青海湖裸鲤颗粒蛋白基因克隆及其参与盐碱适应机制研究  

Cloning of granulin gene in Gymnocypris przewalskii and its involvement in salinity-alkalinity adaptation mechanism

作　　者：岳苗 卫唯 高子韩 卫福磊 梁健 YUE Miao;WEI Wei;GAO Zi-han;WEI Fu-lei;LIANG Jian(State Key Laboratory of Plateau Ecology and Agriculture,Qinghai University,Xining,Qinghai 810016,China;College of Eco-environmental Engineering,Qinghai University,Qinghai,Xining 810016,China)

机构地区：[1]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,青海西宁810016 [2]青海大学生态环境工程学院,青海西宁810016

出　　处：《南方农业学报》2024年第5期1480-1492,共13页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31960741)。

摘　　要：【目的】探究青海湖裸鲤颗粒蛋白A基因(GrnA)和颗粒蛋白B基因(GrnB)的生物学功能,为揭示颗粒蛋白(Grn)在青海湖裸鲤耐盐碱过程中的作用机制提供理论依据。【方法】以青海湖裸鲤为研究对象,通过RACE和PCR克隆青海湖裸鲤GrnA基因cDNA序列和GrnB基因编码区(CDS)序列,利用实时荧光定量PCR检测青海湖裸鲤GrnA和GrnB基因在各组织中的表达情况及在盐碱耐受过程中的表达变化,并分析二者与成骨分化特异性转录因子基因(Runx2)和补体基因C8g间的相关性。【结果】青海湖裸鲤GrnA基因cDNA序列全长653 bp,其开放阅读框(ORF)465 bp,共编码159个氨基酸残基;青海湖裸鲤GrnB基因CDS序列长1560 bp,共编码519个氨基酸残基。青海湖裸鲤与银鲫的GrnA氨基酸序列相似性高达89.61%,基于GrnA氨基酸序列相似性构建的系统发育进化树显示青海湖裸鲤与鲤和鲫的亲缘关系较近;青海湖裸鲤与多鳞白甲鱼的GrnB氨基酸序列相似性高达89.69%,基于GrnB氨基酸序列相似性构建的系统发育进化树显示青海湖裸鲤与多鳞白甲鱼和虎皮鱼的亲缘关系较近。GrnA和GrnB基因在青海湖裸鲤不同组织中均有表达,且以脾脏中的相对表达量最高,显著高于其他组织(P<0.05,下同)。青海湖裸鲤鳃组织中的GrnA基因对盐胁迫浓度增加的响应速度最快,其次是脾脏和肾脏;GrnB基因则表现为在肾脏中的响应速度最快,其次是脾脏和鳃组织,但在鳃组织中能持续响应。在碱胁迫下,青海湖裸鲤GrnA基因在3个组织中呈不规律性表达变化;GrnB基因表现为在鳃组织中的响应最强,其次是肾脏。在盐碱混合胁迫下,GrnA基因在鳃组织中的响应强于脾脏,在肾脏中也有响应;GrnB基因在鳃组织中最先响应盐碱混合胁迫,其次是肾脏和脾脏。在青海湖裸鲤鳃组织中,GrnA和GrnB基因在盐胁迫或碱胁迫下与Runx2基因呈正相关;在脾脏中,GrnA基因与C8g基因在盐胁迫下呈极�【Objective】To investigate the biological functions of granulin A gene(GrnA)and granulin B gene(GrnB)in Gymnocypris przewalskii,and to provide theoretical basis for revealing the mechanism of the role of granulin(Grn)in the salinity and alkalinity tolerance process of G.przewalskii.【Method】G.przewalskii was as research object,cDNA sequence of GrnA gene and the coding region(CDS)sequence of GrnB gene were cloned by RACE and PCR,and real-time quantitative PCR was used to detect the expression of GrnA and GrnB genes in various tissues of G.przewalskii and the changes during salinity and alkalinity tolerance.Correlations among the two genes and osteogenic differentiation-specific transcription factor gene(Runx2)and complement gene C8g were also analyzed.【Result】The cDNA sequence of GrnA gene of G.przewalskii was 653 bp in length,with an open reading frame(ORF)of 465 bp,encoding 159 amino acid residues;the CDS sequence of GrnB gene was 1560 bp in length,encoding 519 amino acid residues.The amino acid sequence similarity of GrnA between G.przewalskii and Silver prussian carp was as high as 89.61%,and the phylogenetic tree constructed on the basis of the GrnA amino acid sequence similarity showed that G.przewalskii was more closely related to carp and crucian carp;and the amino acid sequence similarity of GrnB between G.przewalskii and Scaphesthes macrolepis was as high as 89.69%,and the phylogenetic tree constructed on the basis of the similarity of GrnB amino acid sequences showed that G.przewalskii was closely related to S.macrolepis and Puntius tetrazona.GrnA and GrnB genes were expressed in different tissues of G.przewalskii,with the highest expression in spleen,which was significantly higher than that in other tissues(P<0.05,the same below).The GrnA gene in gill tissue of G.przewalskii showed the fastest response to salt stress,followed by spleen and kidney;while the GrnB gene showed the fastest response in kidney,followed by spleen and gill,but sustained response in gill tissue.Under alkalinity stress,

关 键 词：青海湖裸鲤 颗粒蛋白(Grn) 盐碱胁迫 GrnA基因 GrnB基因 

分 类 号：S965.116[农业科学—水产养殖]