浙东白鹅GAPDH基因的扩增及其在荧光定量PCR中的应用

三磷酸甘油醛脱氢酶(GAPDH)是一种糖酵解酶,在脊椎动物的所有组织均有稳定的表达,经常作为看家基因来研究基因的表达,本研究分析了鸭GAPDH基因序列,设计了一对引物,利用PCR,RT-PCR技术扩增得到了浙东白鹅的GAPDH基因的DNA序列为235 bp,mRNA序列为209 bp,作为参比基因应用于荧光定量PCR来研究目标基因的表达,目标基因在mRNA水平上的表达量可通过参比基因校正cDNA加入量的不同来研究,荧光定量PCR显示了以cDNA为模板,此引物扩增时Ct具有单一的溶解曲线,说明其表达稳定,可以作为参比基因用于浙东白鹅基因的定量表达研究.

Amplification and sequence of eastern Zhejiang white geese glyceraldehyde-3-phophate dehydrogenase for real time PCR applications

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycoytic enzyme, which is active in all vertebrate tissues and frequently used as a housekeeping gene in expression studies. In this study, the mRNA sequence of glyceraldehyde-3-phosphate dehydrogenase in duck was analyzed by designing a pair of primers and ampified in eastern Zhejiang white geese (a native Chinese geese breed) as a control gene in real time PCR. The mRNA levels of the target gene were measured relatively to their respective templates and were normalized to reference genes,to control the amount of cDNA loaded into the reaction. The sequences of genomic DNA and cDNA of goose glyceraldehydes-3-phophate dehydrogenase were 235 bp and 209 bp, respectively. Real time PCR showed a specific Ct using cDNA as template, and the primers showed a single melt peak. So the gene expression study was reliable when glyceraldehyde-3-phosphate dehydrogenase was used as an internal reference gene.