Establishment Of VIGS Protocol And Functional Analysis Of McMYB10、McMYB16 In Malus Crabapple

Color variation of leaves, fruits and other organs during plant growth and development has been the focus of extensive research, especially the coloring mechanism. Brightly colored, beautiful leaf color, rich fruit color, beautiful plant type, Malus crabapple is a good research material to explore the mechanism of plant coloration. Simplicity, rapidity and high silencing efficiency, VIGS is very suitable for high-throughput of plant gene functional analysis. Firstly, VIGS system was established and optimized in spring-red-leaf cultivar ’Red Begonia’. Secondly, to verify the optimized protocol, we silenced McMYB10, McMYB16 gene in ever-red-leaf cultivar ’Royalty’ and ever-green-leaf ’Flame’ plantlets and fruitlets, repectively. Flavonoid contents, for example anthocyanin, McMYB10, McMYB16 and related genes expression were measured. Finally, we verified functions of McMYB10 and McMYB16 in heterologous tobacco and strawberry, repectively. The main results are as follows:1, To construct TRV2-McMYB10 and TRV2-McMYB16 vector, a pair of degenerate primers was designed from conserved regions of McMYB10, McMYB 16, respectively;2, The results of firstly silencing McMYB 10 in spring-red-leaf cultivar ’Red Begonia’ showed that stronger silencing occurred at lower temperatures 19℃ day/18℃ night and after higher vaccum pressure 0.09 MPa, with the condition of 60-80% RH, the light intensity 1800-2000 Lux, photoperiod 16h day/8h night.3, Compared with the control, coloration from green to red significantly delayed in cultivars ’Red Begonia’ and ’Royalty’ and systemic silencing phenotypes in ’Royalty’ fruitlets after silencing McMYB10; McMYB 10 expression and anthocyanin content were significantly decreased.4, Compared with the control, green turned to red in portions of ’Flame’ leaves and fruitlets and McMYB16 expression was significantly decreased, while anthocyanin content was increased after silencing McMYB 16.5, In the silenced tissues, the presence of TRV1 and TRV2 were detected by semi-quantitative RT-PCR, and GFP was detected confocal laser scanning microscope.6, Overexpressing McMYB10 in heterologous tobacco significantly promoted anthocyanin accumulated of tobacco petals; and silencing McMYB16 in heterologous strawberries also promoted to accumulate anthocyanin in strawberries.