卵形鲳鲹耐低温候选基因△6FAD全长cDNA序列的克隆与表达分析

Cloning and Expression Analysis of Full-length cDNA Sequence of a Low Temperature Resistant Candidate Gene △6FAD in Trachinotus ovatus

采用c DNA末端快速扩增(Rapid amplification of c DNA ends,RACE)技术克隆了卵形鲳鲹△6脂肪酸去饱和酶(△6FAD)的c DNA全长序列,并对其基因和蛋白序列结构特征进行了生物信息学分析,同时采用实时荧光定量PCR(Real-time PCR)技术分析了该基因在不同温度条件下的表达差异及低温下的时序表达模式。结果显示,卵形鲳鲹△6FAD基因c DNA序列全长1 908 bp,其中开放阅读框为1 329 bp,3'非编码区431 bp,5'非编码区135 bp,共编码442个氨基酸;其编码蛋白属于疏水性蛋白,无信号肽特征,含有大量蛋白质二级结构和该家族典型的组氨酸富集区及细胞色素b5结构域。系统进化分析表明,卵形鲳鲹△6FAD基因与尖吻鲈、军曹鱼的△6FAD基因在进化关系上最为接近,其次是大菱鲆、蓝鳍金枪鱼和点带石斑鱼,但与斑马鱼、小鼠和人△6FAD基因进化关系较远。实时荧光定量PCR结果表明,该基因的表达与环境温度和胁迫时间存在明显相关性,其表达水平在降温过程中随温度降低而先缓慢下降后又迅速升高,在不同的低温环境下表现出不同的时序表达模式:13℃时,随时间延长该基因的表达量呈现先降低后又升高至原来水平的变化趋势,16℃和19℃时则表现出一种随时间延长逐渐降低的反馈式调节方式。

The full-length cDNA of delta 6 fatty acid desaturase （ △6FAD ） in Trachinotus ovatus was cloned by RACE （ Rapid Amplification of cDNA Ends ） , and the sequence structure of the gene and protein was determined through bioinformatics analysis. RT-PCR （ Real-time PCR ） was employed to analyze its expression differences in tissues under different temperatures and the expression patterns in low temperature. Ultimately, the results indicate that full length of cDNA sequence of △6FAD in T. ovatus is 190g bp, of which has 3＇ non-coding region 431 bp, 5＇ non-coding region 135 bp, and open reading frame 1 329 bp that encodes 442 amino acids. The protein is hydrophobic without signal peptide, and containing abundant protein secondary structure, typical histidine cluster motifs in this family, and cytochrome b5 domain. Phylogenetic results from the BLAST protein database reveal that the deduced amino acid sequence of △6FAD of T. ovatus is highly homologous with Lares calcarifer and Rachycentron canadum ： lowly homologous with Scophthalmus maximu, Thunnusthynnus and Epinephelus coioides ： but little homologous with Danio rerio, Mus musculus and Homo sapiens. RT-PCR results manifest that gene expression of △6FAD is significantly correlated with temperature and stress time in environment, the expression level of the gene at first slowly decreases, then rapidly rises as temperature lowering in the experiment of temperature gradient. However, there is different expression pattern in different low-temperature environment ： for example, at 13℃ the gene expression of △6FAD increases to its original level after first decreasing with time ： and while at 16℃ and 19℃ it is gradually decreasing with time, i.e., feedback adjustment way.