Transformation Of The Chrysanthemums With AFL2 Gene And Prokaryotic Expression Of AFL1 Gene

There were a pair of LFY homologous genes AFL1 and AFL2 which clonedfrom apple, long approximately 1.4kb, have the key function in floralformatting of apple, their alkali base sequences have 90% homology. In orderto the more thorough research the product of AFL gene expresses and themolecular mechanism of floral formation, this research take this pair of genesas materials, has researched the prokaryotic expression and chrysanthemumtransforms of them.First part: By the leaves of three glass cultivars(JA: Late flowered bigchrysanthemum lion; JB: Small early chrysanthemum; JC: In Augustchrysanthemum) of chrysanthemum (Dendranthema morifolium) as materials,studied the effects of pre-cultured way, the pre-cultured time, the infectiontime, the infection conditions, altogether-cultured time on chrysanthemumtransformed and several antibiotics on regeneration of chrysanthemums. Inorder to screen one kind of effective antibiotic that can suppressedAgrobacterium, we use four kinds of antibiotics on test. The result indicatedthat, cultured two day, the infection time 10 minutes, altogether-cultured 2day, the antibacterial cultured 8 to 10 days, the effect is best. The Kanamycin5mg/L can effectively suppressed chrysanthemum regeneration;Carbenicillin 400mg/L can suppressed Agrobacterium regrow, and also lestinfluence to chrysanthemum's regeneration.Finally we got three transgenic plants of chrysanthemum underchrysanthemum regeneration and chrysanthemum heredity transformationexperiment. Although the transgenicplants present differences in early growth stage,but recover soon on later stage.Second part: In this study, the AFL1 gene was cloned into a prokaryoticexpression vector pET28b after enzyme digesting to construct therecombinant plasmid pETAFL1 that based on have got the cloning vector.The recipient cell of Escherichia coli BL21 was transformed with pETAFL1,and then cultured. AFL1 gene was expressed under the induction of IPTG andidentified by SDS-PAGE. The result indicated that AFL1 can expressed aprotein approximately 53KD, with same we estimated, although theexpression level is lower.