Studies On The Different Mechanism Of Pigment Development In 'Nanguo' Pear And 'Nanhong' Pear

Peel color is one of the important indexes to evaluate the appearance quality of the fruit. 'Nanhong' pear is a red mutant of 'Nanguo' pear, and almost all of the 'Nanhong' pear peel is red. It tastes excellent and favored by consumers. Study on the molecular mechanisms of peel coloration in 'Nanhong' pear will provide a theoretical basis for breeding high quality red pear variety. In this study, we cloned red fruit peel-related genes from 'Nanguo' pear and the red mutant'Nanhong'pear, and analyzed the expression levels of these genes in various periods of the fruit development. Our study provide new clues for elucidating the difference of coloring mechanism between 'Nanguo' pear and 'Nanhong' pear from the point of view of physiology and molecular biology.1. Through measuring the content of anthocyanin and chlorophyll in the peel of'Nanguo' pear and 'Nanhong' pear at different development stages, we found that the content of anthocyanin in the peel of 'Nanhong' pear was significantly higher than that of 'Nanguo' pear at all the development stages, espically at 144 DAFB. The anthocyanin synthesis in the peel of 'Nanguo' pear decreased gradually after 60 DAFB which gradually increased in the peel of 'Nanhong' pear. However, there was no significant difference of the chlorophyll content between 'Nanguo' pear and 'Nanhong' pear, indicating that the coloring difference between them is induced by the content difference of anthocyanin between them.2. We identified the differentially expressed genes (DEGs) involved in anthocyanin synthesis between the peel of 'Nanguo' pear and 'Nanhong' pear at 144 DAFB by RNA-Seq. We obtain ten different genes of expression, including structural gene PuCHS?PuF3'H? PuDFR?PuANS?PuANR?PuUFGT and regulatory gene PuMYB10?PuMYB12?PubHLH? PuWD40. Through analyzing the expression levels of the ten genes at different developmental stages, we found that the expression trends of the structural gene PuUFGT and the regulatory genes PuMYB10 and PuMYB12 were consistent with the changes of anthocyanin synthesis in the peel of 'Nanguo' pear and 'Nanhong' pear.3. To determine which caused the expression difference between PuUFGT?PuMYB10 and PuMYB12, we cloned the full sequences and the promoter sequences of the tree genes in the peel of 'Nanguo' pear and 'Nanhong' pear. Nether the full sequences nor the promoter sequences of the three genes between 'Nanguo' pear and 'Nanhong' pear are different, indicating that the coloring difference between 'Nanguo' pear and 'Nanhong' pear is not induced by full sequence and promoter sequence mutation of the anthocyanin synthesis structural gene PuUFGT and regulatory gene PuMYB10?PuMYB12.4. To testify wether there was epigenctic difference between the of'Nanguo'pear and 'Nanhong' pear, we analyzed the methylation status of the promoter regions of these two genes by McrBC-PCR and BSP. The results showed that there was no significant difference in the degree of methylation between PuMYB10 and PuMYB12 promoter regions of 'Nanguo' pear and 'Nanhong' pear, suggesting that the coloring difference between 'Nanguo' pear and 'Nanhong' pear is not induced by methylation in the promoter regions of PuMYB10 and PuMYB12.We have obtained 9 protein kinase related genes. Two of the 9 genes showed higher expression level in the peel of 'Nanhong' pear than that in 'Nanguo' pear at different development stages, speculating that different expression level of protein kinase related genes may play a role in the coloring difference between 'Nanguo' pear and 'Nanhong' pear.In conclusion, the coloring difference between 'Nanguo' pear and 'Nanhong' pear is not induced by sequence mutation of the genes involved in anthocyanin biosynthesis pathway and changes of methylation of PuMYB10 and PuMYB12 gene promoter. We speculated that the different expression level of protein kinase related genes in the peel of 'Nanguo' pear and 'Nanhong' pear may play a role in coloring difference of them.