蓖麻B3转录因子对脂肪酸羟基化酶的调控机制研究

The Ricinus communis L. (castor bean) is a very important non-edible oilseed crop with high levels of hydroxy fatty acid (HFAs) in its seed oil. Although the oleate 12-hydroxylase encoding gene RcFAH12 playing an important role in HFAs biosynthesis in castor bean has been cloned and characterized, little is known about the regulatory mechanisms of accumulation of high levels of HFAs. We have identified three core motifs for B3 transcription factors recognition on the promoter region of RcFAH12. Furthermore, genome-wide gene expression profiling showed three seed-specific expressed B3 transcription factors including RcLEC2, RcABI3 and RcFUS3 co-expressed with RcFAH12 during seed development in castor bean, indicating these B3 transcription factors play a role in regulation of RcFAH12. In this study, we will characterize the regulatory mechanisms of RcFAH12 by RcLEC2, RcABI3 and RcFUS3 based on tobacco transient expression.assay, chromatin immunoprecipitation assay and yeast one-hybrid system. Transgenic castor plants (RcLEC2, RcABI3 and RcFUS3 gene RNAi and over expression) will be used to analyze the relation between RcFAH12 gene expression and the HFAs level. The goal of this research is to decipher the regulatory mechanisms underlying accumulation of high levels of HFAs in castor bean, and provide new genetic resources and the theoretical basis for the seed oil improvement in oilseed crop.

蓖麻作为世界上最重要的非食用油料作物之一，其种子累积大量的羟基化脂肪酸。虽然蓖麻羟基化脂肪酸生物合成的关键功能基因脂肪酸羟基化酶RcFAH12已被克隆，但蓖麻种子大量累积羟基化脂肪酸的调控机制尚知之甚少。前期研究中我们在RcFAH12的启动子上鉴定三个B3转录因子结合元件，且转录组数据分析显示，三个种子特异性表达的B3转录因子（RcLEC2、RcABI3和RcFUS3）在种子发育过程中和RcFAH12共表达，强烈暗示B3转录因子可能调控RcFAH12基因表达。本研究拟运用烟草瞬时表达实验、染色质免疫共沉淀和酵母单杂交分析这三个B3转录因子对RcFAH12基因可能的调控作用，并运用蓖麻转基因技术分别将这三个B3转录因子进行表达抑制和过表达，分析转基因蓖麻种子中RcFAH12的表达及脂肪酸成分，以期阐明蓖麻种子大量累积羟基化脂肪酸的调控机制，为蓖麻遗传改良提供新的基因资源和理论基础。

蓖麻作为世界上最重要的非食用油料作物之一，其种子累积大量的羟基化脂肪酸。虽然蓖麻羟基化脂肪酸生物合成的关键功能基因脂肪酸羟基化酶基因RcFAH12已被克隆，但蓖麻种子大量累积羟基化脂肪酸的调控机制知之甚少。在前期研究中我们在RcFAH12的启动子上鉴定两个B3转录因子结合元件，且转录组数据分析显示，三个种子特异性表达的B3转录因子（RcLEC2、RcABI3和RcFUS3）在种子发育过程中和RcFAH12共表达，强烈暗示着B3转录因子可能调控RcFAH12基因表达。我们通过烟草瞬时表达实验和酵母单杂交实验证明LEC2是RcFAH12表达的正向调控因子，而RcABI3对RcFAH12没有调控作用。我们制备了RcLEC2的特异性抗体，并建立起蓖麻种子染色质免疫沉淀技术体系。分别构建了RcABI3和RcLEC2基因的过表达载体，开展蓖麻遗传转化研究。在项目执行期间培养了2名研究生，发表3篇论文。