Regulation Of Anthocyanin Biosynthesis By MYB In Chinese Bayberry Fruit And The Mechanisms

Studies of anthocyanins biosynthesis pathway were carried out using Chinese bayberry (Myrica rubra Sieb. et. Zucc.) cultivars'Biqi','Dongkui'and'Shuijing'. The relationship between the anthocyanins and the colour of bayberry fruit was analyzed. Structural genes in anthocyanins biosynthesis and MYB transcription factors (TFs) were cloned. Their expression modes in the fruit and pulp of the different cultivars, in different tissues and organs, in different developmental stages and in bagged treatment were analysed by qPCR. Research on reciprocal action mode between MYB and the coding genes in anthocyanins biosynthesis was carried on. The function of MYB transcription factors and their regulation modes on the genes in anthocyanins biosynthesis were confirmed by transient tobacco expression system. The main results are listed as follows:1. The highest anthocyanins content was found in the fruit of'BQ', which was 85.40 mg/100g FW, and the content in the fruit of'DK'was 46.44 mg/100g FW, while no anthocyanin was tested in the fruit of'SJ'. The CIRG values were 7.56,5.27 and 3.84 in 'BQ','DK'and'SJ', respectively. In the analysis of anthocyanin contents in different tissues and organs in'BQ', little anthocyanin was tested in root and young stem, on the contrary, anthocyanin content in the fruits was the highest. In the fruits of the three cultivars on 64d DAFB, no anthocyanin was detected in the fruit of'DK'and'BQ'. The anthocyanin contents and CIRG values increased with the development of fruits. For 'BQ', the anthocyanin content in bagged fruit was only 0.56 mg/100g FW, however, the anthocyanin content in non-bagged fruit was 108.99 mg/100g FW. The CIRG values of the non-bagged fruits were about two fold of those bagged ones.2. From the mature fruit of'BQ', we cloned ten genes, i.e. Mr ACT (227 bp), MrCHS (440 bp), MrCHI (301 bp), MrF3H (436 bp), two DFR members (MrDFR1 221 bp and MrDFR2 221 bp), MrANS (428 bp), MrUFGT (647 bp)and two R2R3 MYB genes (MrMYB1 and MrMYB2). From EST database of bayberry, we obtained two genes, MrF3'H (412 bp) and MrMYB3 (514 bp). The homology of MrCHS and MrF3H with the counterpart genes in other species was as high as 90%. MrDFR1 showed a similarity of 83% with the counterpart genes in Arabidopsis thaliana. However, the similarities between MrUFGT and the counterpart genes in other species ranged from 51% to 70%. The rest genes had similarities from 68% to 82% compared with counterpart genes in other species.3. By 3'RACE (for both members)and 5'RACE (for Mr MYB1 only),966 bp and 996 bp fragments were obtained and named MrMYB1 and MrMYB2. MrMYB1 was a full length fragment. MrMYB1 and MrMYB2 showed a similarity up to 70% with the gene family of R2R3 MYB in Arabidopsis thaliana. Wheras, MrMYB3 showed only a similarity of 55% to AtMYB. MrMYB1 and MrMYB2, all of which contained structural domain of R2R3, showed a high identity of amino acid sequence.4. The expression of MrCHS and MrCHI were similar in fruits of the three cultivars, while that of other genes was higher in the two colored cultivars than those in'SJ'. The transcriptional levels of MrF3H, MrF3'H, MrDFRl and MrUFGT were directly associated with anthocyanin content. In different tissues and organs of'BQ', except for MrCHI whose highest expression level was found in root, the highest expression levels of all other genes were found in ripe fruits. Besides, MrUFGT was expressed exclusively in ripe fruits. In fruits of different developmental stages of'DK'and'BQ', except for MrCHI and MrDFR2, expressions of all other structural genes directly associated with the total content of anthocyanin, and the expression levels in'BQ'fruits were higher than those of'DK'fruits. Bag treatment significantly inhibited the expression of structural genes in anthocyanin biosynthesis.5. The transcriptional level of MrMYB1 directly associated with the total content of anthocyanin. MrMYB1 was exclusively expressed in ripe fruits of'BQ', not in root, stem and leaf. The expressions of MrMYB1 in'DK'and'BQ'increased along with the fruit development and reached the summit in the late developmental stage. The expression of MrMYB1 in bagged fruits was significantly inhibited, suggesting that light could affect the anthocyanin accumulation by MrMYBl expression.6. The results of tobacco transient expression showed that over-expression of MrMYBl resulted in the accumulation of anthocyanin. MrMYBl coordinated with bHLH could activated AtDFR promoters, indicating that MrMYB1 regulated the expressions of structural genes in the pathway of anthocyanin biosynthesis, which further affected the biosynthesis of anthocyanin.7. The full length sequences of MrMYB1 were separately cloned from the fruits of 'BQ','DK'and'SJ'. A cytosine was lost from the+30 position after the start codon of ATG in MrMYB1 (named MrMYB1d) of'SJ', resulting in frameshift mutation, which leaded to failing in normal protein synthesis of MYB1. MrMYB1 gene was normal in'BQ', otherwise, MrMYB1 and MrMYB1d genes were all normal simultaneously in'DK'. Probably, the sequence mutation resulted in which anthocyanin was not able to biosynthesis in fruits of'SJ'.In conclusion, MrMYB1 regulated the expressions of structural genes in the pathway of anthocyanin biosynthesis in Chinese bayberry.