青虾高血糖激素基因全长cDNA序列的克隆及表达分析

Molecular cloning and expression of a full length cDNA encoding crustacean hyperglycemic hormone(CHH) in oriental river pawn(Macrobrachium nipponense)

本研究首次克隆了青虾(Macrobrachium nipponense)高血糖激素(crustacean hyperglycemic homone,CHH)基因全长cDNA序列,并使用荧光定量PCR技术首次检测了CHH基因在青虾不同组织中的表达。青虾CHH基因cDNA全长1 017 bp,包括241 bp的5′UTR,408 bp的开放阅读框(ORF),355 bp的3′UTR,开放阅读框编码135个氨基酸。成熟肽包含6个CHH家族中位置保守的Cys残基。青虾CHH成熟肽C端为GK,确定为ES型CHH基因。蛋白相似度比对显示,所分离的青虾CHH被归为CHH家族I型肽。系统进化树分析表明,青虾CHH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,CHH基因在青虾不同组织中均有表达,其表达量由高到依次为眼柄、精巢、腹神经节、脑、肠、肝、心脏、卵巢。以卵巢为参照其表达量设为1时,眼柄与精巢CHH基因表达量分别为卵巢的500倍和250倍,由此推测CHH基因可能与雄性生殖过程相关。

Crustacean hyperglycemic hormone（CHH） is a neuropeptide hormone that is secreted by a subset of cells in the X-organ of the eyestalk.CHH plays a role in the regulation of ions and energy metabolism,molting, reproduction,and other important physiological processes in crustaceans.The CHH gene has been cloned in a variety of crustacean species,However,little is known about the pattern of expression in different tissues.We cloned the full-length CHH cDNA sequence of the oriental river prawn,Macrobrachium nipponense and used quantitative real-time PCR to detect the expression of CHH in different tissues.We cloned a 1017 bp cDNA of the CHH gene, containing a 241 bp 5′-untranslated region（UTR）,a 355 bp 3′UTR,and 408 bp open reading flame（ORF）.The ORF encoded a 135 amino acid CHH precursor polypeptide.The mature peptide contained six conserved Cys residues in the CHH family.The C-terminus mature peptide was GK,which has the same characteristics as ES-CHH.Based on protein similarity comparison,the separated CHH gene was classified into the type I CHH family of peptides.Phylogenetic tree analysis revealed that oriental river prawn CHH was most closely related to that of Macrobrachium rosenbergii.The CHH gene was expressed in all tissues tested in oriental river prawn.Expression was significantly higher in the eyestalk and testis than in the ovary（500 and 250 higher,respectively）. CHH gene expression was very high in the intestine and ventral nerve cord,but very low in the ovary,liver,brain, and heart.The pattern of expression suggests that the CHH peptide plays a role in male reproduction.Our results provide insight into the role of CHH peptide in the oriental river prawn,and further our understanding of the CHH gene in crustacean species.