凡纳滨对虾繁育相关基因Allatostatin-AR的克隆与表达分析

Cloning and expression of breeding-&development-related gene Allatostatin-A receptor from Litopenaeus vannamei

咽侧体抑制素主要抑制昆虫咽侧体保幼激素的分泌,是昆虫体内重要的神经肽,在甲壳动物体内可能参与繁殖发育的调节。本研究利用RACE PCR技术对凡纳滨对虾(Litopenaeus vannamei)A型咽侧体抑制素受体(Allatostatin-A receptor,AST-AR)基因进行克隆,获得全长为2650 bp的cDNA,包括1425 bp的开放阅读框,1170 bp 5′非编码区,55 bp 3′非编码区,编码474个氨基酸,形成具有7个跨膜区的不稳定亲水性蛋白。同源性和系统发育分析结果表明凡纳滨对虾AST-AR基因与斑节对虾(Penaeus monodon)、美洲龙虾(Homarus americanus)同源性最高,亲缘关系最近。氨基酸多重序列比对结果显示AST-AR氨基酸TM7后C末端精氨酸在不同物种之间高度保守。实时荧光定量PCR结果显示,AST-AR基因在雌、雄凡纳滨对虾多个组织(眼柄、脑、胃、肝胰腺、鳃、性腺、肠、肌肉)中均有表达,除脑组织外AST-AR基因在雌性凡纳滨对虾中相对表达量高于雄性。在幼体发育中,AST-AR基因的相对表达量随发育阶段上升,同时该基因在不同群体仔虾生长对比实验中表达量差异显著。据此推测AST-AR基因参与凡纳滨对虾的繁殖、幼体发育、幼虾生长到成虾,基因表达基本贯穿凡纳滨对虾的一生。

Allatostatin-A receptor gene was cloned from Litopenaeus vannamei by RACE PCR,and the sequence length is 2650 bp,including 1425 bp of ORF,1170 bp of 5′-UTR,55 bp of 3′-UTR,encoding 474 amino acids,forming an unstable hydrophilic protein with seven transmembrane regions,and subcellular localization is mainly located on the cytoplasmic membrane.The results of homology and phylogenetic analysis showed that the Allatostatin-A receptor gene of Litopenaeus vannamei has the highest homology with Penaeus monodon and Homarus americanus,and has the closest genetic relationship.The result of multiple amino acid sequence alignment showed that the C-terminal Arg after Allatostatin-A receptor amino acid TM7 is highly conserved among different species.Real time PCR results showed that the Allatostatin-A receptor gene was expressed in multiple tissues(eyestalk,brain,stomach,hepatopancreas,gill,gonad,intestine,muscle)of female and male Litopenaeus vannamei.Except for brain tissue,the relative expression of Allatostatin-A receptor gene in female Litopenaeus vannamei is higher than that in males.With the development of Litopenaeus vannamei larvae,the relative expression of Allatostatin-A receptor gene increased.The expression level of Allatostatin-A receptor gene was the highest in the hybrid offspring of imported male shrimp and Ecuadorian wild female shrimp.It is speculated that the Allatostatin-A receptor gene may be involved in the reproduction process of Litopenaeus vannamei,and whether it is involved in the growth regulation of Litopenaeus vannamei remains to be further studied.