Cloning And Expression Analysis Of Proanthocyanidin Biosynthetic Genes And Their Promoters In Brassica Juncea

The seed coat color of oilseed Brassica species is controlled by proanthocyanidin content in it. Proanthocyanidin biosynthesis pathway is largely elucidated in the model plants such as Arabidopsis thaliana, but whether the oilseed Brassica species have the same regulatory mechanism for proanthocyanidin biosynthesis pathway as arabidopsis thaliana remains elusive.The BAC sequencing and subsequent sequence analysis showed that4copies each of TT5, TT6and TT18,3copies of TT19,2copies each of TT1, TT2and BAN, one copy of TT3were cloned from genomic BAC library screen of a black-seeded B. juncea variety Purple-leaf Mustard (PM) using primer pairs designed according to released homologous sequence of proanthocyanidin biosynthetic genes.The copy-specific primers were then designed to amplify the homologous sequence from a yellow-seeded B. juncea variety Sichuan Yellow (SY). Comparison of sequences between PM and SY showed that all copies have only minor sequence difference(0-6bp) in coding region except the four copies, i.e., TT1-180A5, TT5-181K10, TT6-22O18and TT18-181K8(26-60bp). Meanwhile, the genome and A-gemone chromosomal position where each copy is located were predicted by BLASTing against B. rapa website using the open reading frame sequences of these copies and end sequences of BACs in which these copies are located. The results showed that the genomes A and B both carry one or more copies of these genes, among which A1chromosome has TT18and BAN, A2TT19, A3TT5, TT6and TT18, A7TT5, A8TT2, and A9TT1, TT3, TT5and TT6. The phylogenetic trees were also constructed using the software MEGA4.0to uncover the evolutionary relationship in TT1gene between B. juncea and other species.RT-PCR analysis of gene expression showed that each copy of TT3, TT18, BAN and two copies of TT19did not express in SY seed coat at15DAP but expressed in its black-seeded near-isogenic line (NIL) NILA and NILB, indicating that these copies is associated with seed coat color in B. juncea. Furthermore, promoter sequences of six copies TT1-180A5, TT3-29J10, TT18-54O2, TT18-91K10, BAN-82J1and BAN-34P21were cloned from SY and PM. The results showed that only TT1-180A5is different in promoter sequence between SY and PM. A MBS element, a MYB binding site, was found-688bp to-693bp upstream transcription start site of TT1-180A5in PM but not in SY. It is proposed that expression of the genes TT3, TT18and BAN and proanthocyanidin biosynthesis could be regulated by complex MBW assisted by interaction between TT1and TT2in B. juncea.