《植物生理学报》 2018, 54(5): 755-762

桃果实中miR393b靶向生长素受体基因TIR1的作用机制分析

张彦苹^1,2,*, 刘照坤³, 朱旭东⁴, 王晨⁴, 李庆魁¹

¹苏州农业职业技术学院, 江苏苏州215008; ²苏州科技大学, 江苏苏州215009; ³苏州市蔬菜研究所, 江苏苏州215000; ⁴南京农业大学园艺学院, 南京210095

通信作者：张彦苹；E-mail: 627306810@qq.com

摘　要：

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PpTIR1的ORF序列。利用RLM-RACE技术以及qRT-PCR方法对Ppe-miR393b作用于靶基因PpTIR1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PpTIR1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PpTIR1, 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PpTIR1的裂解参与生长素信号途径。

关键词：桃; miR393b; TIR1

收稿：2018-02-01   修定：2018-04-17

资助：国家自然科学基金(31601727)、江苏省自然科学基金(BK-20160360)和苏州市农业科技创新项目(SNG201623)。

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

ZHANG Yan-Ping^1,2,*, LIU Zhao-Kun³, ZHU Xu-Dong⁴, WANG Chen⁴, LI Qing-Kui¹

¹Suzhou Polytechnic Institute of Agriculture, Suzhou, Jiangsu 215008, China; ²Suzhou University of Science and Technology, Suzhou, Jiangsu 215009, China; ³Suzhou Institute of Vegetable, Suzhou, Jiangsu 215000, China; ⁴College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China

Corresponding author: ZHANG Yan-Ping; E-mail: 627306810@qq.com

Abstract:

In this study, we accurately determined the precise sequence of peach miR393b by using miR-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-miR393b regulated its target gene TIR1. Observations showed that there was one nucleotide divergences at 5' terminal of miR393b 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-miR393b 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-miR393b. 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 PpemiR393b is involved in the auxin signal process through negatively regulating the expression of its target gene PpTIR1 by guiding cleavage.

Key words: peach; miR393b; TIR1