透明种皮1（TT1）调控油菜种子发育和代谢的机理

Seed is the reproductive organ and economic organ of crops. As the size, structure, and ingredients of the seed influence the yield and quality of crops, dissecting the regulatory mechanisms of seed development and metabolism is of crucial significance. TRANSPARENT TESTA 1 (TT1) of Arabidopsis is a positive regulator of endothelium development and seed coat pigments deposition, and its mutation affects seed size too. Our previous study verified these functions of the TT1 gene family from Brassica napus, but some new important functions were revealed. It was also expressed in the embryo. Its constitutive RNAi generated seeds with thinner outer and inner integuments, lower contents of oleic acid and flavonols, as well as higher contents of linoleic acid and alpha-linolenic acid. Hence, TT1 was speculated to be a regulatory gene of multiple traits in seed development and metabolisms. In this program, research will be carried out on three aspects to dissect the regulatory mechanisms of TT1. In situ hybridization will be done, and overexpression and RNAi with constitutive, seed coat-specific, and embryo-specific promoters, respectively, will be functionally studied, which will help reveal the relationships between the seed coat traits and the embryo traits regulated by TT1. Yeast One-Hybrid and Chromatin Immunoprecipitation-Sequencing will be adopted to screen and identify important target genes of TT1. Bimolecular Fluorescence Complementation and Co-Immunoprecipitation will be done to screen and identify the co-factors interacted by TT1 during its regulation processes.

种子是作物的繁殖器官和经济器官，种子大小、结构、成分影响作物的产量和品质，研究种子发育和代谢的调控机制具有重要意义。拟南芥透明种皮1（TT1）是内种皮发育和种皮色素积累的正调控因子，且突变后种子变小。本项目组的前期研究肯定了油菜TT1具有上述功能，还揭示了一些新的重要功能：TT1在种胚中也明显表达，组成性RNA干扰后外种皮也象内种皮一样显著变薄，种子中油酸、黄酮醇显著下降，而亚油酸和亚麻酸则显著上升，推测TT1为种子发育和代谢多方面性状的调控基因。本项目将从三方面对其调控机理进行解析。开展组织原位杂交，以及组成性、种皮特异性、种胚特异性超量表达和RNA干扰，以揭示TT1调控种皮性状与种胚性状间的关系。基于酵母单杂交和染色质免疫共沉淀-测序，筛选鉴定TT1调控的重要靶基因。基于双分子荧光互补和蛋白质免疫共沉淀，筛选鉴定TT1调控时的重要互作伙伴因子。

种子是作物的繁殖器官和经济器官，种子发育和代谢影响作物的产量和品质，具有重要研究意义。拟南芥透明种皮1（TT1）编码内种皮发育和原花青素（PA）积累的正调控因子，且突变后种子变小。本项目组的前期研究肯定了甘蓝型油菜TT1（BnTT1）具有上述功能，还揭示了一些新功能：BnTT1在种胚中也明显表达，组成性RNA干扰后外种皮也象内种皮一样显著变薄，种子中油酸、黄酮醇显著下降，而亚油酸和亚麻酸则显著上升，推测BnTT1为种子发育和代谢多方面性状的调控基因，于是本项目从三方面对其新功能和调控机理进行了解析。（1）基于启动子-GUS转化拟南芥和烟草的研究，和组成性、种皮特异性、种胚特异性超量表达BnTT1基因转化油菜和拟南芥的研究，揭示了BnTT1调控种皮性状和种胚性状的新功能和二者间的关系。BnTT1同时调控脂肪酸-氧脂素复合途径和苯丙烷-PA复合途径，促进内种皮-糊粉层的生长发育与代谢，还可能间接抑制种胚的生长发育与代谢。BnTT1只有在种皮细胞中才能驱动合成PA，而在种胚中虽然能够上调PA途径一些基因的表达，但不能合成终产物PA。（2）基于DNA亲和纯化测序（DAP-seq）实验和差异转录组（RNA-seq DEGs）分析，筛选鉴定出BnTT1发挥转录调控作用的43个重点候选靶基因，酵母单杂交（Y1H）和双荧光素酶报告基因（LUC）实验点对点分子互作验证表明Bnα-DOX2是其靶基因，它通过结合Bnα-DOX2启动子上的ATAACACCTTG基序来抑制其转录表达。（3）基于酵母双杂交（Y2H）和双分子荧光互补（BiFC）实验，鉴定了BnTT1的一批互作蛋白。BnTT1与自身互作形成同源二聚体，并与类黄酮-PA途径的正调控因子BnTTG2、BnTT2、BnPAP1、BnTT16稳定互作，但与脂肪酸途径的正调控因子bHLH74、L1L、MYB118-3、bZIP67不互作，与决定胚珠发育的转录因子STK也不互作。说明BnTTG2、BnTT2、BnPAP1、BnTT16很可能是BnTT1转录调控时的伙伴因子，是BnTT1转录复合物的组分蛋白。（4）还揭示了BnTT1互作转录因子BnTTG2和一批其它基因调控种子发育与代谢的新功能和新机理。本项目是对植物界TT1和TTG2等基因功能和调控机理的深化，并促进了对植物种子发育与代谢调控机理、种皮与种胚互调关系的认识。