转录因子BnMYB46对甘蓝型油菜角质层蜡质沉积的作用与调控机制研究

As the outmost surface of plants, cuticular wax is related to the adaptation of Brassica napus to biotic and abiotic stresses. However, the key genes regulating cuticular wax deposition in B. napus and their acting mechanism are still unclear. In our previous studies, using Genome-Wide Association Studies (GWAS), a candidate transcription factor regulating cuticular wax deposition in B. napus, BnMYB46, was obtained. Results of subcellular localization showed that BnMYB46 protein was located in the cell nucleus. The transcription level of BnMYB46 was related to cuticular wax deposition in B. napus. Furthermore, the promoter regions of many of the cuticular wax biosynthetic genes contain MYB46-binding cis-regulatory motif M46RE, suggesting that MYB46 directly regulates the genes involved in the biosynthesis of the cuticular wax compositions. In order to reveal the function and molecular mechanism of BnMYB46 as a central regulator for cuticular wax deposition in B. napus, we plan to 1) analyze the role of BnMYB46 in cuticular wax deposition by up regulating and down regulating the expression of BnMYB46 in plant; 2) identify the target genes of BnMYB46 by RNA-seq; 3) confirm that BnMYB46 directly binds the promoter of the target genes and activates the transcription of the cuticular wax biosynthetic genes. The promoter of BnMYB46 will be furtherly cloned and the upstream transcription factor regulating BnMYB46 will be screened. Our aim is to elucidate the transcriptional regulation network and the function of BnMYB46 that controls the cuticular wax in B. napus. The results of the research will provide theoretic foundation and gene resources in increasing the tolerance of B. napus to stresses through genetic manipulation methods.

角质层蜡质与甘蓝型油菜抗（耐）逆性密切相关，但目前控制油菜角质层蜡质沉积的调控因子和作用机制还不清楚。我们利用GWAS从全基因组水平筛选到一个调控甘蓝型油菜角质层蜡质沉积的转录因子BnMYB46，亚细胞定位表明BnMYB46具有核定位功能，其转录水平与甘蓝型油菜叶片蜡质沉积相关；蜡质相关基因启动子分析表明含有MYB46特异性应答元件M46RE，可能被BnMYB46所识别。为了揭示BnMYB46在甘蓝型油菜蜡质沉积中的功能与作用机制，本项目拟通过上调和下调BnMYB46的表达，分析其在蜡质沉积中的作用；进而利用转录组分析明确其调控的靶基因，确定其与靶基因启动子的结合特性及其对靶基因的调控活性；进一步分离BnMYB46基因的启动子，筛选调控BnMYB46的转录因子，从而阐明BnMYB46的上下游调控网络及其在蜡质沉积中的作用和调控机制。研究结果将为提高油菜抗（耐）逆性提供理论依据和基因资源。

角质层蜡质与甘蓝型油菜抗（耐）逆性密切相关，但目前控制油菜角质层蜡质沉积的调控因子和作用机制还不清楚。本研究针对甘蓝型油菜角质层蜡质候选调控因子BnMYB46展开研究，通过基因表达模式分析、转基因功能研究、转录组及DNA与蛋白质互作分析，明确了BnMYB46在甘蓝型油菜蜡质沉积中的功能，初步揭示了BnMYB46调控甘蓝型油菜角质层蜡质沉积的分子机制。主要结果如下：1）BnMYB46主要在木质化程度高的组织器官中表达，其中在种子中的表达量最高，角果与根次之，在花中的表达较低，在高蜡油菜叶表皮中的表达显著高于低蜡材料，MeJA、Nacl和干旱处理后，BnMYB46的表达显著上调，低温处理后显著下调；2）在油菜中过表达BnMYB46，蜡质组分中烷、醛、一级醇以及酮含量显著增加，其中C27-C31烷、C28醛、C29醛等显著高于对照植株；BnMYB46过表达也增加了角质总量，其中十六碳脂肪酸（16：0）含量显著增加；水分损失率测定结果表明，过表达植株的角质层渗透性减弱；3）结合转录组分析与ChIP实验确定了在蜡质生物合成途径中受BnMYB46作用的靶基因，揭示了BnMYB46可能通过调控BnRAP2.4、MYB106等进而调控BnCER1、BnKCS1等蜡质基因的表达，最终影响甘蓝型油菜的蜡质沉积；4）构建了油菜叶表皮cDNA文库，利用酵母单杂交初步筛选获得6个BnMYB46上游调控因子，这些基因参与了细胞壁特性调控、花粉萌发、次生壁生物合成、脂类转运、表皮发育等生物过程。

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