大黄鱼MIPS基因克隆及低温胁迫下的表达分析

摘要

Myo-inositol-1-phosphate synthase (MIPS) is one of the key enzyme in biological inositol metabolism.In order to explore the role of MIPS in the stress response of Larimichthys crocea under cold treatments,MIPS gene was cloned and then its sequence were analyzed and the expression changes were detected under chronic and acute cold stress.The full length of MIPS cDNA (GenBank accession No.MG760436) was 2 599 bp which contained a 1 659 bp open reading frame (ORF) and encoded 553 amino acids,the molecular weight of putative MIPS protein was 60.5 kD.Sequence alignment showed that the amino acid sequence ofL.crocea MIPS shared high similarity (85.5％～88.4％) to other teleost and all of these MIPS had 4 highly conserved core regions.In the phylogenetic tree,L.crocea MIPS gathered with MIPS of other teleost and was most closely related to the MIPS of Fugu rubripes (Takifugu rubripes).During chronic cold stress (water temperature slowed decreased from 12 ℃ to 6 ℃),the MIPS expression in gill,skin and heart significantly increased at first and then significantly decreased (P＜0.05),and the maximum appeared at 8 ℃ which were respectively 13.71,89.50 and 50.83 fold,respectively,compared to 12 ℃.The expression continuously elevated in brain and muscle during chronic cold stress and at 6 ℃ the expression were 99.89 and 110.17 fold higher than before cold stress.And there were no significant changes in intestine,kidney,liver and spleen (P＞0.05).During acute cold stress (from 12 ℃ to 8 ℃ immediately and keeping 8 ℃ for 4 h),the expression level of MIPS were significantly up-regulated (P＜0.05) in all the detected tissues except intestine.The 2 biggest expression changes were appeared in brain and muscle,which were 54.53 and 32.89 fold higher than before cold stress.The expression changes in both chronic and acute cold treatments indicated MIPS participates in the response to cold stress and has possible relationship to cold adaptability of L crocea,brain and muscle are the 2 most important tissues in the response to cold stress.These results provide reference for researching the function of MIPS gene and the mechanism of response to cold stress,as well as breeding low-temperature tolerant cultivars of L.crocea.%肌醇-1-磷酸合成酶(myo-inositol-1-phosphate synthase,MIPS)是生物肌醇代谢调节的关键酶之一,为了解MIPS基因与大黄鱼(Larimichthys crocea)低温胁迫应激响应的相关性,本研究通过cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术克隆了大黄鱼MIPS基因的cDNA全长、进行了序列分析并检测了其在急性和慢性低温胁迫下的表达变化.结果表明,大黄鱼MIPS基因cDNA(GenBank登录号:MG760436)全长为2 599 bp,含1个长度为1 659 bp的开放阅读框,预测编码蛋白含553个氨基酸、分子量为60.5 kD.序列比对显示,大黄鱼与其他硬骨鱼类的MIPS氨基酸序列相似性较高(85.5％～88.4％),均含有4个高度保守核心区.系统进化树中大黄鱼MIPS与其他硬骨鱼类MIPS聚为1个大簇,并与红鳍东方鲀(Takifugu rubripes)MIPS的进化关系最近.慢性低温胁迫中(12℃缓降至6℃),大黄鱼MIPS基因在鳃、皮肤和心脏组织中均呈现先显著上升后显著下降的趋势(P＜0.05),在8℃时达到峰值表达量,分别为12℃时的13.71、89.50和50.83倍;脑和肌肉中MIPS表达量在整个胁迫过程中持续升高,6℃时表达量比降温前分别升高了99.89和110.17倍;肠、肾、肝、脾中MIPS表达量则无显著变化(P＞0.05).急性低温胁迫(由12℃骤降至8℃并持续胁迫4 h)下,MIPS基因在除肠以外的其他组织均出现显著的表达量上调(P＜0.05),以脑和肌肉中变化最为显著,分别比胁迫前升高了54.53和32.89倍.急性和慢性低温胁迫实验的结果提示,MIPS基因参与了大黄鱼的低温胁迫应激响应,并可能与大黄鱼的低温适应性有关,脑和肌肉是大黄鱼应对低温胁迫调节中的重要组织.该研究结果为研究大黄鱼MIPS基因的功能及研究大黄鱼低温耐受性、选育耐低温品种提供了参考资料.