脊尾白虾GAPDH基因的克隆及其内参基因稳定性分析
发布时间：2018-07-13 16:25
【摘要】：为比较甘油醛-3-磷酸脱氢酶(glyceraldehyde 3-phosphate dehydrogenase,GAPDH)、18S rRNA和β-actin基因在脊尾白虾(Exopalaemon carinicauda)作内参基因的优劣,本研究采用同源克隆和RACE技术,克隆了脊尾白虾GAPDH基因全长cDNA序列(GenBank登录号:KX893516),通过实时荧光定量PCR(quantative real-time PCR,qPT-PCR)技术,检测3种基因在脊尾白虾不同组织及不同蜕壳后时间点的表达量变化,在此基础上进行内参稳定性分析。结果显示,脊尾白虾GAPDH基因全长1514 bp,开放读码框1002 bp,编码333个氨基酸,二级结构预测显示GAPDH蛋白具有一个高度保守的NAD~+结合功能域(NAD binding domain)和行使糖运输和代谢的催化功能域。分析qRT-PCR结果并结合ge Norm、Norm Finder和Best Keeper 3种软件的分析发现,在不同组织和不同蜕壳后时间点,3种内参基因的稳定性由高到低依次为18S r RNA、GAPDH、β-actin。因此,在脊尾白虾不同组织和不同蜕壳后时间点的定量分析中,选取单内参基因时,推荐使用18S rRNA为内参基因,双内参时推荐18S rRNA和GAPDH,而18S rRNA、β-actin和GAPDH在其他生理条件下作内参基因的稳定性还有待进一步研究。
[Abstract]:In order to compare the advantages and disadvantages of 18s rRNA and 尾 -actin gene of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal reference gene in Exopalaemon carinicauda, homologous cloning and race techniques were used in this study. The full-length cDNA sequence of GAPDH gene (GenBank accession number: KX893516) was cloned. The expression changes of three genes in different tissues and time points after molting were detected by real-time fluorescence quantitative PCR (quantative real-time PCR-qPT-PCR). On this basis, the stability of internal parameters is analyzed. The results showed that the GAPDH gene was 1514 BP in length and 1002 BP in the open reading frame, encoding 333 amino acids. The secondary structure prediction showed that the GAPDH protein had a highly conserved NAD- binding domain (NAD binding domain) and a catalytic domain for glucose transport and metabolism. By analyzing the results of qRT-PCR and combining with three kinds of software, GE Norm Finder and Best Keeper, it was found that the stability of the three internal reference genes was 18s r RNA-GAPDH, 尾 -actinin from high to low in different tissues and at different time points after molting. Therefore, in the quantitative analysis of different tissues and post-molting time points of white shrimp, 18s rRNA is recommended to be used as internal reference gene when single internal reference gene is selected. The stability of 18s rRNA, 尾 -actin and GAPDH genes under other physiological conditions should be further studied.
【作者单位】： 淮海工学院海洋生命与水产学院江苏省海洋生物技术重点实验室;江苏省海洋生物产业技术协同创新中心;江苏省农业种质资源保护与利用平台;中国水产科学研究院黄海水产研究所;
【基金】：江苏省农业科技支撑计划项目(BE2013363) 江苏省高校“青蓝工程”培养基金项目 江苏省2015年度普通高校研究生科研创新计划项目(KYLX15_1486) 连云港市产学研合作项目(CXY1517) 淮海工学院江苏省海洋生物技术重点实验室研究基金项目(2015HS001)
【分类号】：S917.4
本文编号：2120031
[Abstract]:In order to compare the advantages and disadvantages of 18s rRNA and 尾 -actin gene of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal reference gene in Exopalaemon carinicauda, homologous cloning and race techniques were used in this study. The full-length cDNA sequence of GAPDH gene (GenBank accession number: KX893516) was cloned. The expression changes of three genes in different tissues and time points after molting were detected by real-time fluorescence quantitative PCR (quantative real-time PCR-qPT-PCR). On this basis, the stability of internal parameters is analyzed. The results showed that the GAPDH gene was 1514 BP in length and 1002 BP in the open reading frame, encoding 333 amino acids. The secondary structure prediction showed that the GAPDH protein had a highly conserved NAD- binding domain (NAD binding domain) and a catalytic domain for glucose transport and metabolism. By analyzing the results of qRT-PCR and combining with three kinds of software, GE Norm Finder and Best Keeper, it was found that the stability of the three internal reference genes was 18s r RNA-GAPDH, 尾 -actinin from high to low in different tissues and at different time points after molting. Therefore, in the quantitative analysis of different tissues and post-molting time points of white shrimp, 18s rRNA is recommended to be used as internal reference gene when single internal reference gene is selected. The stability of 18s rRNA, 尾 -actin and GAPDH genes under other physiological conditions should be further studied.
【作者单位】： 淮海工学院海洋生命与水产学院江苏省海洋生物技术重点实验室;江苏省海洋生物产业技术协同创新中心;江苏省农业种质资源保护与利用平台;中国水产科学研究院黄海水产研究所;
【基金】：江苏省农业科技支撑计划项目(BE2013363) 江苏省高校“青蓝工程”培养基金项目 江苏省2015年度普通高校研究生科研创新计划项目(KYLX15_1486) 连云港市产学研合作项目(CXY1517) 淮海工学院江苏省海洋生物技术重点实验室研究基金项目(2015HS001)
【分类号】：S917.4
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