青虾咽侧体抑制激素基因全长cDNA序列的克隆及表达分析

咽侧体抑制激素(Allatostatin, AST)是一类由几至几十个氨基酸构成的神经肽类激素, 在甲壳动物中刺激下颌器官合成甲基法尼酯, 影响甲壳动物的蜕皮和生殖。然而AST基因在甲壳动物中的克隆和表达却罕见报道。研究克隆了青虾的AST基因全长cDNA序列, 在甲壳动物中使用荧光定量PCR技术检测了AST基因在不同组织中的表达。青虾AST基因cDNA全长2995 bp, 包括242 bp的5′非编码区(UTR), 647 bp的3′UTR, 2106 bp的开放阅读框(ORF)。开放阅读框编码701个氨基酸, 可转录翻译出35个AST多肽, 在C末端都具有相同的Y/FXFGL-amide结构, 属于A型-AST。氨基酸序列比对显示保守氨基酸为Tyr、Ala、Phe、Gly、Leu。蛋白相似度比对显示, AST多肽在无脊椎动物的进化中是相对保守的。系统进化树分析表明, 青虾AST多肽与罗氏沼虾聚在一起, 具有最近的亲缘关系。荧光定量PCR检测显示, AST基因在所有被检测组织中均有表达, 由高到低依次为: 肝胰脏>肠道>精巢>脑>心脏>卵巢。对青虾AST基因全长cDNA序列克隆和表达的研究为更进一步的了解AST多肽在青虾中的重要功能奠定了基础。

CLONING, CHARACTERIZATION AND EXPRESSION OF FULL LENGTH cDNA ENCODING ALLATOSTATIN (AST) IN MACROBRACHIUM NIPPONENSE

Allatostatin (AST) is a type of neuropeptide, it is made up of a few to dozens of amino acid. In crustaceans, AST has a significant stimulatory effect on MF synthesis, which involved in moulting and reproduction, so it is suggesting that AST in crustaceans has a positive effect on moulting and reproduction. However, AST gene cloning and expression of crustaceans is rarely reported. To investigate gene cloning and expression of AST cDNA in Macrobrachium nipponense, we cloned AST gene in M. nipponense and used quantitative real-time PCR (qRT-PCR) to detecting the expression of AST gene in various tissues. The cloned AST cDNA was 2995 bp in length containing a 242 bp 5′ untranslated region (UTR), a 647 bp 3′ UTR and a 2106 bp open reading flame (ORF). The ORF encoded a AST precursor polypeptide 701 amino acid residues, which can be hydrolyzed into 35 allatostatins at dibasic cleavage sites. These 35 allatostatins had a C-terminal Y/FXFGL-amide in common, which is the characteristics of A type-AST. On the basis the comparison of the amino acid sequences, five amino acids including Tyr, Ala, Phe, Gly and Leu were considered as conserved amino acids during evolution. According to the phylogenetic tree, the insects and crustaceans were divided into two branches through the comparison between different insect and crustaceans. M. nipponense had a closest relationship with M. rosenbergii. The result of qRT-PCR revealed that AST gene was expressed in all the tested tissues. The tissue' expression levels of AST were in the decreasing order hepatopancreas, intestines, testis, brain, heart, and ovary. Our research on AST gene clone and expression contributes to further researching of the function of AST in M. nipponense.