R/B基因家族调控黑米花青素形成的分子机理研究

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.

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。

花青素是类黄酮物质中的一种，其广泛存在于黑米中，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，Kala4是R/B基因家族中的一员，但其调节黑米色素沉积的机制尚不明确。本课题组利用crispr cas基因编辑技术将黑米（野生型）中的Kala4基因成功敲除，并获得了纯合的突变型水稻材料。通过比较野生型和突变型水稻材料的转录组学差异性，将突变型水稻中的下调基因显著富集在了类黄酮生物合成和植物激素信号转导通路上，而将上调基因显著富集在了葡萄糖、氨基酸、果糖和甘露糖、牛磺酸和亚牛磺酸等代谢或生物合成通路上。通过实时荧光定量PCR对富集于类黄酮生物合成途径中的5个下调基因（CHS、F3H、DFR和2个ANS）进行验证及时空表达差异性研究，发现该5个基因在突变型水稻花后1-3周种皮中的表达量均显著下降。同时，利用高效液相色谱技术对野生型和突变型水稻籽粒中的花色苷进行定性定量检测，发现野生型水稻籽粒中含有的矢车菊素-3-O-葡萄糖苷和芍药素-3-O-葡萄糖苷的含量分别为43.76 μg/g和3.32 μg/g，而突变型材料中未检测到。所有结果表明Kala4基因的敲除，使CHS、F3H、DFR和ANS等基因的表达水平显著下降，并使籽粒中不再积累黑米色素，进一步说明Kala4基因通过调控相关结构基因的表达而诱导并促进黑米色素的生物合成。这些将为深化稻米功能性营养品质理论提供新的研究思路，为体外合成黑米色素提供理论基础。