Studies On Isolation Of Anthocyanin Biosynthesis Related Genes And Their Expression Analysis In Red Chinese Sand Pears

Chinese sand pear (Pyrus pyrifolia Nakai) originated from China and is widely cultivated in East Asia. Ripe sand pears usually appear green, yellow or brown (actually is phellem). Red sand pear also could be found, but not as common as others. In recent years, red skin sand pear, such as’Meirensu’,’Mantianhong’and’Yunhongli No.1’, were well received by customers, showing a great potential of custom market. However, bad color development of red Chinese sand pear greatly influenced the fruit appearance. The study on anthocyanin synthesis mechanism of red Chinese sand pear affected by fruit bagging treatments and different habitats will be of great help to develop a new technology to product high quality fruits.Two red Chinese sand pear cultivars,’Meirensu’(mid-maturing) and’Yunhongli No.1’(late-maturing) in different maturation periods, were used as materials in this study. Two ways were used to isolate the gene sequences, one is used specific primers or degenerate primers which were designed according to plant sequences from available databases, the other is used specific primers based on malus EST database. Combined with RACE methods, two PALS (PpPALl and PpPAL2), four CHSS (PpCHSl, PpCHS2, PpCHS3and PpCHS4), three CHIS (PpCHI1, PpCHI2and PpCH13), one F3H (PpF3H1), two DFRs(PpDFRl and PpDFR2), one ANS (PpANSl) and two UFGTs(PpUFGT1and PpUFGT2) were isolated from red chinese sand pear. Transcription factors PpMYB1and PpMYB10were cloned, and sequence homology analysis showed PpMYB10belong to the subgroup10R2R3MYBs.The coloration of red Chinese sand pears was significantly improved by fruit bagging treatments. Red Chinese sand pear’Meirensu’was used for fruit bagging treatments. Bags were removed15days prior to harvest and the fruits were completely re-exposed to light until harvest. Structural genes and transcription factors that involved in anthocyanin synthesis were analyzed during fruit coloration after the bags removal. Following bag removal, the expression levels of PpPAL1, PpCHSl, PpCHIl, PpF3Hl, PpDFR1, PpANSl and PpUFGTl were markedly enhanced in the sunlit side of ’Meirensu’ fruit; PpCHS1, PpF3H1, PpANSl and PpUFGT1maintained high expression levels in5days after the bags removal (DABG). All seven structural genes were enhanced by sunlight, indicated both genes were involved in the anthocyanin biosynthesis. Light intensity and quality are important for fruit coloration. Anthocyanin amounts increased linearly in the skin of the sunlit side of’Meirensu’ fruits. However, anthocyanin content was barely detected in the skin of the shaded side. The transcript level of anthocyanin biosynthetic genes was markedly up-regulated in the skin of sunlit side of’Meirensu’, but much lower in the skin from the shaded side. The expression level of PpCHSl, PpUFGT1, PpF3H1and PpANSl (1DABR) in sunlit side as compared to shaded side is above2.7folds. PpCHS1, PpF3Hl, PpANSl and PpUFGTl expressed at higher level in the skin of mature fruits than in other tissues, and PpCHSl, PpANSl also showed high expression in young leaves with faint red. The expression level was coordinated with anthocyanin level in different organs. The expression pattern of PpMYB10was similar to other structural genes during the coloration of ’Meirensu’ after the bags removal. However, PpMYB1showed less coordination with other genes.There were family members in structural genes that involved in anthocyanin synthesis. They showed many differences in sequences and expression patterns. The highest percentage for amino acid sequences was found between PpCHSl and PpCHS2(97%), while the lowest one was PpCHIl and PpCHI2(23%). The expression levels of PpPAL1, PpCHSl, PpCHS2, PpCHI1, PpCHI3, PpF3Hl, PpDFR1, PpANS1, PpUFGT1and PpUFGT2was markedly enhanced in the peel of ’Yunhongli No.1’fruit after the bags removal. Other structural genes including PpPAL2, PpCHS3, PpCHS4and PpCHI2showed no response, indicating that they may not involved in anthocyanin synthesis during fruit coloration. The expression pattern of these genes that involved in anthocyanin synthesis shows different ways, transcript levels of PpPAL1, PpCHS1, PpCHI1, PpCHI3, PpF3H1, PpDFR1,PpANSl and PpUFGTl peaked at1DABR. However, PpCHS2and PpUFGT2peaked at3DABR. All these genes might be involved in anthocyanin biosynthesis by being coordinately expressed during anthocyanin accumulation. The formation of anthocyanin was not only genetically determined, but also might be influenced by many environmental factors. Anning is located in Yunnan province, having a warm, high irradiant climate and high day and night temperature difference, while Xingyang is located in Henan province, with a hot, low irradiant climate and low day and night temperature difference. At harvest, red Chinese sand pear’Mantianhong’in Anning showed better coloration, higher concentrations of anthocyanin, carotenoid, total soluble solid (TSS) and soluble sugar but lower chlorophyll in pear peel, fruit weight, firmness and content of starch than those in Xingyang. Significantly increasing in the anthocyanin structural and regulatory genes, especially for PpCHS, PpANS, PpUFGT, PpMYB10and PpbHLH in red Chinese sand pears were found in materials sampled in Anning. The statistical analysis indicated that soluble sugar content was significantly associated with anthocyanin accumulation in both Anning (r=0.96981) and Xingyang (r=0.94433) habitats. Those results indicated high altitude, high-irradiant and high temperature differences in Anning habit were more conducive to the color development and inner quality of red Chinese sand pear, which was probably resulted in the higher concentration of sugar and expression of anthocyanin structural and regulatory genes, especially for the expression of PpCHS, PpANS, PpUFGT, PpMYB10and PpbHLH.