大豆干旱胁迫下miRNA与mRNA荧光定量PCR内参基因的筛选

Selection of reference genes for quantitative polymerase chain reaction of miRNA and mRNA in soybean under drought stress

【目的】通过分析大豆中候选内参基因的稳定性,筛选大豆干旱胁迫处理条件下适合成熟miRNA、前体miRNA及靶基因mRNA荧光定量PCR的内参基因。【方法】以干旱胁迫处理后的大豆根和叶片为材料,选择了5个成熟miRNAs和5个传统的看家基因作为候选内参基因,利用GeNorm和NormFinder程序对10个候选内参基因的稳定性进行评价。【结果】在干旱胁迫下,大豆根、叶片中,成熟miRNA定量最合适的单个内参基因分别为miR156a、miR167a,最合适的内参基因组合分别为miR1520d与miR156a、miR1520d与miR167a。前体miRNA和靶基因mRNA定量最合适的单个内参基因分别为Fbox、Act11,最合适的内参基因组合分别为Act11与Fbox、Act11与EF1A。【结论】筛选出大豆干旱胁迫条件下成熟miRNA、前体miRNA及其对应靶基因mRNA的荧光定量PCR的内参基因,最稳定内参基因数目为2个。

【Objective】This study aimed to select appropriate reference genes for qPCR of mature miRNAs,Pre-miRNAs and their target mRNA genes in soybean by analyzing the stabilities of candidate reference genes under drought.【Method】Roots and leaves of soybean after drought stress treatment were used as materials in this experiment,and five mature miRNAs and five traditional housekeeping genes wereselected as candidate reference genes to evaluate their stabilities using GeNorm and NormFinder procedures.【Result】Under drought treatment to soybean,miR156 aand miR167awere found to be the most suitable reference gene for qPCR of mature miRNA while miR1520d/miR156 aand miR1520d/miR167 a were the most suitable reference gene pairs in roots and leaves,respectively.For qPCR of Pre-miRNAs and their target mRNA genes,Fboxand Act11 were the most suitable reference genes while Act11/Fboxand Act11/EF1 A were the most suitable reference gene pairs in roots and leaves,respectively.【Conclusion】Two most suitable reference gene pairs for qPCR of mature miRNAs,Pre-miRNAs and their target mRNA genes under drought stress were obtained,respectively.