BnbHLH140基因在甘蓝型油菜株型调控中的功能研究

The plant architecture traits of Brassica napus are closely related to rapeseed yield, and it is one of the significant objectives of genetic improvement breeding of rapeseed. In our previous study, we identified a differentially expressed candidate gene BnbHLH140 from the RNA-Seq analysis of the resynthesized B. napus with dramatically increased branches and the natural B. napus plants. Overexpressing BnbHLH140 in B. napus resulted in increased main stems, therefore we assumed that BnbHLH140 might participate in the regulation of rapeseed plant architecture. On this basis, transgenic functional confirmation of BnbHLH140 will be carried out through genetic transformation of the overexpression and CRISPR/Cas9 constructs in B. napus. We will clarify the biological function of BnbHLH140 in plant architecture regulation by performing phenotype identification, histological analysis, and phytohormone content determination of the transgenic plants. Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) and RNA-Seq analysis will be employed to investigate the downstream regulation network of BnbHLH140. Yeast two-hybrid screening, bimolecular fluorescence complementation (BiFC) assays, and co-immunoprecipitation (Co-IP) will be applied to explore and verify the interaction protein of BnbHLH140. Yeast one-hybrid and electrophoretic mobility shift assay (EMSA) will be used to identify the potential upstream regulators of BnbHLH140. The findings will elucidate the molecular mechanism and networks of BnbHLH140 in rapeseed plant architecture regulation. This study will provide theoretical foundation for plant architecture improvement and genetic breeding in rapeseed, thus this work is of great significance in theory and practice.

甘蓝型油菜的株型性状与产量密切相关，是油菜遗传改良育种的重要目标之一。在前期研究中，我们从人工合成多分枝甘蓝型油菜和正常对照的RNA-Seq结果中鉴定出一个差异表达基因BnbHLH140。在甘蓝型油菜中过表达该基因导致转基因植株主茎增多，推测BnbHLH140参与油菜株型调控。本项目拟通过过表达和CRISPR/Cas9敲除对其进行转基因功能验证，对转基因材料进行表型观察、组织学分析和激素含量测定，阐明BnbHLH140在株型调控中的生物学功能；利用ChIP-Seq和RNA-Seq分析BnbHLH140下游调控网络。通过Y2H、BiFC、Co-IP等技术筛选鉴定BnbHLH140互作蛋白，利用Y1H和EMSA等手段鉴定BnbHLH140的上游调控因子，解析BnbHLH140在控制油菜株型中的分子机制和调控网络。研究结果能够为油菜株型改良和遗传育种提供理论依据，具有重要的理论意义和应用价值。

甘蓝型油菜的株型性状是影响油菜产量的关键因素之一，改良株型是油菜增产的有效途径，然而，油菜株型调控的分子机制尚不明确。因此，鉴定油菜株型调控基因有助于理解油菜株型调控的遗传网络和分子机理，促进甘蓝型油菜株型性状的遗传改良。项目前期工作发现BnbHLH140在多分枝甘蓝型油菜材料和对照中显著差异表达，在油菜中过表达该基因使油菜株型发生改变。本项目在此基础上对该基因创建了相关遗传材料并进行转基因功能分析，阐明其在油菜株型调控中的生物学功能。本研究发现BnbHLH140在甘蓝型油菜茎顶端分生组织中特异表达，在油菜中过量表达BnbHLH140能够增加油菜的主茎数目，提高油菜的经济产量。烟草瞬时表达分析结果表明，BnbHLH140定位于细胞核中。酵母生化分析结果显示，BnbHLH140全长蛋白不具有转录激活活性。通过酵母双杂交和双分子荧光互补实验证实BnbHLH140能够与HAT2、PUCHI、LAX2等蛋白互作。利用RNA-Seq对BnbHLH140下游基因进行鉴定，发现大量生长素和细胞分裂素生物合成与信号转导相关基因的表达水平在BnbHLH140-OE植株中发生了显著变化，YUC1、YUC6、YUC8、YUC9等生长素生物合成相关基因上调表达，而大部分细胞分裂素生物合成相关基因下调表达，说明过表达BnbHLH140基因能够通过影响生长素和细胞分裂素的合成以及信号转导过程，从而调控油菜顶端分生组织的形成，进而影响油菜株型。此外，研究发现，BnbHLH140可能通过与2个ERF基因启动子区域结合从而使其表达上升。本研究阐明了转录因子BnbHLH140在甘蓝型油菜株型调控中的作用，初步揭示了BnbHLH140通过影响生长素和细胞分裂素等植物激素的生物合成和信号转导，从而影响油菜的株型建成，为解析油菜株型调控的分子机制提供了理论基础。