日本对虾(Penaeus japonicus)性腺差异表达基因GD13之cDNA5’端克隆

Cloning of cDNA 5’End of Gonad Differential Expression Gene GD13 in Penaeus japonicus

采用基于SMART(SwitchingMechanismAt5'endofRNATranscript)原理的cDNA末端快速扩增技术(RapidAmpli ficationofcDNAends:RACE),对日本对虾性腺差异表达基因cDNA5'端进行克隆,即在逆转录过程中通过SMARTⅡoligo的介导,在cDNA第1条链的3'端接上接头,然后根据已知序列设计的基因特异性引物(GSP1和GSP2)和接头引物(UPM和NUP)进行Nested PCR特异性扩增,并对特异产物进行克隆和序列分析,最终获得全长cDNA序列.测序结果表明,在卵巢内表达量高于精巢的GD13基因全长为656bp,编码164氨基酸,经NCBI检索,此基因与文昌鱼、家鼠的核糖体蛋白L24及黄牛的核糖体蛋白L30氨基酸序列同源性分别高达58%、56%和56%,可断定该基因为编码日本对虾核糖体蛋白L24亚基的基因.为进一步认识对虾生长发育和繁殖的调控机制奠定基础.

In this paper,the method of SMART Rapid Amplification of cDNA ends (RACE) was employed to clone the 5’ end of cDNA of GD13,which is a gene differentially expressed between ovary and testis of Penaeus japonicus.The SMARTII Oligo nucleotide was used as a template to stretch the cDNA 5’end when the first strand cDNA was synthesized.Then a Nested-PCR was carried out to specially amplify the interested cDNA with the adaptor primers (UPM and NUP) and the gene special primers (13GSP1 and 13GSP2).As a result,we got a 656 bp full length cDNA of GD13 which encodes 164 amino acid.Homologous comparison of amino acid sequence demonstrated that this gene shared significant homology with the ribosome protein L24 gene of Branchiostoma belcheri,Mus musculus and L30 gene of Bos Taurus.Further work investigating why this ribosome protein gene differentially expressed in testis and ovary of shrimp is going on.