甘蓝型油菜生长素极性运输异常导致茎秆弯曲的分子机制

Lodging is a key factor in reducing seed yield and quality and limiting the mechanized production of rapeseed (Brassica napus). In previous study, a stem lodging mutant with stem bending phenotype at the end of bud stage has been identified and named stb1. Genetic analysis demonstrated that the mutant was controlled by a single autosomal recessive gene, which has been mapped to a 1.995-Mb region of chromosome A01. RNA-Seq analysis showed that genes involved in polar auxin transport were significantly down-regulated in the stem of stb1 mutant. We proposed that the stem bending phenotype of stb1 mutant was probably caused by the slower velocity of polar auxin transport than the elongation speed of stem, which leading to abnormalities in auxin accumulation and distribution. To validate the hypothesis, the F2 segregating population will be constructed and used for map-based cloning of STB1 gene. Quantitative reverse transcription-PCR (qRT-PCR), in situ hybridization and promoter analysis will be used to reveal the temporal and spatial expression patterns of this gene. The biological function of STB1 will be elucidated by further studies on the complement plants, STB1-silencing and -overexpressing transgenic plants. The downstream genes regulated by STB1 will be screened with RNA-Seq, ChIP-Seq or RIP-Seq analysis. Proteins interacting with STB1 will be fished out by immunoprecipitation combined with mass spectrometry analysis and validated by yeast-two hybrid system. The STB1-centric regulatory network of the velocity of polar auxin transport will be summarized based on abovementioned studies. Our researches not only lay a theoretical foundation for the lodging resistance breeding of rapeseed, but also provide a novel research strategy and model for investigation on the molecular regulatory mechanism of the velocity of polar auxin transport.

倒伏是影响油菜产量、品质和机械化的关键因素。本项目前期发现一个蕾薹末期茎秆弯曲的茎倒突变体stb1；分析表明其受常染色体上的隐性单基因控制，定位于A01染色体的1.995 Mb范围内；RNA-Seq分析表明stb1茎秆中生长素极性运输相关基因显著下调。我们推测stb1的曲茎表型可能与生长素极性运输速率低于新生茎秆伸长速率，导致生长素异常积累和分布有关。本项目将继续构建F2精细定位群体，图位克隆STB1基因；通过qRT-PCR、原位杂交和启动子分析，明确STB1的表达模式；对转基因植株进行深入研究以解析STB1基因功能；RNA-Seq、ChIP-Seq或RIP-Seq鉴定下游调控基因；免疫共沉淀结合质谱分析及酵母双杂交筛选互作蛋白，构建以STB1基因为核心的生长素极性运输速率调控网络。研究结果将为油菜抗倒育种提供分子理论基础，为生长素极性运输速率调控机制研究提供新的研究思路和模型。

本项目对曲茎突变体stb1和ZS11进行了表型、组织化学染色和扫描电镜比较分析，比较了stb1茎尖生长点和弯曲茎秆以及ZS11的茎尖生长点和正常茎秆的转录组数据，结合STB1基因的QTL定位区间，确定STB1候选基因。随后对候选基因进行克隆，构建过表达和干扰载体，转化拟南芥和油菜进行功能验证。研究结果为挖掘曲茎性状控制基因，阐明生长素极性运输（polar auxin transport，PAT）与茎秆弯曲表型的关系，揭示油菜曲茎性状的分子调控机制。主要研究结果如下：.表型观察结果显示，stb1突变体在初花期弯曲性状最为明显，盛花期重新恢复直立生长。stb1突变体株高、每角果粒数、角果长和抗倒伏性均显著性降低，导致突变体产量显著降低。SEM扫描电镜观察和组织化学染色显示，stb1茎秆木质部、导管数量和薄壁细胞发达程度远低于对照ZS11；内茎中负责生长素运输的维管系统没有外茎发达。ZS11、突变体stb1的茎尖生长点和茎秆转录组比较发现，下调基因主要集中在细胞过程，生物合成过程，辅酶代谢过程和生长素运输过程。筛选得到与PAT相关的下调基因28个，结合精细定位标记区间和项目组筛选的另外两个以EMS诱变获得的曲茎突变体stb2和stb3的三组RNA-seq结果分析，stb1精细定位区间内有2个与生长素合成、信号转导和极性运输相关的差异表达候选基因，最终，将BnSIP3和BnPAP2确定为STB1的候选基因。.候选基因亚细胞定位发现BnPAP2定位于细胞核内，BnSIP3同时定位在细胞核和细胞膜。对BnPAP2过表达载体转基因油菜田间表型进行观察，发现互补植株仍然发生弯曲，确定BnPAP2不是STB1候选基因。利用表型观察、外源激素处理、打顶处理和qRT-PCR检测等方法对BnSIP3的T3代转基因拟南芥进行分析发现，BnSIP3更可能与曲茎性状有关。酵母双杂发现CIPK6可与F-BOX蛋白互作。qRT-PCR发现PIN1的表达量与SIP3呈正相关，大部分的PIN蛋白存在于质膜，结合候选基因亚细胞定位结果猜想突变体植株中BnSIP3在油菜中编码形成的CIPK6蛋白，在细胞膜上通过与F-BOX PROTEIN互作调控PIN1的表达量，以实现对PAT调控。