1. 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 连云港 222005;
2. 江苏省海洋生物产业技术协同创新中心, 江苏 连云港 222001;
3. 江苏省农业种质资源保护与利用平台, 江苏 南京 210014;
4. 中国水产科学研究院 黄海水产研究所, 山东 青岛 266071
薛蓓(1992-),女,硕士研究生,研究方向为海洋生物繁殖与遗传育种学.E-mail:malvsy@163.com
S917
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）.
XUE Bei
Jiangsu Key Laboratory of Marine BiotechnologyMarine Life and Fisheries College, Huaihai Institute of Technology, Lianyungang 222005, ChinaZHANG Pei
Jiangsu Key Laboratory of Marine BiotechnologyMarine Life and Fisheries College, Huaihai Institute of Technology, Lianyungang 222005, ChinaLI Zhihui
Jiangsu Key Laboratory of Marine BiotechnologyMarine Life and Fisheries College, Huaihai Institute of Technology, Lianyungang 222005, ChinaZHAO Lian
Jiangsu Key Laboratory of Marine BiotechnologyMarine Life and Fisheries College, Huaihai Institute of Technology, Lianyungang 222005, ChinaLAI Xiaofang
Jiangsu Key Laboratory of Marine BiotechnologyMarine Life and Fisheries College, Huaihai Institute of Technology, Lianyungang 222005, ChinaGAO Huan
Jiangsu Key Laboratory of Marine BiotechnologyMarine Life and Fisheries College, Huaihai Institute of Technology, Lianyungang 222005, ChinaYAN Binlun
Jiangsu Key Laboratory of Marine BiotechnologyMarine Life and Fisheries College, Huaihai Institute of Technology, Lianyungang 222005, China1. Jiangsu Key Laboratory of Marine Biotechnology;Marine Life and Fisheries College, Huaihai Institute of Technology, Lianyungang 222005, China;
2. Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Lianyungang 222001, China;
3. The Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm, Nanjing 210014, China;
4. Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China
为比较甘油醛-3-磷酸脱氢酶（glyceraldehyde 3-phosphate dehydrogenase，）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geNorm、NormFinder和BestKeeper 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 rRNA、-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。
Quantitative real-time PCR (qRT-PCR) is a powerful and commonly used method for in-depth analysis of gene expression that offers increased sensitivity and specificity over other methods. However, in order to obtain accurate results when using qRT-PCR to study gene expression, one or several internal control genes for normalization are needed. Housekeeping genes are known as such a class of genes that their expression levels are expected to remain constant in the cells or tissues in response to any environmental or physiological stress. But, in fact, no any housekeeping gene always stably expressed under all physiological conditions as ideal reference genes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a classic key glycolysis enzyme presented in all tissues, is one of the most common housekeeping genes used in the analysis of comparing gene expression levels as reference genes. Nowadays, the role of as the reference gene was being questioned and challenged by accumulated experiment evidences. To investigate the stability of as a reference gene, the full-length cDNA sequence was cloned from the ridgetail white prawn, , which mRNA was measured in different tissues and at different post-molt times. The obtained full-length cDNA of was 1514bp, including 69 bp of 5'-untranslated region (UTR), 1002 bp of open reading frame (ORF), 443 bp of 3'-UTR containing a canonical polyadenylation signal sequence AATAAA prior to a poly A tail. The ORF of encoded 333 amino acids without signal peptide analyzed by SignalP software which is highly conserved across the phylogenetic scale. The molecular mass was calculated to be 35.71 kDa, and the pI was estimated to be 6.61. By alignment, the amino acid sequence of binding domain (amino acids 3-149) and the catalytic domain (amino acids 154-311). In order to compare the expression stability of three endogenous candidate genes (18S rRNA, ) in qRT-PCR analysis in different tissues and different post-molt times, eight tissues (eyestalk, gill, heart, hepatopancreas, ovary, stomach, instestines and abdominal muscle) and 4 different post-molt times (1, 5, 10 and 15 min) of E. carinicauda were collected for qRT-PCR. Comprehensive analysis of the results using delta Ct method and the software packages geNorm, NormFinder and BestKeeper revealed that 18S rRNA was the most stable reference gene in both different tissues and different post-molt times, then was the -actin in decreasing order. In conclusion, the best choice for single reference gene is 18S rRNA, and 18S rRNA and E. carinicauda.
薛蓓,张培,李志辉,赵莲,赖晓芳,高焕,李健,阎斌伦.脊尾白虾GAPDH基因的克隆及其内参基因稳定性分析[J].中国水产科学,2017,24(5):1003-1012
XUE Bei, ZHANG Pei, LI Zhihui, ZHAO Lian, LAI Xiaofang, GAO Huan, LI Jian, YAN Binlun. Cloning, expression and stability analysis of the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in Exopalaemon carinicauda[J]. Journal of Fishery Sciences of China,2017,24(5):1003-1012
