Function Analysis Of Black Pericarp Gene In Rice Variety ’Tianheimi’

Recently, the transcriptional control of Anthocyanin biosynthesis has been studied extensively. Based on the previous mapping results, we did complemental test and functional analysis of the candidate gene Ra.Firstly, both Ra and its allele Ru (with GT insertion) were cloned into pCAMBIA1390for overexpression. Transient expression showed that the dominant Ra gene can promote the deposition of pigment in the aleurone layer of Nipponbare, whereas, both Ru gene and the control plasmid were incapable to do so. This indicated that Ra gene involves in the synthesis of anthocyanin, GT insertion in Ru gene probably disrupt the normal function. In addition, with Agrobacterium tumefaciens-mediated rice transformation, we also obtained over-expression and RNA interference lines in Nipponbare and Tianheimi background, respectively. The investigation of phenotype in each transgenic lines is still underway.Secondly, according to transient expression in rice protoplast, we found that the bHLH protein, encoded by Ra gene located in nucleus, as well as the Ru gene, which means that the GT insertion in Ru gene didn’t change its feature as a transcription factor.In order to identify the bHLH-MYB-WD40complex involved in regulation, we cloned corresponding genes and proved the interactions between these subunits to figure out the formation of the complex. As results showed, bHLH protein encoded by Ra gene can interact with R2R3MYB protein encoded by OsMYB3and OsMYB4, and also the WD40protein. The GT insertion did affect the formation of complex.Finally, we collected45rice varieties, including white, red and purple or black rice, and identified the genotype both in Ra and Rc sites. The results showed that raraRcRc was present in all the red rice, however, RaRarcrc and rararcrc were exist in all the tested black and white rice varieties, respectively. This indicated that Ra and Rc may regulate different structure gene to implement the synthesis of different flavonoid components. Thus, We analyzed the gene expression in the anthocyanin and proanthocyanidin pathway in typical white (Nipponbare), red (Kasalath) and black (TianHeiMi) rice varieties. In black rice, each gene showed high expression except for LAR and ANR, whereas, the expression of ANS was predominately reduced in red rice, which means that Ra gene can promote the accumulation of anthocyanin by high expression of ANS in black rice, however, proanthocyanidin was the major product in red rice because Rc gene can’t promote the expression of ANS. This conclusion is consistent with the content of pigments in black and red rice varities.The study about the regulation mechanism in anthocyanin synthesis would enhance the understanding of rice pigmentation, which would shed light on the improvement of rice cultivars in rice breeding.