MADS-box家族转录因子JcMADS1调控木本植物小桐子成花转变的分子机理

Jatropha curcas seeds are an excellent biofuel feedstock, although the seed yield limited by the poor abilities of flowering. Thus, exploring the molecular mechanisms controlling flowering is critical for genetic improvement of Jatropha seed yield. Fortunately, a transcription factor of MADS-box family was isolated from Jatropha flower EST and nominated as JcMADS1. Overexpressing of JcMADS1 induced extremely early flowering in transgenic Jatropha. JcMADS1 amino acid sequence shows the highest similarity with both AtAP1 and AtFUL. However, JcMADS1 didn’t recover the phenotypes of ap1 or ful mutant. This suggests JcMADS1 is a novel key factor in regulating floral meristem identity rather than a homologue of AP1 or FUL. In the further study, we are trying to explore the molecular mechanism of JcMADS1 promoting flowering by molecular and genetic biotechnology. Concretely, the target genes of JcMADS1 will be scanned by Chromatin Immunoprecipitation (ChIP) sequencing assay using GFP antibody and transcriptome analysis. Furthermore, the binding of JcMADS1 and its target genes will be confirmed by yeast one hybrid and transient expression assay in tobacco cytoplasm. The function of these candidate genes will be testified by overexpressing and knock-out in Jatropha. In brief, this research is helpful to uncovering the flowering molecular mechanism of Jatropha and promoting the Jatropha becoming a new woody model plant. The early flowering plants will be useful for generating superior varieties of high-yielding transgenic Jatropha.

小桐子种子是一种适合加工生物柴油的原料。然而，受到开花习性的限制，小桐子产量不能满足工业需求。因此对小桐子开花机制的研究有助于提高种子产量。在前期的研究中，我们发现一个小桐子MADS-box家族的转录因子JcMADS1，超量表达该基因的小桐子植株出现极度早花的表型。该基因编码的氨基酸与拟南芥AP1和FUL具有最高相似性；然而，系统进化树结果显示该基因不与AP1和FUL聚类到一起。互补实验结果显示该基因并不能互补ap1和ful突变体，说明该基因是一个新的调控小桐子开花的关键基因。本研究通过转录组测序、染色质免疫共沉淀、酵母单杂交、瞬时表达等方法寻找JcMADS1的下游靶基因。然后利用超量表达和基因敲除技术验证候选基因功能，初步了解JcMADS1调控小桐子开花的分子机理。本研究有助于阐明小桐子开花机制和推动小桐子成为新的木本模式植物；也为利用基因工程方法培育高产、优质小桐子品种奠定基础。

通过对JcMADS1:GUS转基因植株染色分析发现JcMADS1启动子在茎顶端和花序芽中有活性。亚细胞定位的结果表明，JcMADS1可能作为一个转录因子在细胞核内行使功能。我们成功通过RNA干扰和CRISPR/Cas9沉默了JcMADS1在小桐子里的表达。将JcMADS1被沉默的幼芽和野生型的幼芽同时嫁接到超表达JcFT的转基因砧木上，结果JcMADS1被沉默的嫁接植株开花时间更晚，这进一步表明JcMADS1参与了小桐子成花转变，并且在开花信号通路中位于JcFT下游。通过进一步研究，发现一个锌指蛋白JcZHD2能结合JcMADS1启动子，并且JcZHD2蛋白能和JcZHD11蛋白相互作用，表明在小桐子里JcZHD2和JcZHD11可能以二聚体形式调节JcMADS1的表达。酵母双杂交、双分子荧光互补以及pull-down 实验证明JcMADS1蛋白与JcSEUSS或JcASHR2蛋白也分别存在相互作用。采用野生型和35:JcMADS1转基因小桐子的顶端进行转录组测序，分析结果表明大量促进开花的基因在转基因植株中表达增加，同时抑制开花的基因表达降低。为了通过ChIP-Seq找到JcMADS1的直接靶基因，将35S:JcMADS1-Myc转入小桐子并获得了早花的转基因植株，以35S:JcMADS1-Myc转基因小桐子为材料的ChIP-Seq实验结果表明JcMADS1结合元件是CArG motife，其直接调控的基因包括JcAGL16, JcAP3, JcSEP1, JcFLC, JcAGL25, JcAGL15, JcAGL6, JcAGL1, JcSOC1, JcSVP, JcWRKY12, JcWRKY38, JcAGL63, JcTCP2, JcAGL13, JcAG等。JcMADS1调节小桐子开花的分子机理研究对于深入了解小桐子以及其它木本植物的成花转变有重要意义，同时也为培育童期短、产量高的优良木本作物奠定了理论基础。