摘要
采用抑制消减杂交(suppression subtractive hybridization,SSH)技术,研究了青海湖裸鲤(Gymnocyprisprzewalskii)在高盐碱胁迫下转录水平上的基因表达差异,实验获得差异表达序列91条,其中成功注释60条。以同是鲤科鱼类的斑马鱼(Danio rerio)的基因ID作为参照,用DAVID软件对这些序列进行功能分类,结果从正向文库获得28条功能基因片段,反向文库获得14条。按功能将其分为10类,其中,催化活性、细胞、部分结合相关基因表达上调,结合、免疫相关基因表达下调。分析发现,高盐碱胁迫会改变鱼类的渗透压和酸碱平衡,对免疫系统产生一定的抑制作用,对此,鱼类通过增强离子调控、提高血清尿素氮浓度、合成应激蛋白等,来维持渗透压和酸碱平衡,缓解应激反应。
Saline-alkali water areas are widely distributed in China,but the utilization ratio is very low.High salinity and high carbonate alkalinity are considered to be the major stressors for aquatic animals living in saline-alkali waters.Gymnocypris przewalskii,scale-less carp,is an important economic fish in Qinghai Lake,the largest inland lake in China,which has a low concentration of dissolved oxygen,strong alkalinity and high salinity.The saline-alkali tolerance of G.przewalskii is very high.The study is to determine genome-wide gene expression profiles and to better understand how saline-alkali stress influences gene expression in G.przewalskii.Suppression subtractive hybridization method was applied to construct directional cDNA libraries and to obtain differential expression genes.450 positive clones from libraries were randomly selected to validate with PCR,and 318 clones showed a single channel result.The results showed that the quality of libraries were perfect.Consequently,195 positive clones were selected for analysis through Blast.Altogether 91 genes were identified from the forward and reverse libraries,69 of them were well annotated.These differentially expressed genes could be divided into a number of biological gene ontology groups related to catalytic activity,binding,cell,immune system process,transporter activity,structural molecule activity,cellular process,development,reproduction,biological regulation.Catalytic activity,binding and cell genes were up-regulated,while immune system process genes were down-regulated.Quantitative real-time PCR showed that the expression of Na＋,K＋-ATPase β1b subunit and glutamine synthase increased significantly at 72h after exposure.Compared with the control group,the expression of Na＋,K＋-ATPase β1b subunit increased 5.6 fold,glutamine synthase increased 2.3 fold.High saline-alkali water could change the osmotic pressure and acid-base balance in fish,injure surface mucous membrane of gill and kidney,and inhibit the immune system to a certain extent.Therefore,G.przewalskii may activate and enhance partial signal path to activate the metabolism,transporter and tolerance of gene expression.G.przewalskii improved the expression and activity of catalyticase to increase the ion exchange with the environment and maintain osmotic equilibrium;raised the expression of glutamine synthase to increase the content of glutamine and serum urea nitrogen,thus rebuilt the acid-base balance;synthetized heat shock protein and annexin to increase the defense ability,thus reduced stress response.
出处
《海洋渔业》
CSCD
北大核心
2012年第2期137-144,共8页
Marine Fisheries
基金
中央级公益性科研院所基本科研业务费专项资金(中国水产科学研究院东海水产研究所,2009M03)
公益性行业(农业)科研专项(200903001-05-02)资助
关键词
青海湖裸鲤
高盐碱胁迫
抑制消减杂交
基因表达
Gymnocypris przewalskii
high saline-alkali stress
suppression subtractive hybridization
gene expression
