白菜花药发育特异基因msLTP的表达调控及其调节发育的分子机制

Genetic regulation of anther development plays vital role in plant reproductive development. In our previous study of a genome-wide transcriptional profiling of the flower buds of the male meiotic cytokinesis (mmc) mutant and its wild type of Brassica campestris L. ssp. Chinensis, we identified a novel anther-specific gene msLTP that is silenced as the result of the mutation of MMC gene. msLTP is exclusively expressed in the fertile line, its down-regulation by RNAi results in the aberrant development of anther and pollen. We also have isolated the promoter of msLTP by TAIL-PCR and named it as BC15. Intriguingly, the expression of Bc15: :GUS can only be detected in anthers. We therefore suggest that msLTP is a novel crucial player in the expressional and regulatory network of anther development. However, the precise regulatory mechanism of msLTP expression is largely unknown. In this proposal, firstly, we planned to identify the anther-specific and cis regulatory elements in the Bc15 by using a series of 5' flanking sequence deletion mutation and site-directed mutagenesis. Then, we will employ yeast one hybrid system combined with EMSA (electrophoretic mobility shift assay) and subcellular localization method to identify transcription factors that may interact with Bc15. The selected transcription factors will be verified in both over-expression and silencing transgenic lines by analysis of msLTP expression. Thirdly, we will use ChIP-Seq and target gene sequencing and real-time RT-PCR to elucidate the transcription factor binding sites within Bc15 and the target genes on chromatin level. Finally, we are going to study the cooperative regulatory effect of Bc15 and transcription factors on msLTP expression and the consequent role in the regulation of anther development and pollen formation. The Success of this study may not only provide a new mechanism of the anther-and pollen-specific regulatory networks in Brassica campestris ssp. Chinensis,but also have a potential application in the realization of breeding for heterosis and quality breeding in Chinese Cabbage and other crops.

花药发育关键基因及其调控网络是植物生殖发育调控机制的重要内容。我们在白菜中发现了减数分裂胞质分裂突变中表达沉默的基因msLTP，该基因在可育系花蕾中特异表达，其表达沉默则导致花药和花粉的形态及功能异常，我们推测msLTP是调控白菜花药发育的新基因，但其表达及沉默的分子机制尚不明确。用TAIL-PCR法我们克隆到msLTP的启动子Bc15，GUS表达等方法证明Bc15在花药中特异表达。本课题首先通过缺失突变及点突变等方法明确Bc15的关键调控序列；再以酵母单杂交和凝胶迁移等方法分析其转录调控因子；用过表达和干涉的方法研究转录因子对msLTP的表达调控；用ChIP-seq技术在染色质水平研究这些转录因子对靶基因的表达调控作用。阐明msLTP的表达调控及其在花药发育和花粉形成过程中的分子机制，为白菜花药发育的表达调控网络提供新的理论支撑，并在白菜等蔬菜作物杂种优势育种中有潜在的应用价值。

利用生物信息学在线工具、系列缺失表达载体结合基金枪轰击洋葱表皮细胞及拟南芥中的稳定表达分析，发现msLTP的启动子Bc15序列中包含花粉发育的重要调节序列如GGTT和GTGA元件，-1~ - 131bp的5‘侧翼序列区段已足以驱动报告基因GUS的表达，而-1~-731区段GUS活性最强。启动子Bc15驱动的GUS基因在花器官中从阶段6开始一直持续表达到成熟花蕾（阶段15）的花药中，树脂切片分析结果进一步证实了Bc15显著地表达在花药中。.酵母单杂交文库的构建及筛选结果显示：共筛选到40个阳性克隆，编码了37种蛋白。经测序比对结合DNA结合结构及功能域的分析，筛选出可能与启动子Bc15结合的5个蛋白：编号为29的E3泛素蛋白连接酶、编号为30的E3泛素蛋白连接酶、编号为35的多胺氧化酶、编号为40的钙调素及编号为47的果胶酸裂合酶。通过CDS基因全长的克隆，过表达、反义、敲除载体转化菜心，分析四个调控蛋白基因（29, 30, 40, 47）的功能鉴定及转化植株中msLTP的表达模式。利用农杆菌介导的烟草体内瞬时表达体系，四个基因的亚细胞定位分析发现29定位于细胞核和细胞质、30定位于细胞核、40, 47定位于细胞膜。利用烟草瞬时表达体系进行四个基因的双荧光素酶检测，验证四个调控蛋白基因与Bc15启动子之间的互作。结果表明47对Bc15启动子活性无明显调控作用， 29、30（E3泛素蛋白连接酶）及40（钙调素）能负调控Bc15启动子活性。.新增了雪里蕻和甘蓝中LTP基因的克隆与表达分析，发现它们可能与花粉发育有关。此外项目组在研究过程中发现植物ß-半乳糖苷酶和植物纤维素合酶基因与植物雄性不育密切相关。.项目的研究结果为白菜花药或花粉发育特异基因表达调控网络构建提供新的理论支撑，也为在白菜等蔬菜作物生产中利用杂种优势来提高作物的产量和品质奠定了基础。