虎皮鱼PtScd1基因序列、功能与表达分析  

Analysis of Sequence,Function and Expression of PtScd1 Gene in Tropical Stenothermal Fish Tiger Skin Barb Puntius tetrazona

作　　者：刘丽丽 张蓉 王晓雯[1] 朱华[1] LIU Lili;ZHANG Rong;WANG Xiaowen;ZHU Hua(Beijing Key Laboratory of Fishery Biotechnology, Beijing Fisheries Research Institute, Beijing 100068, China)

机构地区：[1]北京市水产科学研究所,渔业生物技术北京市重点实验室,北京100068

出　　处：《水产科学》2020年第6期820-828,共9页Fisheries Science

基　　金：北京市自然科学基金资助项目(6194032);现代农业产业技术体系北京市观赏鱼创新团队项目(BAIC03-20200210).

摘　　要：将体长5~7 cm的虎皮鱼饲养在30 L的养殖缸中,每缸30尾。水温自27℃起,每24 h降温4℃,第4 d再降温2℃,即各试验组的水温分别为23、19、15、13℃,对照组水温保持27℃。每组3个平行。每组水温维持24 h后,每缸随机取3尾鱼,用间氨基苯甲酸乙脂甲磺酸盐溶液麻醉后,解剖脑、鳃、肝脏和肌肉组织,液氮速冻,-80℃超低温冰箱保存备用。利用CODEHOP软件设计简并引物,PCR扩增到一段虎皮鱼硬脂酰辅酶A去饱和酶(SCD)基因(PtScd1基因)保守序列,用RACE克隆和序列拼接获得PtScd1基因cDNA序列。试验结果显示,PtScd1基因cDNA序列全长1338 bp,ORFfinder预测开放阅读框长度981个核苷酸,编码326个氨基酸。根据保守结构域数据库预测PtSCD1蛋白氨基酸序列含有高度保守的脂肪酸去饱和酶结构域,Swiss-Model同源建模发现蛋白质三维结构存在2个高度保守的锌离子结合位点(可能与双铁离子结合),2个配体结合位点,但是其序列保守性很差。PtSCD1蛋白可能在脂肪酸链delta9位置引入双链,催化生成单不饱和脂肪酸。实时荧光定量PCR技术检测到PtScd1转录本在虎皮鱼脑、鳃、肝脏和肌肉组织中表达,表达水平依次为肝脏>脑>肌肉>鳃;低温胁迫下虎皮鱼脑、鳃、肌肉和肝脏组织PtScd1转录本丰度上调,且在一定范围内脑组织PtScd1转录本相对表达水平与温度呈负相关趋势。试验结果表明,PtScd1基因在虎皮鱼组织中特异性表达,表达水平具有一定的温度依赖性,预测PtScd1基因在虎皮鱼应对低温胁迫过程中发挥重要作用。Tiger skin barb Puntius tetrazona with body length of 5—7 cm was housed in a 30 L breeding tank at a rate of 30 fish per tank at water temperature of 27℃(control group),and then exposed to water temperature of 23℃,19℃,15℃,and 13℃on the fourth day,by dropping 4℃and 2℃every 24 hours from the next day in the 4 groups.After 24 hours of treatment,3 fish were randomly sampled from each tank,and anesthetized by MS-222 solution,and the brain,gills,liver and muscle tissues were dissected,and were quickly frozen in liquid nitrogen.Using CODEHOP software to design degenerate primers,a conserved sequence of tiger skin fish Scd1(PtScd1)gene was amplified by PCR,RACE cloning and sequence splicing to obtain tiger skin fish stearoyl-CoA desaturase(SCD)PtScd1 gene cDNA sequence.A 1338 bp PtScd1 cDNA sequence was obtained by rapid-amplification of cDNA ends and sequence assembly,and the open reading frame was predicted with 981 bp,encoding 326 amino acids.PtSCD1 protein was predicted with a fatty-acid desaturase conservation domain according to the Conserved Domain Database.Swiss-Model showed that there was a highly conserved binding site for two zinc ions,with more likely to be a diiron center,in the dimensional structure of PtSCD1,with two unconserved ligand sites.The protein model indicated that PtSCD1 catalyzed the synthesis of monounsaturated fatty acids leading to introducing a double band into the delta-9 position of fatty acids chain.The real time quantitative PCR revealed that the descending abundance of PtScd1 transcripts in various tissues and organs of adult tiger barb fish was described as liver>brain>muscle>gill,with negative correlation between the relative expression level of PtScd1 transcripts in brain tissue and the exposure temperature,and tissue-specific expression of PtScd1 transcripts in a temperature-dependent way.The findings indicated that PtScd1 gene was expected to play a vital role for tiger skin barb coping with cold stress,which would help promote the research of tiger skin barb surviv

关 键 词：狭温热带鱼 耐低温基因 硬脂酰辅酶A去饱和酶 蛋白质结构预测 组织特异性表达 

分 类 号：Q786[生物学—分子生物学]