The Study On Transformation Of ARF (Auxin Response Factor) Gene Into Tabacco

Auxin effects all the growth development periods of plants. The promoters of auxin early or primitive reaction gene generally include the minimal auxin reaction element sequence (TGTCTC). Auxin responsive factor (ARF) combines with this kind of auxin reaction elements uniquely and control the expression of auxin reaction gene. It has been reported that ARF gene expression was not effected by auxin concentration. We expect that the transgenic tobacco plant become a bioreactor and develop a new way to produce auxin protein.In this paper, tobacco aseptic seedling leaf were used as explants, and the gene was transferred into tobacco by Agrobacterium LBA4404 mediation which has been inserted with the plant expression vector pBin438 that contain ARF promoter and laid a foundation for scale production of ARF from transgenic plant. The main results are as follows:1. Regeneration system of plants.The regeneration system of tobacco was established. The adventitious shoots were induced from leaf explants of tobacco based on MS basal medium supplemented with 0.8 mg/L 6-BA and 0.05mg/L NAA. Then the regenerated plants were rooted on 1/2MS medium containing 0.02 mg/L NAA and 0.02 mg/L IBA. And the frequency was about 96.9%, 96.7% respectively.2. Transformation and selection of plants.Leaf explants had been pre-cultured for 2 days, then immersed in Agrobacterium suspension for 5-6minutes. The co-cultivation had been carried out for 2-3 days. After that, the transformants were obtained by transferring explants to selection medium containing 75mg/L kanamycin and rooting medium containing 75mg/L, 100mg/L kanamycin. And its rooted frequency was 66.1%.In comparison with CK, obvious morphological variation was observed in the transgenic tobaccos. Their leaves became greener and thicker, chief veins turned bolder and thicker than those of CK.3. Detection of transgenic plants.The PCR assay of plants showed that the target gene had been integrated...