外源生长素类物质毒莠定（PIC）调控百子莲胚性细胞成胚潜力的分子机制

Picloram (PIC), as a plant growth regulator similar to auxin, plays an essential role in the transition of Agapanthus somatic cell to embryonic cells and the maintenance of the embryogenic potential in long-term subculture of somatic embryogenesis. It has been proved that the lose of embryogenic potentials is a key scientific problem, however, its mechanism has been uncleared. In the present proposal, the samples include callus, EC (Embryonic callus) and non-embryogenic callus(NEC) directly induced on medium supplemented with from 1.0 to 2.0mg.L-1PIC after subculture for three to six months. Cell morphological features and the numbers of somatic embryos induced from one gram of fresh weigh of EC will be used for evaluation of EP. In order to clarify the physiological and biochemical mechanism of ROS stress and programmed cell death (PCD) on EP of EC, the contents and distributions of endogenous hormones (IAA, ABA and ethylene), the changes in energy metabolism and polar structures of EC will be analyzed using HPLC-MS, laser confocal scanning microscope（LSCM) and immunohistochemical localization, respectively. Based on association analysis of DNA methylation, miRNA sequencing, RNA sequencing and iTRAQ (Isobaric tag for relative and absolute quantitation) from different insights, including epigenetics, transcriptomics and proteomics, the molecular network of how PIC regulates EP of EC will be constructed. Finally, these research results will be tested reversely by adding DNA methylation inhibitors and PCD inhibitors into EC proliferation medium.

生长素类物质毒莠定（PIC）对百子莲体细胞向胚性细胞转变及长期继代过程中维持胚性细胞成胚潜力（Embryogenic potential，EP）至关重要，而EP丧失是一个早已被科学家证实的重要科学问题。本研究以1.0～2.0mg.L-1PIC诱导百子莲愈伤组织（Callus，Ca）和继代3～6个月的胚性愈伤组织（Embryogenic Callus, EC））、非胚性NEC为对象，以细胞形态特征及1克鲜重EC产生体细胞胚的数量作为EP评价指标，HPLC-MS、激光共聚焦和免疫组化定量、定位分析内源激素（IAA，ABA和乙烯）和能量代谢物质的变化与EC极性结构，明确PIC诱导的活性氧簇（Reactive Oxygen Species，ROS)）氧化胁迫与细胞程序性死亡（PCD）对EP影响的生理生化基础；利用DNA甲基化、miRNA测序、RNA-seq与iTRAQ关联分析在表观遗传学、转录组学和蛋白组学层面构建PIC调控EC-EP的分子网络；最后添加DNA甲基化及PCD抑制剂反向验证上述研究结果的正确性。

以百子莲愈伤组织（Callus, Ca）为对照，不同浓度（1.0，1.5，2.0 mg/L）PIC培养的胚性愈伤组织（Embryogenic callus, EC）为实验材料。形态学观察发现，百子莲愈伤组织细胞较大、核较小，胞内富含淀粉；而PIC继代培养后的EC细胞较为致密。.采用MSAP技术对百子莲Ca、EC进行DNA甲基化水平研究表明，在Ca转化为EC的过程中总甲基化率降低，其中1.5 mg/LPIC中EC的总甲基化位点数和甲基化率均最低，分别为110个和25.46%。淀粉、糖组分和5种内源激素（IAA、ABA、CTK、ETH、BR）在胚性的启动、保持中发挥重要作用。3种活性氧ROS组分（O2·-、OH·和H2O2·）的产生和清除ROS的抗氧化酶（SOD、POD和CAT）活性间的动态平衡，抑制了细胞程序性死亡（Programmed Cell Death，PCD），促进了百子莲细胞胚性的保持和成胚能力的获得。成胚能力与淀粉、可溶性糖、H2O2、CTK与BR含量，SOD活性呈显著正相关，相关系数为0.741*~0.999*；与产生OH∙能力、IAA含量及CAT、POD活性呈明显负相关，相关系数为-0.818*~ -0.958*，表明保持高水平的淀粉、可溶性糖、CTK、BR、H2O2含量和SOD活性，以及低水平的IAA浓度和CAT、POD活性，有利于细胞保持胚性。.在1.0mg/L与1.5mg/L PIC继代的EC间共有16条显著富集（Qvalue<0.5）的miRNA pathway，其中，1.0mg/LPIC 继代EC的转录因子明显高于1.5mg/L PIC处理的样品；而两样品间生长素信号上调表达，赤霉素与油菜素内酯信号下调表达。比较转录组分析结果表明，Ca与EC间差异表达基因DEGs总数为9485，其中上调表达为7342，下调表达为2143。挑选三大代谢通路中的32个关键基因进行qRT-PCR表达分析验证结果表明，与Ca和其他浓度PIC继代培养的EC相比，1.5 mg/L PIC继代的EC中GH3和ARF1、淀粉合酶、蔗糖合成酶和POD酶基因显著上调表达；LEC在Ca中不表达，而在1.5 mg/L PIC继代的EC中表达量最高，可作为胚性获得和保持的标记基因。通过对成胚潜力生理生化、分子组学的研究，构建了外源生长素类物质调控百子莲胚性获得的分子网络。