Functional Verification Of Methylation Regions On MdMYB1 Promoter And Effect On Pigmentation Of Different Color Type Apple Peel

Apple(Malus domestica)peel color is one of the most important index,which effects it`s commodity value.Synthesis and accumulation of anthocyanin which is effected by regulatory gene MdMYB1 is closely related to peel coloring.Researches had found that the methylation modification on MdMYB1 promoter has inhibitory action on pigmentation.In this study we used three key methylation regions(A:-1062～-964 bp,B:-2026～-1870 bp,C:-1898～-1633bp)analyzed from the green apple ‘Granny Smith’ to structure three promoters which lack of these regions.Then,we verified the function of region A,B and C by infecting tobacco leaves and ‘Pink Lady’ peel with Agrobacterium.Based on this,we used ten different color type varieties(‘Starkrimon’,‘Yue Shuai’,‘Qin Yang’ and ‘Hua Guan’ are red;‘Yin Du’ and ‘Dai Lv’are green;‘Jin Fu’,‘Jin Zhong’,‘Rui Xue’ and ‘Wang Lin’ are yellow ones)as materials,after bagging treatment,we determined their chromaticity values,pigments contents and analyzed methylation level of the three experiment regions on MdMYB1 promoter to further clarify how the methylation modification of MdMYB1 promoter effects the anthocyanin synthesis and peel color formation.The main results are as follows:1.We successfully completed the deletion of three methylation regions and obtained the Agrobacterium carried the corresponding recombinant MdMYB1 promoters.After infection of tobacco leaves and apple peel,we analyzed the GUS activity and infection performance and we founded that the lack of region A and B decreased the activity of promoters,with worse staining effect on leaf and non-pigmentation of peel,while there`s no significant differences whether MdMYB1 promoter lack of region C.Therefore,the overexpression of MdMYB1 and MdMYB1-C also raised the synthesis and accumulation of anthocyanin in apple peel with more consistencies on GUS activity and staining performance making coloring degree of experiment position higher than control part.We suggested that MdMYB1-C had similar function with MdMYB1,and region C might not be the key methylation region in MdMYB1 promoter.2.Through the analysis on methylation modification level of region A,B and C in MdMYB1 promoter of different color type of apple varieties we found that the key methylation regions of different apple varieties are different: the three target regions are critical ones to green varieties,while there`s no significant relativity to yellow ones.What`s more,region A is related to the anthocyanins` synthesis of red apples,but region C might not be the key one to them.Besides,region B is the key methylation region in MdMYB1 promoter of ‘Starkrimon’ and ‘Hua Guan’,but it has no significant correlation with ‘Qin Yang’ and‘Yue Shuai’.