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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]

ANS genes were cloned from Brassica nigra by homology - based clone strategy. 2 copies named BnANS1 and BnANS2 were obtained. BnANS1 sequence was 1 366bp with a 76bp - intron. BnANS2 sequence gene was 1 367bp with 76bp intron. Sequence analysis showed polymorphisms in coding regions, intron, 5 ' - and 3 ' - noncoding regions of the two genes, including 3 polymorphic bases and a 4 - base deletion in 5 ' - noncoding region, 26 polymorphic bases in coding region, 2 polymorphic bases in intron, 18 polymorphic bases and a 3 - base insertion in 3 ' - noncoding region. The two copies encoded deduced a polypeptide of 356 amino acids, having the same conserved domain of ANS as other species, belonging to 2OG - Fe (II) oxygenase superfamily. BnANS1 protein was predicted to be 40 448. 58Da with pI of 5. 05, while BnANS2 was 40 468. 52Da with pI of 5. 04. Nine polymorphisms were found in their protein sequences. RT - PCR result showed that BnANS1 and BnANS2 expressed in seed coats and embryos in B. nigra. Compared with published ANS gene sequences of Brassia species, a pair of primer was obtained based on B genome - specific nucleotide variations loci, which could effectively identify B - genome origin of ANS in Brassia by allele - specific PCR. [ABSTRACT FROM AUTHOR]