草鱼CYP1A和ACT基因cDNA片段的克隆与鉴定

Cloning and Identification of Grass Carp CYP1A and ACT cDNA Fragment

以Trizol法分别提取BNF诱导和对照处理草鱼的肝组织总RNA并合成cDNA第一链,以此为模板利用1对ACT特异性引物和(8条)6对CYP1A简并引物进行扩增。结果显示,引物对F0-R0在对照和诱导草鱼中均扩增得到预期ACTcDNA片段,而引物对F4-R4在诱导草鱼中获得预期CYP1A cDNA产物。这两个cDNA片段分别进行克隆、测序和比对,BLAST结果表明草鱼ACTcDNA片段(800 bp)与GenBank中ACT基因(登录号M25013)同源性为99.1%,推导氨基酸序列同源性为99.2%;草鱼CYP1A cDNA片段(439 bp)与鲤鱼同源性最高,为92.5%,推导氨基酸同源性为96.6%。上述序列提交GenBank,获得登录号分别为DQ211096和DQ211095。通过Mega 3.1软件的Neighbor-joining程序对CYP基因的部分cDNA序列和氨基酸序列进行比对分析并绘制进化树,根据CYP1A部分蛋白的系统发育关系,在进化上可以将参与比对的真骨鱼划分为4个主要的分支。

Total RNA of BNF-treated and control grass carp liver were extracted respectively,using Trizol reagent,and reverse transcribed to first strand cDNA as templates in PCR reaction.PCR were performed using 1 pair of specific primer for ACT cDNA and 6 pairs of degenerated primers for CYP1A cDNA,respectively.As a result,primer pair F0-R0 could amplify the expected ACT cDNA fragment both in the BNF-treated and control grass carp,but F4-R4 could amplify the expected CYP1A cDNA fragment only in the BNF-treated grass carp.These two cDNA fragments were cloned,sequenced and blasted respectively.ACT cDNA fragment（800 bp） had identity of 99.1% with the sequence in GenBank（accession number： M25013）,and the identity of deduced amino acids was 99.2%.The new-cloned grass carp CYP1A cDNA fragment（439 bp） had identity of 92.5% with that of common carp in GenBank（accession number： AB048939）,and the identity of deduced amino acids was 96.6%.These two cDNA sequences were submitted to GenBank and acquired accession numbers DQ211096 and DQ211095,respectively.Partial cDNA and amino acid sequences of teleost CYP genes were aligned and plotted phylogenetic tree using the Neighbor-joining method by the Mega 3.1 softwares.According to the phylogenetic relation of partial CYP1A protein,the aligned teleosts were divided into four main branches.