梅PmmiR319a与靶基因PmTCP2的验证与表达分析

Identification and Expression Character Analysis of PmmiR319a and Target Gene PmTCP2 in Prunus mume

为验证PmmiR319a与PmTCP2基因的靶标关系,并研究PmmiR319a在梅花芽发育过程中的作用,本研究以梅品种‘大嵌蒂’为试验材料,克隆梅PmmiR319a前体序列及其靶基因PmTCP2,采用5'RACE技术对两者之间的靶标关系进行验证,并进一步分析在花芽发育过程中的表达模式。结果表明,梅PmmiR319a前体序列含有茎环结构,通过序列比对和系统发育分析发现,不同物种的miR319a前体序列的茎环发卡结构和成熟体区域表现出较高的保守性。梅PmTCP2基因全长1 500 bp,编码499个氨基酸。荧光定量PCR分析结果表明,PmmiR319a在梅花发育的前期表达量高,而PmTCP2在梅花发育的中期和后期表达量较高,PmmiR319a与预测靶基因PmTCP2的表达模式呈负相关,表明二者存在负调控的关系,与预测的靶标关系一致。RLM-5'RACE技术验证结果表明,PmmiR319a对其靶基因PmTCP2的mRNA进行了切割,切割位点位于第10和第11个碱基之间。由此可知,PmmiR319a通过调控靶基因PmTCP2影响花的发育。本研究结果为阐明梅花中miR319a的功能提供了一定的理论依据。

In order to verify the relationship between PmmiR319 a and its predicted target gene PmTCP2,and study the role of PmmiR319 a in flower development. The Prunus mume cultivar Daqiandi was used as the experimental material to isolate the Pmm TR319 a precursor sequence and the predicted target gene PmTCP2,and their expression during the flower bud development were analyzed. The relationship between PmmiR319 a and its target gene PmTCP2 was verified with RLM-5＇RACE. The results indicated that the PmmiR319 a precursor sequence contained stem and loop structure and the sequence alignment and phylogenetic analysis of PmmiR319 a with miR319 a of other species showed that the sequences of miR319 a precursor were different,and the mature sequences of miR319 a were very conservative. The PmTCP2 gene was 1 500 bp in length and encodes a protein of 499 amino acids. Real-time PCR analysis indicated that PmmiR319 a was highly expressed in the early stage of flower bud development in Prunus mume,while PmTCP2 was higher in the middle and late stages,and the expression of PmmiR319 a was negatively correlated with the expression of the predicted target gene PmTCP2 expression. PmmiR319 a guided-cleavage and target site for the putative PmTCP2 mRNA was validated using RLM-5＇ RACE. The cleavage site was between the 10 thand 11 thbase of its interaction region. These results indicated that PmmiR319 a plays a role in flower bud development by regulating the target gene PmTCP2. The results of study provide a theoretical basis for the function of miR319 a to the development of Prumes mume