麻疯树开花关键基因DOF/CO/FT的表达分析和功能研究

Jatropha curcas, a perennial photoperiod-insensitive plant belonging to the family Euphorbiacea, is widespread in tropical and subtropical areas. Its seed has a high oil content （40-60％） that can be processed as biodiesel. However, the prospect of utilizing J. curcas for extracting biodiesel is extremely limited by low yields because the energy from vegetative growth under erosion and reclaim land transit continuous flowering (unlimited developmental inflorescence). In contrast to Arabidopsis and rice, little is known about the molecular mechanisms of photoperiodic responses in J. curcas. Three groups of key genes JcDof4-6、JcCOL1-6 and JcFTL1-3, involving in flowering time, were isolated from J. curcas genome database （http://www.kazusa.or.jp/jatropha/). We plan to investigate the expression levels of all of them under different light conditions, different developmental phases and different organs for building up their expression patterns in response to light signals. Yeast one hybrid systems, yeast two hybrid systems and subcellular localization assays will be used to analyze the transcripton factor features of JcDof4-6, the transcription regulator features of JcCOL1-6 and discover some special proteins interacting with them,respectively. Based on the results above, some of 35s:: JcDofs,35s:: JcCOLs and 35s:: JcFTLs will be transformed into long-day Nicotiana sylvestris (Ns) and the short-day Maryland Mammoth （MM）tobacco , and the day-neutral Nicotiana tabacum (Nt),respectively. The different tansgene lines (T1) will be measured the number of leaves, the height of plant after sowing 40 days for elucidating the functions of genes in photoperiod-sensitive and -insensitive vareties. In particular, we aim to discover their function under the process flowering for providing database information to J.curcas molecular breeding that is to manipulate the regulation of flowering time to increase the economic potential of this species.

麻疯树为大戟科麻疯树属植物，是一种对光周期不敏感的多年生油料树种，表现出所积累的营养生长能量转化为无限的生殖开花，造成花期长而果量少的现实矛盾。根据目前追逐果实产量的生产需求，我们结合前期研究基础和新公布的数据库信息，拟从启动营养生长向生殖生长转换的关键基因入手，选择了JcDof4-6、JcCOL1-6和JcFTL1-3（共12条）在开花启动中最核心、最上游的三类基因开展相关研究。力图分析它们在不同光照长度、光质、发育阶段和组织中的表达，建立相应的光响应表达模式；根据三类基因的不同特点，分别进行酵母单杂、双杂，酵母cDNA文库双杂实验，确定它们的基因特性和寻找互作蛋白，建立上下游调控网络；有选择的转化三种烟草品种（长日、短日和日中性），比较它们对光周期敏感和不敏感烟草开花时间的影响，明确它们在开花过程中的作用，为提高产量而开展的麻疯树开花可控育种研究奠定基础。

摘要：麻疯树为大戟科麻疯树属植物，多年生油料树种，表现出在荒地上积累的营养生长能量转化为无限的生殖开花，从而造成花期长而果量少的现实矛盾。根据目前追逐果实产量的生产需求，结合数据库和前期研究结果，从启动营养生长向生殖生长转换的关键基因入手，选择了包括光周期、GA途径和年龄（AGE）途径相关31个基因分别进行了以小时、月份、树龄、叶龄为单位的转录表达研究，结果显示与光周期有关的基因虽然表达呈现节律变化，但是月份变化差异不显著，推测不是麻疯树控制开花的关键因子；而与GA和AGE相关的基因呈现出显著的发育变化差异性，特别是miR156和miR159随年龄增加逐渐减少，而miR172a和JcSPL3，9逐渐增加，有利于开花；但是JcDella1,3,4逐渐增加，JcGID1A，JcGA20OX1和JcSPL4逐渐减少，不利于开花；推测麻疯树最终转向生殖生长与GA 和AGE相关基因的协同作用关系密切。选择miR172和JcSPL3构建过量表达载体，农杆菌转化麻疯树，获得多株F0，利用CRISPR/Cas9系统构建敲除miR156转化拟南芥 （公司进行）和转化麻疯树（正在进行）。望后续转基因结果能阐明相关基因在麻疯树开花过程中的重要作用。. 另外，经多元线性回归分析显示种子贮藏时间只与萌发率和种仁含水率负相关，与油脂酸值正相关，即用于扩繁再生的种子最好贮藏一年以内，控制低湿度；作为原料油再加工的种子至少可贮藏5年，应尽量干燥。