桃果实中miR393b靶向生长素受体基因TIR1的作用机制分析

The interactive mode analysis between miR393b and its target gene TIR1 in peach fruit

本研究以水蜜桃品种‘小白凤’为试材,利用miR-RACE技术验证了桃miR393b的精确序列,克隆得到了其靶基因生长素受体Pp TIR1的ORF序列。利用RLM-RACE技术以及q RT-PCR方法对Ppe-miR393b作用于靶基因Pp TIR1的作用模式及作用频度进行了分析。结果表明,Ppe-miR393b的精确序列与预测序列在5′端存在1个碱基的差异,其靶基因Pp TIR1编码584个氨基酸且N端含有1个高度保守的FBOX DNA结合域。RLM-5'RACE结果显示,Ppe-miR393b以裂解的方式作用于其靶基因Pp TIR1,且在Ppe-miR393b 5′端的第10和11位碱基之间以及第8和9位碱基间均存在裂解位点,但前者的裂解频度显著高于后者。以上结果表明Ppe-miR393b通过介导靶基因Pp TIR1的裂解参与生长素信号途径。

In this study, we accurately determined the precise sequence of peach mi R393 b by using mi R-RACE PCR reactions, the target gene PpTIR1 was also cloned with homology-based cloning and reverse transcription-PCR（RT-PCR） from Prunus persica ‘Xiao bai feng＇. RLM-5＇ RACE and real-time RT-PCR were employed to validate the mode and frequency that Ppe-mi R393 b regulated its target gene TIR1. Observations showed that there was one nucleotide divergences at 5＇ terminal of mi R393 b between predicted and validated sequence. The open reading frame of PpTIR1 was encoding a protein of 584 amino acids. Sequence alignment showed that PpTIR1 contained a highly conserved FBOX DNA domain at N terminal. The RLM-5＇ RACE result showed that Ppe-mi R393 b could negatively regulate expression of its target genes by cleavage, and the target cleavage site were between the tenth and eleventh nucleotide and also between the eighth and ninth nucleotide at the 5＇-end of the Ppe-mi R393 b. The cleavage frequency between the tenth and eleventh nucleotide site was significantly higher than the site between the eighth and ninth nucleotide. These results suggested that Ppemi R393 b is involved in the auxin signal process through negatively regulating the expression of its target gene PpTIR1 by guiding cleavage.