黄鳝脑芳香化酶基因cDNA的克隆及组织表达特异性分析

Molecular Cloning and Tissue-specific Expression Analysis of Brain P450arom in Ricefield Eel(Monopterus albus)

根据NCBI数据库报道的黄鳝芳香化酶基因的gDNA序列,设计了一对特异性引物。用Tr-izol试剂盒提取黄鳝脑总RNA,反转录获得cDNA第一条链,进行PCR扩增。扩增产物经过回收、连接、测序后得到了一条长1514 bp的芳香化酶基因cDNA序列。同源性比较表明该基因属于脑型P450aromB,与其他鱼类脑型P450aromB的同源性较高(>70%),与性腺型P450aromA的同源性较低(<60%),与自身性腺型芳香化酶同源性为59.5%。采用RT-PCR的方法对该基因在雄性黄鳝的各组织表达情况进行了分析,结果表明,该基因的转录本较高的表达于脑和精巢中,较低的在皮肤中表达,而在肝脏、心脏、小肠、肌肉等组织中没有检测到该基因的表达。

Based on one P450arom genomic DNA sequence （EU840259）, a pair of gene-specific primers was designed and synthesized. After extraction of brain total mRNA using Trizol and eDNA synthesized with M-MLV, PCR was done to amplify the cDNA of the brain P450arom. Subsequently, the PCR product was purified and cloned into pMD-18T vector. Sequencing results showed that the rice field eel brain P450arom was 1514bp in length, containing the full open reading frame of the gene. Sequence and phylogenetic analysis revealed that the P450aromB belonged to brain P450arom subfamilies. The putative P450aromB amino acid residues shared more than 70% sequence affinity with other fish brain P450arom, and shared less than 60% sequence affinity with ovarian P450arom of other fish. RT-PCR analysis revealed that the P450aromB mRNA was expressed at a high level in the brain and gonad, but less in skin, and not detection in other tissues.