closely correlated with the activity of muscle of P. trituberculatus,and might play an essential role in the molting.

关 键 词：三疣梭子蟹 腺苷酸转移酶 克隆 能量转运 表达水平 

分 类 号：S917.4[农业科学—水产科学] Q78[生物学—分子生物学]