| Blackberry (Rubus L.) belongs to the Rubus genus and is among one of the three Rubus populations which were used in horticultural productions. It is included in the thrived’third generation’fruits, due to the fact that blackberry fruits are rich in anthocyanin, proanthocyanidin and several mineral element contents. It has become most popular in both European and America, so does in China in the recent years. Fruit extractions of blackberry exhibited a lot of health positive effects in the aspects of anti-cancer, anti-inflammation, and against cardiovascular disease. Hence, studies involved in chemistry and pharmacy were widely carried out around the world. However, biosynthesis of the extraction fractions, especially the anthocyanin and proanthocyanidins, was seldom reported. It has been demonstrated that there are not any wild blackberry resources distributing in China. Most of our cultivars were imported from abroad, in which most excellent blackberry traits were selected. In such a background, molecular manipulations aiming to improve or create new germplasm are necessary here in China. In the present study, we initially investigated and determined the anthocyanin and proanthocyanidin content changes in blackberry fruits. Based on the conserved sequences of other plants, we successfully isolated6key structural genes in the flavonoid biosynthesis pathway. The transcription factor, RuMYb10, which specially regulates the anthocyanin branch, was also obtained. Finally, expression patterns of all these genes were assayed by quantitative RT-PCR. The major results were as follows:(1) Total anthocyanin content was estimated using the pH differential assay. Proanthocyanidins were determined using a standard method based on the specificity reaction between flvanol and DMACA reagent. Our results indicated that blackberry fruits were rich in both anthocyanin and proanthocyanidin contents. Anthocyanin in fruits could count1.426mg·g-1fresh weight fruits. The major anthocyanin was synthesized in the late developing stage, when fruits turn black from red in the outter appearance. On the contrary, proanthocyanidins peaked in the early green fruits. When the fruits get mature, PA gradually diminished and reached a level that could not be detected. (2) Based on the knowledge that genes were similar in the phylogenetic relatives, degenerate primers were designed and used to amplify the key structural genes in the pathway. We managed to characterize6genes from blackberry fruits, including genes encoding chalcone synthase, RuCHS, dihydroflavonol4-reductase, RuDFR, leucoanthocyanidin dioxygenase, RuANS, leucoanthocyanidin reductase, RuLAR, anthocyanidin reductase, RuANR, and a glycosyltransferase gene RuGTl. All these genes were deposited in Genbank with accession number JN602374, JF764809, JF764807, JQ068826, JQ068825and JF764808, respectively.(3) A fragment sequence encoding the conserved domain of MYB transcription factor was amplified using genomic DNA as templates. Then SON-PCR technique was used to amplify both flanking sequences of the gene. The full coding sequences of RuMYB10were obtained and tested in cDNA templates. Sequence analysis suggested that the gene,1837bp in length, encoding a polypeptide consisting of216amino acids,24863Da in molecular weight. The peptide possessed the R2R3domain of MYB transcription factors. When compared with Rubus ideaus RiMYB10, the most striking diversities were found in the noncoding regions, including the intron length and the intron sequence divergence. The second intron of RuMYB10ranked750bp, constitute of as much as40.8%of the full length sequence. In the R3repeat region, the motif ’[DE]Lx2[RK]x3Lx6Lx3R’ which was implicated in its bHLH cofactors was perfectly matched. Of the three deduced amino acid residues in anthocyanin-promoting MYBs, the alanine was substitute by a serine residue.(4) Total RNA of high quality was isolated from blackberry fruits at different maturation stage using our newly adopted CTAB-based protocol. Expression levels of all the above isolated genes were investigated by quantitative RT-PCR. RuANS, which channels materials into anthocyanin and proanthocyanidin branch, showed coordination with anthocyanin production. The highest level was observed in the turning stage, from red to black fruits. RuLAR and RuANR, presenting the specificity enzymes in the PA pathway, were strictly concomitant to the accumulation of PAs in the fruits. The most abundant transcriptions of RuLAR and RuANR were present in the initial green fruits or even earlier in the flowers. Along with the degradation of PA in the developing process, their expression levels gradually decreased. RuCHS, as one of the early genes in the pathway, and RuDFR, which acted as the key step in the middle of anthocyanin or PA synthesis, had two expression peaks. The first peak appeared just before the time when fruits got red, and the second one existed in the last stage, when fruits were black, accumulating large quantitative of anthocyanin content. Expression of RuMYB10was similar to that of RuANS gene. It was coordinating to the synthesis of anthocyanin in the blackberry fruits, indicating its specific role in the anthocyanin branch. Pearson’s coefficient analysis indicated that the RuANS was the most important enzyme in blackberry anthocyanin biosynthesis. As for PA production, RuLAR was more crucial when compared with RuANR.(5) The isolated RuGT1might be a candidate proanthocyanidin-related glycosyltransferase. The expression patterns of the isolated RuGTl were totally different to that of the previous reported flavonoid glycosyltransferase gene. The highest expression levels appeared in the fruits of green stage, when PA was the dominant metabolites. Afterward, transcripts decreased till the last stage with a slight increasing in the black berries. This was much similar to that of the UGT72L1in the Medicago truncatula, which catalyze the glycosylation of epicatechin. However, the subtract specificity and enzymatic kinetics need further investigation. |
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