关键词: 龙眼, miR166初级体, 启动子, 实时荧光定量PCR, 表达特性

Abstract: In order to understand the structural characteristics of primary miR166 S53 gene （Pri-miR166 S53）and the expression pattern of its precursor and mature miRNA in the early stage of somatic embryogenesis（SE）in longan，SMARTTM RACE kit and PCR amplification were used to clone the pri-miR166 S53，to confirm its transcription start site and predict its potential ORF；longan genome database was used to extract its promoter sequence and predict cis-acting element；real-time fluorescence quantitative PCR was used to analyze the expression pattern of miR166 gene precursor（Pre-miR166 S53）and mature（miR166a.2）in longan early SE and in embryogenic callus under different hormone treatments. The results showed that the primary sequence of Pri-miR166 S53 gene was obtained with a length of 317 bp，encoded 13 amino acid sequence of miPEP（MLCFVDALFLIST）. Analysis of promoter sequence of Pri-miR166 S53 gene using bioinformatics software revealed that in addition to TATA/CAAT-box，it also contains auxin，abscisic acid，ethylene，salicylic acid，methyl jasmonate，spl，HSE，and other specific elements. Real-time fluorescence quantitative PCR showed that pre-miR166 S53 and mature miR166a.2 showed a down trend in the early stage of 2,4-D-regulated longan SE during the process from friable-embryogenic callus（EC）into globular embryos；however，pre-miR166 S53 and miR166a.2 showed different expression patterns in the early stage of SE without 2,4-D regulation. In addition，pre-miR166 S53 was down-regulated in EC at different concentrations of ABA and ethylene treatment，but no response to 2,4-D；miR166a.2 was down-regulated in EC under different concentrations of 2,4-D，ABA，but up-regulated under ethylene treatment. The above results suggest that pre-miR166 S53 and miR166a.2 are not simple linear correlation patterns in the response to exogenous hormone，there may be multi-level，multi-directional regulation.

Key words: Dimocarpus longan, miR166 primary, promoter, real-time fluorescence quantitative PCR, expression characteristic