桑树中miR319的克隆及在雄花发育中表达分析

为探究miR319及靶基因在调控桑树雄花发育的分子机制,本研究通过石蜡切片技术判断桑树雄花芽发育时期及在线网站预测miR319的靶基因。结果表明,桑树雄花芽发育分为未分化期(A1)、分化初期(A2)、花序分化期(A3)和总苞形成期(A4)4个不同的阶段;桑树中miR319家族存在miR319a、miR319b和miR319c 3个成员,其中miR319b与miR319c位于同一条前体序列上,预测并获得了miR319 6个靶基因。运用5'-RACE技术验证了miR319a的4个靶基因,其切割位点主要集中在与miRNA互补位点的第10和第11位碱基之间。采用RT-qPCR技术检测miR319a及靶基因在雄花发育不同阶段的表达水平,发现miR319a在雄花发育A2-A3阶段表达水平迅速降低,靶基因Morus012208、Morus008229、Morus008100与Morus005893在此阶段表达水平迅速升高,茉莉酸(JA)含量也迅速升高。同时,JA合成关键基因PLDα1和LOX5在雄花发育A2-A3阶段表达上调,表明miR319通过调控靶基因影响JA合成来调控桑树花序分化。本研究为揭示桑树miR319在JA途径中调控花发育中的分子调控机制奠定了一定的理论基础。

Cloning of miR319 in Mulberry and Its Expression Analysis in Male Flower Development

To explore the molecular mechanism of miR319 and its target genes during male flower development in mulberry, histological technology was used to analyze the stage of flower bud development and the target genes of miR319 were predicted through on-line software. We found that make flower development under went 4 different stages: undifferentiated period (A1), early differentiation stage (A2), inflorescence differentiation period (A3) and total pod formation period (A4). Three miR319 members (miR319a、b、c) were found in mulberry genome, miR319b and miR319c were located on the same precursor sequence. A total of 6 target genes were found to be the targets of miR319 in mulberry. Which were further validated via 5’-RACE, and the cleavage site was between the 10^th and 11^th base. RT-qPCR was used to detect the expression of miR319 and it’s targets during male flower development. The expression level of miR319 reduced rapidly during A2-A3 stage, and target genes (Morus012208, Morus008229, Morus008100 and Morus005893) showed the opposite expression pattern. Meanwhile, the content of JA increased rapidly in this stage. JA synthetic component genes (PLDα1 and LOX5) also increased rapidly in the stage. It showed that miR319 regulated the male flower development through influencing JA synthesis pathway by regulating the target gene. This study can help to reveal the molecular regulation mechanism of miR319 regulating JA pathway during flower development.