| Apple(Malus domestica Borkh.)is a fruit tree with high economic value.Apple of the red color is liked by all the people.Therefore,it is of great significance to isolate genes related to fruit coloring and perform further functional verification.Earlier,the research team found that the expressions of MdbHLH51 and MdbHLH155 in ‘Delicious’ apples were significantly related to the anthocyanin content.In this study,MdbHLH51 and MdbHLH155 were cloned from ‘Delicious’ and the overexpression vectors MdbHLH51-GFP and MdbHLH155-GFP were constructed.To elucidation of the functional role of apple MdbHLH51 and MdbHLH155 in anthocyanin biosynthesis,subcellular localization analysis in the onion epidermal cell,transient expression in fruit skin and ‘Gala’ apple callus transformation experiment were conducted.In addition,MdbHLH51 and MdbHLH155 were transformed into tobacco and Arabidopsis thaliana by Agrobacterium-mediated.The main results are as follows:1.MdbHLH51(gene sequence number: MD01G1155000)and MdbHLH155(gene sequence number: MD11G1252200)were cloned by RT-PCR that utilize the cDNA of ‘Delicious’ apple skin as a template.The number of amino acids of MdbHLH51 is 279,and the open reading frame is 837 bp.The number of amino acids of MdbHLH155 is 416,and the open reading frame is 1248 bp.Bioinformatics analyses show that MdbHLH51 and MdbHLH155 are predicted to be localized in the nucleus.The phylogenetic tree indicates that MdbHLH51 are more closely related to the ZmB and ZmLC involved in anthocyanin biosynthesis in maize,MdbHLH155 are more closely related to the MdbHLH3,AtTT8 and PhAN1.Sequence analysis of MdbHLH51 and MdbHLH155 with the bHLH protein that regulates anthocyanin synthesis in other species showed that MdbHLH51 and MdbHLH155 are typical bHLH transcription factors.2.The onion epidermal subcellular localization study showed that MdbHLH51 and MdbHLH155 were located in the cell nucleus,it gives the same result as the bioinformatics analyse.Overexpression of MdbHLH51 and MdbHLH155 promoted anthocyanin accumulation in ’Golden Delicious’ apple skin around the injection sites.Expression levels of anthocyanin synthetic and regulatory genes including MdCHI,MdCHS,MdF3 H and MdUFGT were extensively up-regulated.Overexpression of MdbHLH51 and MdbHLH155 promoted the coloring of callus in ’Gala’ apple under strong light,and its anthocyanin content is 3.4 times compared with the control.3.In order to further verify the function of MdbHLH51 and MdbHLH155,MdbHLH51 and MdbHLH155 were overexpressed in tobacco and Arabidopsis.The results showed that MdbHLH51 and MdbHLH155 enhanced the accumulation of anthocyanins on the veins from Arabidopsis and up-regulated the expression levels of the genes-related anthocyanins biosynthesis(AtF3H,AtCHS,AtCHI and AtDFR),which indicated that MdbHLH51 and MdbHLH155 played a positive regulatory role in anthocyanin synthesis. |
