澳洲坚果实时荧光定量PCR分析中内参基因的筛选

澳洲坚果是重要的热区经济作物,目前国内外尚无关于澳洲坚果实时荧光定量PCR分析内参基因的报道。选择合适的内参基因是提高实时荧光定量PCR分析准确性的先决条件。为筛选澳洲坚果实时定量PCR最适内参基因,以澳洲坚果的根、茎、叶、果皮、果仁为材料,利用实时荧光定量PCR技术,对18S rRNA,Actin,CYP,EF1a,EF1b,GAPDH,MDH,TUBa,TUBb,UBQ,UBC等11个常用的内参基因在澳洲坚果不同组织中的表达稳定性进行了分析。geNorm软件分析的最适内参基因数目为2,最稳定内参组合为MDH和EF1b,TUBa基因的稳定性最差。BestKeeper分析结果认为,MDH基因表达最稳定,EF1b次之,与geNorm结果一致。NormFinder软件稳定性分析显示,GAPDH最稳定,其次是CYP基因;TUBa基因表达最不稳定。ΔCt算法结果表明,18S基因表达最稳定,其次是GAPDH基因,MDH和EF1b排第3和第4。RefFinder综合排序为：MDH>18S>GADPH>EF1b>CYP>UBC>EF1a>Actin>TUBb>UBQ> TUBa。因此,MDH基因在澳洲坚果不同组织中表达最稳定,初步确认可以作为实时荧光定量PCR分析的校正内参基因,在澳洲坚果的基因表达模式分析中具有重要意义。

Screening of Stable Reference Genes for qRT-PCR Analysis in Macadamia integrifolia

Macadamia nut is an important economics crop in subtropical areas of China. The selection of a suitable reference gene is an important prerequisite for successful gene expression analysis by real-time fluorescence quantitative PCR (RT-qPCR). In order to select the appropriate reference genes, we investigated the expression stability of 11 candidate genes (18S rRNA, Actin, CYP, EF1a, EF1b, GAPDH, MDH, TUBa, TUBb, UBQ, UBC) in RT-qPCR experiments in different tissues, including kernel, peel, roots, stem, leaf from Macadamia with geNorm, NormFinder, BestKeeper, ΔCt, RefFinder program software packages. As determined by geNorm, MDH/EF1b were the most stable reference genes, TUBa was the least stable gene. BestKeeper revealed that MDH was the most stables reference gene, and EF1b ranked the second. The rank in BestKeeper was similarly with that in geNorm. The result by NormFinder showed GAPDH was the most stable gene, CYP ranks the second, the least stable gene was TUBa. ΔCt algorithm demonstrated that18S was the most stable gene, GAPDH ranks the second, MDH and EF1b ranked the third and fourth. To obtain a consensus result of the most stable reference genes according to the RefFinder approach, the geometric mean of the four algorithms corresponding rankings for each candidate gene were calculated: MDH>18S>GADPH>EF1b>CYP>UBC>EF1a>Actin>TUBb>UBQ>TUBa. The result showed that MDH was the most suitable reference gene for macadamia in different tissues.