| Cerasus humilis, as one kind of dwart shrubby fruit tree of rosaceae, is also called ’calcium fruit’. Cerasus humilis can be used to prevent wind, regularize sand and conserve water and soil with the characteristics of developed root system and strong adversity resistance. The nutrient-rich fruit with unique flavor can be eaten fresh or processed into juice, fruit wine, preserved fruit, etc. The roots and seeds can be used as medicine. The MYB transcription factors as one of the largest families in plant transcription factors play significant roles in regulating the secondary metabolism, especially in regulating the biosyntheses of anthocyanins and flavonoids. In order to provide a certain basis for the future research on the function of MYB transcription factors in the aspect of regulation of anthocyanin biosynthesis in Cerasus humilis, four of MYB family genes ChMYB10α, ChMYB10b, ChMYB4 and ChMYB114 were cloned by using the fruit of Cerasus humilis ’Nongda No.4’ as experimental material, and the relative transcript levels of ChMYB10 (including ChMYB10αa and ChMYB10b) and ChMYB4 in skin and pulp during fruit development were analyzed by using "Nongda No.41 and "Nongda No.5’ as experimental material. In addition, the expressing vectors of the four MYB genes above were constructed. Finally, the four MYB genes were transformed into tobacco. The main results for this study were as follows:(1) Four of MYB family genes ChMYB10α, ChMYB10b, ChMYB4 and ChMYB114 were cloned from Cerasus humilis ’Nongda No.4’ fruit during color-changed period by using homology cloning and electronic cloning technique. Both ChMYB10α and ChMYB10b encoded 239 amino acid molecules, while ChMYB4 and ChMYB114 encoded 202 and 222 amino acid molecules, respectively. Sequence analysis showed that all of the four MYB genes were typical R2R3-MYB transcription factors. The phylogenetic analysis indicated that the four MYB proteins had closer evolutionary relationship to the MYB genes of subgroup 6 in Arabidopsis thaliana.(2) The relative transcript levels of ChMYB10 and ChMYB4 in skin and pulp during fruit development were analyzed by using ’Nongda No.41 (red skin red pulp) and ’Nongda No.51 (yellow skin yellow pulp) as experimental material. The result of Real-time Quantitative PCR showed that the expression quantity of ChMYB10 and ChMYB4 in skin and pulp of ’Nongda No.4’were higher than those of ’Nongda No.5’ during the fruit growing periods.(3) The expressing vectors of four MYB genes ChMYB10α, ChMYB10b, ChMYB4 and ChMYB114 were successfully constructed, named PMV-ChMYB10α, PMV-ChMYB10b, PMV-ChMYB4 and PMV-ChMYB114, respectively. Results of Agrobacterium-mediated transient expression analyses of the four MYB genes in tobacco leaves showed that the transient expression of the four MYB genes had no obvious effect on the accumulation of anthocyanins at injection sites, respectively. The four MYB genes were transformed into tobacco via Agrobacterium mediation using leaf disc method and the regenerated tobacco plants were obtained, respectively. It had no obvious accumulations of anthocyanins in the PCR-positive regenerated tobacco plants of all the four MYB genes. |
