桃果实中miR160a与其靶基因ARF的鉴定及对IAA的响应分析

以水蜜桃品种‘小白凤’果实为试材,利用miR-RACE技术验证了桃mi R160a的精确序列,克隆了其3个ARF靶基因(PpARF18、PpARF17和PpARF18-like)的ORF序列。利用RLM-RACE技术以及qRT-PCR方法对Ppe-miR160a作用于3个靶基因的模式及频度进行了分析。结果表明,Ppe-miR160a的精确序列与预测序列在5′端和3′端均存在1个碱基的差异,其3个靶基因ARF均含有高度保守的B3和生长素响应DNA结合域。RLM-5′RACE结果显示,Ppe-miR160a以裂解的方式作用于3个ARF靶基因,且裂解位点均位于Ppe-miR160a5′端的第9和第10位碱基之间。IAA响应分析表明,与对照相比,IAA处理后的桃果实中Ppe-miR160a以及3个靶基因3′端裂解产物的表达量均显著升高。以上结果表明桃果实中的Ppe-miR160a通过介导其3个靶基因的裂解参与生长素信号途径调控。

Identification of miR160a and Its Target Gene ARFs in Peach Fruit and the Response Analysis of IAA

Using peach（Prunus persica）cultivar‘Xiaobaifeng’as experimental materials，we accurately determined the precise sequence of peach miR160a by using miR-RACE PCR reactions，three target genes PpARF18，PpARF17 and PpARF18-like were also cloned. RLM-5′ RACE and qualitative real-time RT-PCR were employed to validate the mode and frequency that Ppe-miR160a regulated its three target genes. Observations showed that the accurate sequence of Ppe-miR160a was different from the predicted sequence by one nucleotide at the 5′ end and 3′ end. All the three target genes（ARFs）contained highly conserved B3 and auxin resp DNA binding domains. The RLM-5′ RACE result showed that Ppe-miR160a could negatively regulate expression of its target genes by cleavage，and the target cleavage site was between the ninth and tenth nucleotide at the 5′-end of the Ppe-miR160a. Moreover，the qRT-PCR analysis revealed that compared with controls，the expression of Ppe-miR160a，three target genes and the cleavage production of three target genes at 3? ends were significantly hoisted in peach fruit by IAA. Taken together，these findings showed that Ppe-miR160a in peach fruit was involved in auxin signaling pathway by mediating the cleavage of its three target genes ARFs.