三疣梭子蟹AACT基因克隆及其在卵巢发育中的表达分析  

Cloning and expression analysis of acetyl-CoA C-acetyltransferase( AACT) in Portunus trituberculatus

作　　者：王凤皎 朱冬发[1] 邱锡尔[1] 谭义川 周彦琦 柳志业 谢熙[1] 

机构地区：[1]宁波大学海洋学院,浙江宁波315211

出　　处：《水产学报》2015年第6期790-798,共9页Journal of Fisheries of China

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

摘　　要：乙酰Co A酰基转移酶(acetyl-Co A C-acetyltransferase,AACT)是甲羟戊酸途径的初始酶,为了研究该酶在甲壳动物卵巢发育调控中的作用,用RT-PCR和c DNA末端快速扩增(RACE)技术,克隆得到三疣梭子蟹AACT(Pt-AACT)的c DNA全长(Gen Bank登录号:KM033231)。这段序列全长1 630 bp,包括1个101 bp的5'端非编码区,1个395 bp的3'端非编码区和1个1 134 bp的开放阅读框,编码377个氨基酸。对其氨基酸序列进行生物信息学预测,发现Pt-AACT属于疏水性蛋白,无跨膜结构域,存在2个硫解酶特异性保守区。与其他已公布物种的AACT氨基酸序列进行比对,发现与金小蜂、埃及伊蚊等同源性最高(均为72%)。系统进化树结果显示,Pt-AACT与昆虫AACT聚为一支,用实时荧光定量PCR(qRTPCR)技术,分析三疣梭子蟹AACT的组织表达差异及在卵巢发育中的表达变化,结果表明AACT在大颚器中表达量最高,在其余组织中表达量较低,差异显著;在卵巢发育Ⅰ期表达量最高,与其他期存在极显著差异。表明AACT可能在三疣梭子蟹卵巢发育中具有一定调控作用。Acetyl-CoA C-acetyltransferase （AACT）is the starting enzymes in mevalonate pathway of terpenoids biosynthesis. To reveal the function of this enzyme during the ovarian development of the crustaceans, we used reversed transcript PCR （RT-PCR） and rapid amplification of cDNA ends （RACE） techniques to clone the AACT（ GeneBank accession number： KM033231 ）. The full length of Pt-AACT is 1 630 bp,including a 5＇-untranslated region（UTR）of 101 bp, a 3＇-UTR of 395 bp and an opening reading frame（ORF） of 1 134 bp encoding 377 amino acid protein. The structures of AACT proteins have two special conserved sequences of thiolase,belonging to stable hydrophobic proteins without an obvious transmembrane region. The deduced amino acid sequence of Pt-AACT exhibited the highest identity （72%）with AACT of Nasonia vitripennis and Aedes aegypti. The phylogenetic tree analysis showed that Pt-AACT was clustered in insect AACTs. Tissue distribution by quantitative real-time PCR（qRT-PCR） illustrated that the Pt-AACT was most highly expressed in the MOs. During the ovarian development, expression of Pt-AACT in MOs significantly remained the highest level at stage I . Therefore, AACT plays an important role in ovarian development regulation of P. Trituberculatus.

关 键 词：三疣梭子蟹 乙酰CoA酰基转移酶 基因克隆 卵巢发育 表达水平 

分 类 号：Q785[生物学—分子生物学] S966.1[农业科学—水产养殖]