赤霞珠葡萄发育后期RT-PCR内参基因的筛选和验证

Screening and validation of reference genes for real-time fluorescence quantitative PCR during grape berry development of Cabernet Sauvignon

葡萄果实发育后期基因表达水平的变化对果实品质的形成具有重要影响,选择合适的内参基因是提高实时荧光定量PCR分析准确性的首要条件。本试验以赤霞珠葡萄发育后期不同取样时间的果皮为材料,通过qRT-PCR分析了常用候选内参基因β-actin、EF-1α、GAPDH和SAND的表达变化,借助ge Norm、Norm Finder和BestKeeper程序筛选出在葡萄发育后期qRT-PCR分析的理想内参基因。结果表明,β-actin的稳定性最好,其次是SAND,而GAPDH和EF-1α的稳定性相对较低;同时,用筛选的内参基因β-actin和SAND分析葡萄白藜芦醇合成途径中二苯乙烯合酶基因的表达水平,其表达规律一致。说明,β-actin和SAND是研究赤霞珠葡萄发育后期基因表达的理想内参基因。

The change of gene expression level during berry ripening is crucial for the formation of grape fruit quality. The selection of a suitable reference gene is an important factor for accurate gene expression analysis by real-time fluorescence quantitative PCR（ qRT-PCR）. In this study,the pericarp of wine grape（ Cabernet Sauvignon） was taken as material at different time during berry ripening to detect the expression levels of four commonly used housekeeping genes β-actin,EF-1α,GAPDH and SAND by qRT-PCR,so as to select reliable reference genes using softwares ge Norm,NormFinder,and Best Keeper. The stability was found to the best in β-actin,followed by SAND. The expression of STS gene in the pathway of grape resveratrol anthocyanin biosynthesis was analyzed,and the results indicated that the variation dynamics of STS using β-actin as reference gene was exactly consistent with that using SAND as reference gene. Therefore,β-actin and SAND are ideal reference genes for gene expression level analysis during grape berry development.