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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

DOI: 10.13560/j.cnki.biotech.bull.1985.2015.07.017, PP. 115-123

Keywords: 卵形鲳鲹,△6脂肪酸去饱和酶,cDNA克隆,低温胁迫

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Abstract:

采用cDNA末端快速扩增(RapidamplificationofcDNAends,RACE)技术克隆了卵形鲳鲹△6脂肪酸去饱和酶(△6FAD)的cDNA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-timePCR)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cDNA序列全长1908bp,其中开放阅读框为1329bp,3'非编码区431bp,5'非编码区135bp,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。

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