Homologous Transformation And Functional Verification Of Chrysanthemum CmDREBa-2 Gene

Chrysanthemum is widely loved as one of the top ten flowers and one of the four gentlemen flowers in China.In this study,the leaves of Chrysanthemum variety‘Zaoyupan'sterile seedlings were used as experimental materials,based on the establishment of its high-frequency regeneration system,the factors influencing the genetic transformation process were analyzed and screened.The CmDREBa-2 gene was transformed into‘Zaoyupan'using agrobacterium-mediated method,a high efficient genetic transformation system of‘Zaoyupan'was established,and the resistant plants were screened and functionally verified.This study lays a foundation for understanding the biological function of CmDREBa-2 gene.The main test results are as follows:1.The regeneration ability of the upper leaves of the sterile seedlings of the Chrysanthemum variety‘Zaoyupan'was stronger than that of the middle and lower leaves.2.Chrysanthemum variety‘Zaoyupan'adventitious bud differentiation medium is MS+0.6mg·L^-16-BA+0.2mg·L^-1NAA;The most suitable medium for rooting is 1/2MS+0.3mg·L^-1NAA.‘Zaoyupan'kanamycin screening adventitious buds concentration is 15mg·L^-1;Screening concentration of kanamycin adventitious shoots is 15mg·L^-1;Cefotaxime inhibits the Agrobacterium screening concentration is 200mg·L^-1.Genetic transformation system is:pre-culture for 2d,bacteria concentration is OD₆₀₀=0.6,the dilution factor is 50 times and infected for 7min,then co-cultured for 2d and delayed time for 2d.This variety obtained 8resistant plants,and PCR test showed 3 positive plants.3.The PCR-positive transgenic plants were detection and verification for drought resistance and white rust of Chrysanthemum disease resistance.Test results show,under dehydration in nature water stress,the relative water deficits of three transgenic plants?CmDREBa-2-OX4,CmDREBa-2-OX5,CmDREBa-2-OX8?were significantly lower than those of wild-type plants?WT?.At 6 days of water stress,the water saturation deficit coefficient of WT is 1.5 times that of CmDREBa-2-OX4,2.2 times that of Cm DREBa-2-OX5and 1.4 times that of CmDREBa-2-OX8,among them,CmDREBa-2-OX5 has the strongest resistance to water stress.The water loss rate of wild type was 1.6 times that of CmDREBa-2-OX5 at 4h of water loss,was 1.45 times that of CmDREBa-2-OX5 at 5h of water loss.The wilting index of wild-type plants was higher than that of Cm DREBa-2-OX5 at2d,4d and 6d on water stress,the transgenic plants showed stronger resistance to stress than the wild type plants.Under the stress of white rust of Chrysanthemum pathogen,CmDREBa-2gene is over expressed in CmDREBa-2-OX5,6h after inoculation reached the peak expression of about 3 times the control group.