Cloning Of MAPK Gene In Dunaliella Salina And Transformation To Tobacco

Mitogen-activated protein kinase belong to Ser/Thr protein kinases and exist in eucaryota. It transmit upstream signal to downstream by means of Phosphorylation of Ser/Thr position, playing a vital role in regulating the operation of cell cycle and gene expression.Recently,more and more experimental proofs and datas indicated that it has very important effects on plant growth and development, as well as stress resistance. Plant growth is severely affected by stress environment, which makes stress resistance research more important. MAPK gene was successfully cloned from Dunaliella.salina by tranditional homogene cloning method.And the recombinant plant expression vector pBI121-MAPK was constructed and introduced into Agrobaterium tumefacines EHA105. Subsequently, MAPK gene was transformed to tobacco through Agrobaterium-mediated system. Transgenic plants were tested to study the transformation of MAPK gene in tobacco. It provides important foundation to other forages. The results of the experiment were as follows:1. Cloning of MAPK gene in D. salinaThe traditional homogene cloning method was used to clone a cDNA fragment of MAPK from D. salina. The full-length cDNA of MAPK was constructed by the RACE technique based on this sequence.2 . Construction of plant expression vector pBI121-MAPKThe expression vector pBI121-MAPK was constracted using DNA recombination technology and introduced into Agrobacterium tumefacines EHA105 by freeze-thaw method .3. Regeneration system of tobaccoThe adventitious shoots were induced from leaf explants of tobacco based on MS +2.0 mg/L 6-BA + 0.1 mg/L NAA and rooted on MS + 0.2 mg/L NAA to gain completely regenerated plants. And the frequency was about 52.4%,88.5% respectivly.4.Selection of transgenic plantsTobacco leaf explants were pre-cultured for 2 days, then immersed in Agrobacterium suspension for 10 minutes, and co-cultivated for 2 days. The...