R/B基因家族调控黑米花青素形成的分子机理研究

项目名称： R/B基因家族调控黑米花青素形成的分子机理研究

项目编号： No.31500246

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 生物科学

项目作者： 邵雅芳

作者单位： 中国水稻研究所

项目金额： 20万元

中文摘要： 黑米富含多种花青素，有很强的抗氧化活性，对多种疾病具有预防作用。R/B基因家族能通过激活结构基因的表达而调节花青素的合成，其中Ra和Rb起了关键性作用，但在黑米花青素合成网络中的调控机理尚不明确。前期研究，我们利用发夹RNA（hpRNA）干扰技术特异性沉默Ra，发现沉默后黑米花青素含量显著下降但未完全消失。据此我们推断这可能是由于Ra的沉默不完全或其他有同源结构域的蛋白发挥作用而导致的。本项目拟采取人工微小RNA（amiRNA）和hpRNA干扰技术对Ra、Rb及相关蛋白的碱性螺旋-环-螺旋（bHLH）同源结构域进行沉默，研究其对花青素合成的调控机制。同时体外合成Ra和Rb蛋白，研究bHLH结构域和关键功能基因启动子的DNA结合能力，揭示其在花青素合成中的调控作用和相互关系。通过本项目的实施，对阐明水稻花青素合成代谢网络的分子调控机制和深化稻米功能性营养品质的应用理论研究均具有重要意义。

中文关键词： R/B基因家族；花青素类；调控网络；黑米

英文摘要： Anthocyanins, which are present in black grain diversely, have high antioxidant activities, and are good to many chronic diseases such as cardiovascular diseases, type II diabetes, obesity and some cancer. It is reported that R/B gene family, Ra and Rb, could activate transcription of some structural genes in the pigmentation of rice grain. However, it is not clear about the regulatory mechanisms of Ra and Rb that take part in the anthocyanins biosynthetic pathway in black rice. In previous study, we silenced Ra gene specifically by using short hairpin RNA interference. The content of anthocyanins in the transgenic black rice grain decreased significantly but did not disappear. Therefore, we deduced that it might be due to the incomplete silence of Ra or other homologous proteins which have the same functional domain. In this project, both artificial microRNA (amiRNA) and hairpin RNA (hpRNA) will be used to silence Ra, Rb, and the associated homologous gene sequence of basic helix-loop-helix (bHLH) protein domain, in order to study the effects of Ra, Rb, bHLH domain, and the interaction between Ra and Rb on the anthocyanins accumulate pattern. We will also express Ra and Rb protein in Escherichia coli to study the DNA binding ability between target proteins and the promoters of some structural genes by electrophoretic mobility shift assay. This work will provide the new insight into the regulatory mechanism of Ra and Rb and their interactions in anthocyanins biosynthetic pathway in black rice. It is also useful for the application and theoretical research of the functional nutrition quality in rice.

英文关键词： R/B gene family;anthocyanins;regulatory network;black rice (Oryza sativa L.)