小麦特异小分子RNA TamiR2001及其目标基因TR的分离克隆及功能鉴定

MiRNAs are single-stranded non-coding RNAs with sizes most often in the range of 20–24 nucleotides and play very important roles in morphogenesis, development, flowering time and stress resistance by degradating target mRNAs or repressing translation targeted genes on posttranscriptional level in plant. In our previous study, wheat miRNAs including conserved in other species and wheat specific miRNAs have been identified from various tissues. One of the wheat specific miRNAs is TamiR2001 which has been proved to be response to abiotic and biotic stress and express differently in various tissues. This project aims to clarify the biological function of wheat specific miRNA TamiR2001 and its targeted genes which is predicted to encode tropinone reductase family (TR) in respect of development and stress resistance. Firstly, we are going to identify and clone the putative target genes regulated by TamiR2001; secondly, the expression pattern of TamiR2001 and its target genes across varoius tissues during wheat development will be examined by Northern or qRT-PCR. Moreover, VIGS and transgenic technology will be used to analyze the biological function of TamiR2001 and its target genes.

miRNA是长度为21-24 nt的非编码RNA，它通过调控目标基因的转录和翻译在植物生长发育和逆境防御等生命过程中发挥非常重要的作用。申请人前期筛选出一个小麦特异的小分子RNA TamiR2001，不仅表达丰度较高，而且在不同组织不同逆境中差异表达，预测其候选目标基因可能编码次生代谢有关的莨菪酮还原酶（TR），本研究在此基础上提出开展TamiR2001对其目标基因的表达调控机制及生物学功能研究。首先获得受TamiR2001调控的目标基因，明确TamiR2001对目标基因转录本的剪切抑制调控模式，系统查明TamiR2001及其调控目标基因在小麦不同发育时期、不同组织器官、高温、干旱和白粉菌胁迫下的表达特点；并采用VIGS技术和转基因策略完成小麦TamiR2001及其目标基因在发育和抗逆方面生物学功能鉴定。

已有的研究表明植物miRNA通过调控靶基因参与多种胁迫应答，尤其是真菌诱导后很多非保守的miRNA表达水平发生改变，而且利用转基因技术改变植物体内miRNA表达水平后引发了植物抗病敏感性的改变。本研究首先克隆了一个小麦特异的miRNA TamiR2001前体序列，利用生物信息学预测到了两个候选目标基因，后续5' RACE和烟草瞬时表达实验证明TamiR2001在翻译水平调控TaFBL的表达。在不同小麦器官中考察了TamiR2001及TaFBL的表达量，发现TamiR2001在旗叶、幼穗和发育种子中表达量较高，而TaFBL则在根、穗下节中表达量较高。TamiR2001在小麦幼苗中受条锈菌诱导，而且VIGS实验证明TaFBL沉默后植物抗病性降低，转基因小麦初步结果显示TamiR2001-TaFBL调控途径参与了植物抗病性响应。本研究有助于理解miRNA调控途径在植物响应逆境胁迫过程中发挥的作用，为进一步利用miRNA途径改良作物性状提供参考。