蓖麻WRI1转录因子特异地调控种子油脂累积的机理研究

Castor bean (Ricinus communis L.) is one of the most important inedible oil crops worldwide. Due to its strong adoption ability castor bean is able to grow well in most marginal lands. In particular, the seed oils containing rich hydroxylated fatty acid are broadly used as chemical materials in industry, as ideal feedstock for biodiesel production as well. As a typical case of endosperm seeds, it is necessary for castor genetic improvement to reveal the molecular basis of lipid accumulation in castor bean seeds. Although previous studies based on functional genomics have identified and characterized many key enzyme genes involved in the lipid biosynthesis in castor bean, little is known about the molecular mechanism underlying the regulation of those lipid genes at the transcriptional level, which may play critical roles in activating lipid gene expression and controlling oil content in seeds. According to our previous transcriptome data, it was found that transcription factor WRINKLED1 (WRI1) was specifically and highly expressed in the endosperm when a large amount of seed oil was accumulated. However, little is known about how WRI1 plays a role in castor bean in vivo. In this proposal, our research is focused on: 1) identifying the binding site and direct target genes in the genome using chromatin immunoprecipitation combined with high throughput sequencing (ChIP-seq) technique; 2) dissecting the molecular mechanism underlying WRI1 regulating the expression of lipid genes from ChIP-seq analysis, and 3) over-expressing the castor WRI1 gene and detecting the putative influences on lipid accumulation. This research will not only hopefully provide novel insights into understanding the molecular basis of lipid accumulation genetic improvement of castor bean, but also is also of importance data and reference for compare the regulatory differences between different types of seeds.

蓖麻(Ricinus communis L.)是世界上最重要的非食用油料作物之一。蓖麻适应性强、种子富含羟基化脂肪酸，其种子油脂是重要的化工原料和优质的生物柴油原料。解析典型的胚乳型蓖麻种子中油脂累积的分子基础是其遗传育种的重要内容。虽然目前已鉴别了很多影响蓖麻种子含油量的脂类合成基因，但是这些脂类基因的转录调控机理仍很不清楚。我们前期转录组数据发现蓖麻WRI1转录因子特异地在油脂大量累积时期的胚乳中丰富地表达，而WRI1在蓖麻中体内如何特异地发挥功能仍知之甚少。本项目拟运用染色质免疫共沉淀和高通量测序技术在基因组水平上鉴定WRI的直接结合位点和靶基因，结合分子实验深入解析蓖麻WRI1对脂类合成基因的调控规律，并检验过表达WRI1基因对种子油脂累积的影响。本项目为解析蓖麻种子油脂累积的分子机理和遗传改良提供理论依据，以及为理解不同类型种子油脂累积的调控机理提供重要数据和借鉴。

蓖麻（Ricinus communis L.）是世界上最重要的非食用油料作物之一。蓖麻种子富含羟基化脂肪酸，其油脂是重要的化工原料和优质的生物柴油原料。解析典型的胚乳型蓖麻种子中油脂累积的分子基础是其遗传育种的重要内容。我们前期转录组数据发现蓖麻转录因子RcWRI1特异地在油脂大量累积时期的胚乳中丰富地表达，但是RcWRI1在蓖麻体内如何特异地发挥功能仍知之甚少。本项目首先建立了适宜高油脂种子材料的染色质免疫沉淀（ChIP）的研究体系，然后结合高通量测序技术（ChIP-seq）在基因组水平上全面地鉴定了RcWRI1的结合位点和靶基因。ChIP-seq数据分析表明在全基因组上有1948个显著的富集峰，其中21.66%的峰位于256个靶基因启动子上，启动子基序分析表明RcWRI1结合位点不仅包括经典的AW-box，还包括5个新的结合基序。这些靶基因不仅编码参与油脂合成的酶，如甘油-3-磷酸脱氢酶、Omega-6去饱和酶（内质网）、磷脂酶 C、乙酰-辅酶A酰基转移酶、乙酰-辅酶A合成酶、乙酰-辅酶A羧化酶β亚基等，还包括参与光合作用、淀粉代谢的酶。这些靶基因的鉴定表明RcWRI1不仅能够通过控制碳流动而调控油脂的生物合成，同时也可能在光合作用等其它方面发挥重要的调控功能。酵母单杂和烟草瞬时激活实验进一步验证了RcWRI1通过结合RcBCCP2和RcEAR1启动子中的AW-box而激活它们的表达。RcWRI1需要2个AP2 domain的同时存在来发挥转录调控功能。通过叶盘法在烟草中过表达RcWRI1基因，气相色谱分析显示，转基因烟草叶片中总脂肪酸含量为3.7304-5.1186 mg/g，是对照的1.23-1.69倍。多种脂肪酸含量呈现不同程度的增加，其中棕榈酸和亚麻酸增加最多。通过酵母双杂交筛选到107个与RcWRI1互作蛋白，互作蛋白的筛选暗示RcWRI1可能通过互作蛋白共同调控下游靶标基因，并发挥多种功能。本项目的研究结果解析了蓖麻RcWRI1调控种子油脂累积的分子机理，为后续的遗传改良提供了理论依据。在项目执行期间，协助培养了1名博士研究生，发表了2篇SCI论文，1篇论文在准备中。