雌核发育银鲫四种孵化酶基因全长cDNA的克隆及其特征分析

Cloning and Characterization of Four Full Length cDNAs of Hatching Enzyme Genes in Gynogenetic Gibel Carp

用抑制性差减杂交结合SMARTcDNA合成和RACE PCR技术 ,克隆得到雌核发育银鲫 (Carassiusauratusgibelio) 4种孵化酶基因的全长cDNA。 4种孵化酶基因分别被命名为GHE1、GHE2、GHE3、GHE4。序列分析表明 ,4种孵化酶基因cDNA的开放阅读框均为 795bp ,都编码 2 6 5个氨基酸。它们推断的编码氨基酸序列同源性在 79 6～95 1 %之间。种系分析表明 ,GHE1、GHE2、GHE3、GHE4的进化地位位于日本鳗鲡(Anguillajaponica)孵化酶和青 (Oryziaslatipes)孵化酶之间。不同发育时期胚胎的RT PCR分析表明 ,银鲫GHE1、GHE2、GHE3、GHE4 4种孵化酶基因从尾芽期到幼苗期都检测到表达 ,且GHE3和GHE4在神经胚期也检测到表达。各种组织的RT PCR表明 。

Four full length cDNAs of hatching enzyme genes were cloned from gynogenetic gibel carp ( Carassius auratus gibelio) by suppression subtractive hybridization, SMART cDNA synthesis and RACE PCR. They were designated as gibel carp hatching enzymes GHE1?GHE2? GHE3 and GHE4 Sequence analysis indicated that they all have an open reading frame of 795 nucleotide acids for encoding 265 amino acids. The identity of their deduced amino acid sequences was 79 6%～95 1%. Phylogenetic analysis indicated that GHE1?GHE2? GHE3 and GHE4 belong to the subgroup of hatching enzymes within the Astacin family. The evolutionary position of GHE1?GHE2? GHE3 and GHE4 was between the hatching enzymes of Japanese eel ( Anguilla japonica ) and medaka (Oryzias latipes) . RT PCR analysis showed that all of them were detected from tail bud stage to larvae during embryonic development. GHE3 and GHE4 were also detected in neurula stage. And no mRNAs of the four genes were detected in adult fish.