油菜miR1140靶基因鉴定及其调控油菜分枝的生理功能

Branch is the important indicator of the Brassica napus plant type traits,expression of the gene related to branches determines the plant branching phenotype. Preliminary studies show that miR1140 expressed in the leaf axils and petioles of Brassica napus, overexpression of miR1140 perform double main inflorescence, speculate that miR1140 involved in the development of axillary meristems by regulating the expression of its target genes, thus affecting the generation of branching. But miR1140 targets is still unknown, the project is planed to discover the targets of miR1140 by deep sequencing the fragments of miRNA mediated mRNA degradation; with RACE to confirme miR1140-directed cleavage and detect changes of target transcripts, and analysis consistency in both spatial and temporal expression; construct miR1140 target vectors and RNAi vectors and obtain the positive transgenic Brassica napus plants; analysis the role of miR1140 and its targets in the development of Brassica napus branch by studying on the expression of miR1140 targets, cell morphology and physiologic changes among the different transformants and wild plants, to state molecular mechanisms of miR1140 regularating branch development, which can provide the theoretical basis for Brassica napus plant type breeding.

分枝是油菜株型性状中重要的指标，分枝相关基因的时空表达决定了植株分枝表型。前期研究表明，油菜miR1140在油菜体内主要在叶腋及叶柄表达，过表达miR1140油菜株系表现为双主序，推测miR1140通过调控其靶基因的表达参与了叶腋分生组织的发育，从而影响分枝的产生。但是miR1140的靶基因还未知，本项目拟通过对miRNA介导mRNA降解的剪切片段进行深度测序分析，找到miR1140的靶基因；用RACE验证靶基因在miR1140识别位点处被切割的状况，检测靶基因转录本的变化，并分析二者在时空上的一致性；在此基础上构建靶基因过表达及RNAi载体转化油菜，通过对不同转化体及野生型植株中靶基因表达、细胞形态学及生理学指标变化研究，分析miR1140及其靶基因在油菜分枝发育中的作用，阐明miR1140调控分枝发育的分子基础，为油菜株型育种中的应用提供依据。

miRNAs(microRNA)是一类在进化上保守的长约21nt的非编码单链RNA，在基因表达调控的网络中处于核心位置，高效的调控其靶基因的翻译或表达，在生物体生长发育、抵抗生物或者非生物胁迫以及在生物体代谢途径中起着非常重要的作用。本研究对甘蓝型油菜(Brassica napus)栽培种Westar材料进行小RNA测序及降解组分析，鉴定了芸薹属独特的miRNA家族Bna-miR1140的靶标基因，并进行了其靶标的全长克隆和植物表达载体构建。为了进一步研究Bna-miR1140的功能，人工合成了Bna-miR1140的短串联靶标(short tandem target mimic, STTM)，构建了油菜miR1140的干扰载体pBWA(V)HS-STTM1140(d)。通过将构建的Bna-miR1140植物过表达载体35S::Bna-miR1140和干扰载体pBWA(V)HS-STTM1140(d)转化油菜，研究了Bna-miR1140表达调控机制。.主要结果如下：.1.通过油菜miRNA测序和降解组分析，找到了Bna-miR1140的靶基因为一种糖基转移酶（glycosyltransferase）和响应调节因子（Arabidopsis response regulator ARR8-like protein）。.2.扩增获得了油菜Bna-miR1140基因前体片段，构建了油菜Bna-miR1140基因植物过表达载体，通过农杆菌介导侵染油菜子叶柄法转化过表达载体35S::Bna-miR1140，获得5株转基因阳性株系，过表达Bna-miR1140油菜株的株高、主花序长度、主花序结角数和千粒重都与对照相当，而分枝部位较对照低，分枝数增多，致使全株有效角果数增多，从而使单株生产力较对照高26%。调查了株型特异的5个株系T1代表型分离情况，经卡方检验发现其中有4个株系的株型变异遗传符合3:1的孟德尔分离规律。.3.通过5’RACE和3’RACE进行油菜Bna-miR1140靶标糖基转移酶基因（glycosyltransferase）全长的克隆，获得油菜Bna-gla基因全长为2039bp，该序列具有完整的读码框，长度1563bp。在gene bank 中已进行登记（Accession: KP100641.1），并构建了35S::Bna-gla的植物表达载体。