论文部分内容阅读
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。
In order to investigate the role of this enzyme in the regulation of ovarian development in crustacea, acetyl-Co A C-acetyltransferase (AACT) is the initial enzyme for the mevalonate pathway. RT-PCR and c DNA end-rapid (GenBank accession number: KM033231) was cloned by using RACE technique. This sequence is 1 630 bp in length and consists of a 101 bp 5 ’untranslated region, a 395 bp 3’ noncoding region and a 1 134 bp open reading frame encoding 377 amino acids. Bioinformatics prediction of its amino acid sequence showed that Pt-AACT belongs to the hydrophobic protein and has no transmembrane domain. There are two conserved regions of thiolase specificity. Compared with AACT amino acid sequences of other published species, it showed the highest homology (72%) with Aedes aegypti and Aedes aegypti. The results of phylogenetic tree showed that Pt-AACT and insect AACT clustered together. Using real-time quantitative PCR (qRTPCR) to analyze the difference of tissue expression and the expression of AACT in Portunus tritubus, the results showed that AACT was large The highest expression in the jaw, the lower expression in the rest of the organization, the difference was significant; the highest expression in ovarian stage Ⅰ, there are very significant differences with other periods. Indicating that AACT may play a role in regulating ovary development of Portunus trituberculatus.