脊尾白虾GAPDH基因的克隆及其内参基因稳定性分析

Cloning, expression and stability analysis of the reference gene glyceraldehyde-3-phosphate dehydrogenase(GAPDH) in Exopalaemon carinicauda

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。

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 normali- zation 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 refer- ence genes. Nowadays, the role of GAPDH as the reference gene was being questioned and challenged by accu- mulated experiment evidences. To investigate the stability of GAPDH as a reference gene, the full-length cDNA sequence was cloned from the ridgetail white prawn, Exopalaemon carinicauda, which mRNA was measured in different tissues and at different post-molt times. The obtained full-length cDNA of GAPDH was 1514bp, includ- ing 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 GAPDH encoded 333 amino acids without signal peptide analyzed by SignalP software which is highly conserved across the phyloge- netic 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 E. carinicauda GAPDH contains two major domains, the NAD＋ 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, β-actin and GAPDH） 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 rain） 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 GAPDH and β-actin in decreasing order. In conclusion, the best choice for single reference gene is 18S rRNA, and 18S rRNA and GAPDH can be together as double reference genes if needed in E. carinicauda.