Cloning And Expression Analysis Of COMT Gene And MYB Transcription Factor In ’Dangshansuli’ Pear And Its Mutant ’Xiusu’

Skin color is an important characteristic for the economic value evaluation in fruit commodities. A sport was found in the 50-years-old tree of ’Dangshansuli’ pear in Dangshan County, Anhui provience in 2001. The mature fruit of’Dangshansuli’pear was yellow-green while the mutant branch yielded fruit with russet color, temporarily named ’Xiusu’ pear. After having observed for three years, the variation character of mutant was stable. At the same time, the results of high-graft test and molecular remarker analysis also indicated that’Xiusu’ pear was a genetic variation. The reason of russet skin formation have been reported, but less molecular mechanism was investigated. In order to study the mechanism of russet skin formation of ’Xiusu’ pear, key genes and transcription factors, PbCOMT1, PbCOMT2, PbMYBR, and PbMYB31 involved in lignin biosynthesis were cloned from the pericarp of ’Xiusu’ at 150 days after full bloom (DAFB). The relative levels of expression of PbCOMTl, PbCOMT2, PbMYBR and PbMYB31 gene at different stages in ’Dangshansuli’ and ’Xiusu’ pear were analyzed by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR).The main results achieved were as follows:1. Full-length sequences of COMT1, COMT2, MYBR and MYB31 genes were obtained from the pericarp of pear based on suppression subtractive hybridisation (SSH) and named as PbCOMTl, PbCOMT2, PbMYBR and PbMYB31, respectively. Bioinformatics analysis indicated that full length cDNA sequence of PbCOMTl gene was 1371 bp containing an open reading frame (ORF) of 1098 bp, encoding 365 putative amino acids (AA); its protein molecular formula was presumed to be C1802H2808N456O522S23, molecular weight was 39,950.2 Da, and an isoelectric point was at value of pI 5.52. The sequence of PbCOMT2 gene was 1405bp, which contained an ORF of 900bp encoding 299 putative AA, its protein molecular formula was presumed to be C1483H2268N392O411S20, molecular weight is 32805.9 Da, and isoelectric point was at value of pI 6.59. The sequence of PbMYBR gene is 1579 bp with a sequence coding region (CDS) of 1386 bp, which encoded a peptide of 461 AA. The formula of PbMYBR protein was C2146H3403N645O716S18, and isoelectric point of was at value of pI 6.74. The cDNA sequence of PbMYB31 gene was 1198 bp, from the base to a first coding region of 1131 bp encoding 376 AA, the protein formula was presumed to be C1802H2810N534O586S12, isoelectric point of was at value of pI 6.12.2. The results of semi-quantitative RT-PCR amplification showed that PbCOMT2 gene could expressed in pericarp, pulp and leaves of pear plant, and relatively high level of the expression in pericarp has demonstrated its tissue specific profile. Using the house keeping gene glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) as an internal referenced gene, PbCOMT1 genes was analysed by real-time qRT-PCR amlification. The results showed that the expression of PbCOMT1 has been detected The relative level of PbCOMTl gene expression in the pericarp of’Xiusu ’and’ Dangshansuli’pear was lower at 150 DAFB than at 25 or 75 DAFB. As the fruit matured, the relative level of PbCOMTl gene expression in the pericarp of ’Xiusu’ pear exhibited a downward trend, while that of ’Dangshansuli’pear increased first and then declined. At 25 DAFB, the level of PbCOMTl gene expression pericarp of in ’Xiusu’ pear was about ten-times higher than that in ’Dangshansuli’ pear. This probably was a reason of the russet skin formation in ’Xiusu’ pear.The results of semi-quantitative RT-PCR amplification showed that PbCOMT2 gene could only expressed in pericarp but nearly not in pulp and leaf of ’Dangshansuli’ and ’Xiusu’ pear. The results of real-time qRT-PCR amplification showed that the relative level of PbCOMT2 gene expression in’Xiusu’ pear was higher than in’Dangshansuli’pear at different stage of fruit development. The relative level of PbCOMT2 gene expression in ’Xiusu’ pear was about ten-times higher than that in’Dangshansuli’ pear at 25 DAFB, there was no significant difference between the relative level of PbCOMT2 gene expression in the pericarp of’Dangshansuli’and ’Xiusu’ pear at 75 DAFB, but it in ’Xiusu’ pear was almost two times higher than that in ’Dangshansuli’ pear at 150 DAFB. These results probably proved why the russet skin formed in ’Xiusu’ pear.3. The relative levels of PbMYBR and PbMYB31 gene expression were also analysed by real-time qRT-PCR using GAPDH as an internal referenced gene. Expression of PbMYBR and PbMYB31 were detected in pericarp, pulp and leaf of pear plant, and relative levels of PbMYBR gene expression was the highest in leaf, and that of PbMYB31 gene expression was the highest in pericarp. PbMYBR and PbMYB31 genes were both expressed in the pericarp of ’Dangshansuli’ and ’Xiusu’ pear at 25 DAFB, but the relative level of PbMYBR gene expression in ’Xiusu’ pear was ten-times higher than that in’Dangshansuli’ pear. The relative level of PbMYB31 gene expression in ’Xiusu’ pear was four-times higher than that in ’Dangshansuli’ pear. The results indicated that the difference between the expression of PbMYBR and PbMYB31 might be another important factor in the skin formation of pear fruit.4. The promoters in PbCOMTl and PbCOMT2 gene were analysed by online service using the software PLACE (plant cis-acting regulatory DNA elements). The result showed that PbCOMT1 and PbCOMT2 contain multiple general structures of eukaryotic promoters such as CAAT-box and TATA-box, which indicating that the two kinds of promoters are strong promoters. The cis-acting element, including MYB1AT, MYBPLANT, and MYBCORE were also found in MYB transcription factors.