小麦倒春寒响应小RNAtae-miR167d及其靶基因ARF12功能分析

Spring frost is one of the major causes of yield losses in common wheat (Triticum aestivum L.). MicroRNAs (miRNAs) are a class of small non-coding RNAs which play important roles in abiotic stresses by regulating their target genes. It is important to identify the responsive miRNAs to spring frost and its molecular mechanisms. In our previous study, we performed both small RNA and degradome analysis with samples under normal conditions and low temperature stress in wheat reproduction stage, and 39 differentially expressed miRNAs were found. RT-PCR showed the expression of tae-miR167d was down regulated after cold stress treatment, while its target gene ARF12 was up-regulated. In order to study the mechanisms of tae-miR167d responding to spring frost in common wheat, the present study is intended to analyze the relationship between expression patterns of tae-miR167d and its target gene ARF12 with the spring frost resistance. Study the relationship between the expression level of ARF12 and downstream pathway genes with the wheat spring frost resistance by tae-miR167d over-expression and RNAi. These results may provide valuable information to understand the molecular mechanisms of tae-miR167d in response to spring frost, and providing new ideas and theoretical basis for the wheat breeding.

倒春寒是造成小麦产量损失的主要灾害之一，植物miRNAs在逆境响应中发挥重要作用，分析倒春寒胁迫响应miRNAs，对研究小麦耐倒春寒的分子机理具有重要意义。我们在前期工作中，分析了在小麦生殖发育阶段低温胁迫前后小RNA和降解组的变化，发现了39个差异表达miRNAs。RT-PCR结果显示tae-miR167d冷胁迫处理后表达下调，其对应的靶基因ARF12则上调表达，二者呈现负调控关系。为深入研究tae-miR167d在小麦倒春寒响应过程中的作用机制，本项目拟研究冷胁迫下tae-miR167d及靶基因ARF12表达模式与倒春寒响应的关系，同时利用转基因技术来提高和降低tae-miR167d的表达量，分析其转基因后代中靶基因ARF12及下游级联通路上基因的表达变化与小麦耐倒春寒的关系，明确tae-miR167d响应小麦倒春寒的分子机理，为耐倒春寒小麦品种选育提供新思路和理论依据。

本研究分析了参与小麦冷响应miRNA及靶基因的调控网络，发现一些参与冷响应miRNA的靶基因同时参与花发育过程。以参与花发育过程的tae-miR167d及靶基因ARF12为研究目标，分析了两种个倒春寒耐受能力不同的小麦品种济麦22和西农889，在不同发育时期相同器官以及相同器官不同发育时期的tae-miR167d和靶基因ARF12的表达差异，发现两品种在雌雄蕊发育时期，叶鞘和茎中tae-miR167d和靶基因ARF12表达水平存在显著差异。选用8个小麦品种，分析了各品种冷胁迫前后叶片和幼穗中tae-miR167d和靶基因ARF12的表达差异。克隆了济麦22tae-miR167d的前体序列和靶基因ARF12的基因序列，分析A、B和D基因组中序列差异。根据tae-miR167d的前体序列分析结果，分别建了tae-miR167d的过表达（OE）载体和降低tae-miR167d表达的STTM载体。利用农杆菌介导的方法分别获得了tae-miR167d的OE和STTM转基因株系，并筛选出阳性植株和阴性植株分离比例为3:1的转基因株系。将获得的STTM和OE转基因株系与对照进行相比发现，STTM转基因植株表型变化不明显，而OE转基因植株的叶片夹角变大，茎秆粗壮，株高变矮。分析STTM和OE株系在正常条件下、0℃条件下处理48h和浓度为100 mg/L IAA喷施后48 h时转录组和激素表达水平差异，探讨tae-mi167d和靶基因ARF12参与调控的信号通路，解析小麦耐倒春寒过程中小RNA参与的调控机制，为小麦抗逆育种提供新的思路和理论依据。