中国对虾蜕皮抑制激素全长cDNA的克隆及序列分析

Molecular Cloning and Sequence Analysis of Full Length cDNA Encoding Molt-inhibiting Hormone from Fennropenaeus chinensis

对虾的蜕皮活动由蜕皮抑制激素和蜕皮激素调控 ,蜕皮抑制激素是甲壳动物CHH家族神经肽的成员之一 ,通过抑制Y器官蜕皮激素的合成而调节蜕皮。以中国对虾 (Fennropenaeuschinensis)眼柄总RNA为材料 ,采用cDNA末端快速扩增 (RACE)方法 ,首次得到蜕皮抑制激素的全长cDNA (GenBank登录号 :AF4 6 9187)。该全长cDNA大小为 6 97bp ,是由 32 0bp的 3′RACE产物和 4 6 8bp的 5′RACE产物拼接而成。Blast搜索结果显示 ,该全长cDNA与甲壳动物的MIH基因序列具有较高的相似性 ;用ClustalX进行多序列比较结果表明 ,由该全长cDNA推导的氨基酸序列与对虾类的MIH的氨基酸序列同源性最高 ,其中与日本对虾、斑节对虾、刀额新对虾MIH的同源性分别为95 1%、83 1%、79 1%。根据以上数据 ,推断该 6 97bp的全长cDNA为编码中国对虾MIH前体的cDNA。进一步序列分析表明 ,编码中国对虾MIH前体cDNA包括 312bp的开放阅读框、81bp的 3′UTR和 30 2bp的 5′UTR ;编码 10 3个氨基酸的MIH前体分子包括信号肽和成熟肽 ,信号肽由 2 8个氨基酸组成 ,成熟肽由 75个氨基酸组成 ,成熟肽中6个半胱氨酸非常保守。

Molt inhibiting hormone (MIH) is a neuropeptide member belonging to the eyestalk CHH family.Molting in shrimp is controlled by MIH and ecdysone.By inhibiting the synthesis of ecdysone in the Y organ,MIH indirectly suppress the molting activity of shrimp.A 697 bp full length encoding molt inhibiting hormone precursor cDNA,which has been accepted by GenBank (accession number:AF469187 ),was firstly amplified from the total RNA of eyestalk from Fennropenaeus chinensis by the 3′ and 5′ rapid amplification of cDNA ends (RACE) method.The 697 bp full length cDNA encoding MIH precursor was assembled with a 320 bp 3′ RACE product and 468 bp 5′ RACE product.Results derived from searching by Blast revealed the 697 bp cDNA had high similarity with MIH gene of crustacean.By using Clustal X program,alignment of the amino acid sequence deduced from the 697 bp cDNA with amino acid sequences of 7 MIHs revealed that the deduced amino acid sequence had very high identity with amino acid sequences of MIHs of shrimps.The identities between the deduced amino acid sequence with that of MIH of Marsupenaeus japonicus,Penaeus monodon and Metapenaeus ensis were respectively 95 1%,83 1% and 79 1%.On the base of all the data,we concluded that the 697 bp full length cDNA was the cDNA encoding MIH precursor of F.chinensis. Sequence analysis of the 697 bp cDNA revealed a 312 bp open reading frame,and 81 bp 5′ untranslated region,and a 302 bp 3′ untranslated region.The deduced 103 amino acid polypeptide consisted of a 28 amino acid region of signal peptide and a 75 amino acid region of mature peptide.The six cysteine residues were very conserved in the mature peptide.