关键词: 梨, novel_miRNA, 克隆鉴定, RNAfold, qRT-PCR

Abstract:

Differentially expressed novel miRNAs related to calyx persistence were identified in a previous experiment using small RNA（Illumina） sequencing. The expression of the novel miRNAs ranked among the top 20 in the small RNA sequencing. There were significant differences in novel miRNA expression between samples with a deciduous calyx and those with a persistent calyx and between ovaries and sepals. The miRNAs were cloned using stem-loop methods and then qRT-PCR analysis was conducted. The target genes of mature novel miRNAs were predicted and the secondary structures of pre-miRNAs were analyzed. The mature sequences of nine novel miRNAs and the qRT-PCR expression of four novel miRNAs were consistent with the results of small RNA sequencing. Many functional target genes were predicted. Novel miRNAs were cloned and their structure was analyzed. This work lays the foundation for further elucidation of the mechanism by which miRNA regulates calyx persistence.

Key words: pear, novel_miRNA, clone and identification, RNA fold, qRT-PCR