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青海湖裸鲤TOB1和TOB2基因的克隆与表达分析
梁 健, 陈雪妍, 卫 唯,等
青海大学 省部共建三江源生态与高原农牧业国家重点实验室
摘要:
【目的】对青海湖裸鲤TOB1和TOB2基因进行克隆及表达分析,为揭示其在青海湖裸鲤生长和生理过程中的作用奠定基础。【方法】采用PCR和RACE技术克隆青海湖裸鲤中TOB1和TOB2基因的cDNA全长序列,对其特性进行了生物信息学分析。以β-actin为内参基因,采用实时荧光定量PCR法(qRT-PCR)检测TOB1和TOB2基因在3龄成鱼肾脏、肠、鳃、脑、心脏、肌肉等组织及发育12,72,120,168,216 h胚胎中的表达水平。【结果】青海湖裸鲤TOB1 cDNA全长为1 734 bp,5′非编码区为466 bp,3′非编码区为296 bp,开放阅读框长972 bp,编码323个氨基酸。TOB2 cDNA全长为2 785 bp,5′非编码区为51 bp,3′非编码区为1 582 bp,开放阅读框为1 152 bp,编码383个氨基酸。TOB1和TOB2在N端均具有明显的BTG家族结构域,且系统进化分析显示2个TOB蛋白与鲤科鱼类同源性较高,亲缘较近。qRT-PCR结果表明,TOB1和TOB2在3龄成鱼的肾脏、肠、鳃、脑、心脏、肌肉等组织中均有表达;TOB1在3龄成鱼的心脏组织中表达量最高,其次为脑和肌肉组织,在鳃组织中的表达量最低;TOB2在3龄成鱼的脑组织中表达量最高,其次为肌肉。TOB1及TOB2在不同发育时期胚胎中均有表达,且都在12 h胚胎中表达量最高。【结论】TOB1及TOB2在3龄成鱼各个组织及胚胎中均有表达,但表达量有较大差异,表明2个基因均具有时空表达特异性。
关键词:  TOB基因  基因克隆与表达  青海湖裸鲤
DOI:
分类号:
基金项目:青海省科技项目“青海湖裸鲤耐盐碱基因的筛选和机理的初步研究”(2016-ZJ-940Q)
Cloning and expression analysis of TOB1 and TOB2 genes in naked carp (Gymnocypris przewalskii) in Qinghai Lake
LIANG Jian, CHEN Xueyan, WEI Wei,et al
Abstract:
【Objective】Cloning and expression analysis of two TOB genes of naked carp (Gymnocypris przewalskii) in Qinghai Lake were conducted to provide basis for revealing their role in the growth and physiological processes.【Method】The full-length cDNA sequences of two genes in TOB family of naked carp were cloned by PCR and RACE.Their characteristics and structure function were studied by bioinformatics method,and the expression in different tissues and different developmental stage of enbryos (12,72,120,168,216 h) was analyzed by qRT-PCR.【Result】The full-length cDNA sequence of TOB1 in naked carp was 1 734 bp,the 5′ non coding region was 466 bp,the 3′ non coding region was 296 bp,and the open reading frame was 972 bp long,encoding 323 amino acids.The full length cDNA sequence of TOB2 was 2 785 bp,the 5′ non coding region was 51 bp,the 3′ non coding region was 1 582 bp,and the open reading frame was 1 152 bp,encoding 383 amino acids.Both TOB1 and TOB2 had BTG family domains at the N-terminal.The phylogenetic analysis showed that both TOB proteins had close relationship with Cyprinidae species.The real-time PCR showed that TOB1 and TOB2 were expressed in kidney,intestine,sputum,brain,heart,muscle and other issues of 3rd instar adult fish.TOB1 expressed the highest in heart tissue of 3rd instar adult fish,followed by brain and muscle tissues,and the lowest expression was in gill tissue.TOB2 expressed the highest in brain tissue of 3rd instar adult fish,followed by muscle.TOB1 and TOB2 were also expressed in different developmental stages of embryos with the highest levels in 12 hour embryos.【Conclusion】TOB1 and TOB2 were expressed in all tissues of 3rd instar adult fish and embryos with significant differences in expression levels,indicating spatiotemporal expression specificity.
Key words:  TOB gene  cloning and expression of gene  Gymnocypris przewalskii

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