油菜抗倒伏主效MYB转录因子的性状鉴定和调控机理研究

The rapeseed lodging happens frequently, which not only reduces the yield, but also affects the seeds quality. Until now, some researches for the rapeseed lodging resistance were focus on the morphological, physiological, biochemical and molecular marker. It is very important for the rapeseed lodging research by means of molecular mechanism analysis, function gene cloning and so on. It is well known that the MYB genes associated with the rapeseed lodging resistance by effects the secondary wall thicken and lignin accumulation. Our research include: 1) Identify the root and stem specified or up regulated MYB gene by differential expression analysis among the different plant tissues or lodging/(lodging resistant) plants in Brassica napus. 2) Clone the full length target MYB gene, subcellular localization and construct the over-expression and RNA interference vectors to transform B. napus followed by the PCR, RT-PCR identification and pollen culture to select the homozygous transgenic lines. Identify characters of lodging and Sclerotinia sclerotiorm resistant of target genes, and speculate the molecular mechanism. 3) Study on binding proteins of target genes by yeast one hybrid, two-dimensional electrophoresis, mass spectrometer, and speculate the regulation mechanism and the relationship between lodging resistance and disease resistance. The results of this research provides valuable information to obtain a new path to improve the ability of lodging resistance or disease resistance of Brassica napus.

油菜倒伏常年多发，严重降低产量，影响菜籽品质。迄今仅限于形态、生理、生化、经典遗传、分子标记等不多的研究。开展油菜抗倒伏调控机理解析、功能基因克隆等研究意义重要且紧迫。鉴于抗倒性与维管束发育、次生壁加厚相关，而MYB转录因子主要影响次生壁加厚和木质素积累，研究主要内容有：1)基于电子克隆、器官组织间和抗倒/倒伏材料间的RT-PCR，以及根、茎秆切片RNA原位杂交，鉴定油菜根、茎秆中木质部优势或特异表达、与抗倒伏密切相关的候选基因1-2个；2)克隆目的基因全长，亚细胞定位后，构建超量表达和RNA干扰载体，遗传转化油菜并通过分子检测，花粉培养后获得纯合的目标基因表达的优株系；抗倒伏、抗病性状等功能验证，推测分子调控机理。3）酵母单杂交、双向电泳、质谱分析，进行蛋白质组水平研究，推测调控机理及抗病和抗倒关系。研究结果可为获得一条新的油菜高抗倒伏或抗病能力的路径提供有价值的信息。

倒伏是影响我国油菜生产的重要因素之一，不仅严重降低油菜产量，同时也影响菜籽品质和收获指数。油菜倒伏分为根倒和茎倒，倒伏与茎秆木质素排列、茎机械程度、细胞细胞壁厚度、根系发育等都有关。而MYB转录因子主要影响次生壁加厚和木质素积累，因而我们筛选了和油菜根、茎秆中木质部优势或特异表达、与抗倒伏密切相关的候选基因1-2个，克隆目的基因全长，亚细胞定位细胞核表达后，构建超量表达和RNA干扰载体，遗传转化油菜并通过分子检测，自交3-4代后检测获得纯合的目标基因表达的优株系；并验证了抗倒伏、抗病性状等功能，通过转录组测序、酵母双杂、关键基因表达结果确定了基因的调控机理。 .研究结果：1.克隆BnMYB69和BnMYB43的gDNA、cDNA 分别各四条基因序列，生物学信息学分析分别与AtMYB基因同源性很高，亲缘关系较近，都在茎表达较高。逆境胁迫发现其成员对其有强烈响应。2.建立反义和正义载体，遗传转化油菜分别获得BnMYB69和BnMYB43的RNAi干扰植株，考察BnMYB69抑制表达转基因植株及后代农艺性状发现地上部分植株变矮，茎增粗，叶片、叶夹角增大，节数降低，抗折力、种子单株粒重、单株角果数、种子大小、生物产量下降。BnMYB43抑制表达植株其株形矮小，茎纤细，生物产量、根冠比下降。3.细胞学观察BnMYB69干扰植株发现叶片表皮毛增加，细胞壁变薄，细胞增大，木质部减少，须根增加，根基部增粗，根细胞学观察其木质部也有明显变化，测试其纤维素、木质素、果胶都有显著降低，BnMYB43干扰后木质素、细胞数量减少， 纤维素降低而果胶含量上升，BnMYB69抑制植株中IAA含量、过氧化氢含量、还原性糖含量显著降低、ABA含量、过氧化氢酶含量增加。4.关键基因表达分析木质素途径、IAA合成途径、纤维素代谢途径关键基因下调；菌核病抗性分析发现BnMYB69和BnMYB43与抗病性有直接相关关系。5.通过IAA处理BnMYB69干扰植株发现叶绿素代谢直接由光合反应影响，而株高由IAA含量影响，转录组和关键基因表达分析BnMYB69通过影响磷酸戊糖途径、莽草酸代谢、色氨酸代谢、苯丙氨酸代谢这些途径的IAA合成、木质素合成、花青素合成从而影响油菜植株的生长发育、株型及抗性。整个研究信息提供了一条新的油菜抗倒伏、抗病性基因功能研究及机理分析的途径，具有科学价值。