Cloning of PAP2 genes in Brassica nigra and Brassica carinata and functional validation of PAP2 in the B genome.

PAP2 (Production of anthocyanin segment 2) encodes MYB transcription factor, and regulates anthocyanin synthesis by forming MYB-bHLH-WD40 complex with bHLH and WD40. In this study we cloned PAP2 gene in Brassica, and preliminarily verified the function of PAP2, providing a reference for further understanding the regulatory mechanism of anthocyanin synthesis in Brassica. Two and one copies of PAP2 gene were cloned from Brassica carinata and Brassica nigra, respectively, and named as BcaB.PAP2, BcaC.PAP2, BniB.PAP2. These three genes are composed of 3 exons and 2 introns, encoding 247 amino acids. Based on the PAP2 gene sequences cloned in this study and other reported PAP2 gene sequences of Brassica, three pairs of primers were designed to detect the PAP2 gene of A, B and C genomes respectively, and the genomic origin of PAP2 of six species in the U's Triangle of Brassica could be distinguished by allele specific PCR. The BjuB.PAP2 gene from the B genome of Brassica juncea was selected to construct an overexpression vector and transformed into Arabidopsis thaliana. The transgenic Arabidopsis leaves turned purple, indicating that PAP2 gene is regulating anthocyanin synthesis. After shading treatment for 10 days, the purple color of Brassica carinata lightened, and the anthocyanin content decreased by 41.22%. Quantitative PCR and transcriptomic studies showed that the expression of BcaB.PAP2 and BcaC.PAP2 in the leaves of shading plants decreased. The expression of structural genes for anthocyanin synthesis, such as chalcone synthase gene (CHS), chalcone isomerase gene (CHI), flavanone 3-hydroxylase gene (F3H), dihydroflavonol reductase gene (DFR), anthocyanin synthase gene (ANS), flavonoid 3-glucosyltransferase gene (UFGT) and other genes were also decreased. This study completed the cloning of PAP2 gene of Brassica carinata and Brassica nigra, and carried out their evolutionary analysis. Allele specific PCR primers were designed according to the sequences of PAP2 gene cloned in this experiment and other reported genes to provide molecular markers for the genomic transmission identification of PAP2 gene in Brassica interspecific hybrids. Through the transformation of BjuB.PAP2 gene into Arabidopsis plants, it was found that PAP2 gene was involved in the accumulation of anthocyanins. It was found that the expression of BcaPAP2 gene and anthocyanin synthesis structure gene were induced by light after shading. This study cloned the PAP2 gene in Brassica and preliminarily verified its function, providing a reference for further understanding the regulatory mechanism of anthocyanin synthesis in Brassica plants. [ABSTRACT FROM AUTHOR]