Transferring Canz Into Tabacoo Mediated By Agrobacterium Tumefaciens

CaNZ is the CaN plus-sense gene. CaN is a Ca2+/CaM-depended Ser/Thr proteinphosphotase existing in the eucaryons, which includes a catalytic subunit—calcineurin A and a Ca2+ combined'subunit—calcineurin B. The activity of calcineurin A is regulated by CaM. According to the researching methods for animals, through the purpose gene transfered by Agrobacteritunm, fusion protein of expected expression is excreted out of cell and combin with CaM with the signal peptide and bearer protein of CaM combined peptide. So the function of CaM would be restrained, thereby constructing the CaM's antisense plants. By observing the phenotype change of CaM's antisense plants, the function of CaM on the plants'growth and development was suggested.(1) First, the regeneration system of somatic embryogenesis is established for alfalfa genetic transformation. The 14-16 days old tabaco leaf with high regenerability was selected as the plant material. Based on the MS medium, a series of medium were devised and selected by effect. The medium of inducing differentiation of explants is MS+IAA 0.5mg·L-1+6-BA 1.5mg·L-1,the medium of including rootage of sprouts is 1/2MS+NAA0.1mg/L.(2) Kanamycin is used to select transigenic explants. When Kanamycin concentration is below 50mg/L, the tobacco explants grow well;when Kanamycin concentration is 75mg/L, a majority of explant became white; when concentration is 100mg/L,all explant become white,so Kanamycin concentration of 100mg/L could be regarded as the selection-pressure. The rootage of sprouts is sensitive to antibiotic. So it is enough to select explant,when Kanamycin concentration is 50mg/L. The factors influencing Agrobacterium-mediated tabacco transformation were studied in systematism, When three days pre-cultured, Agrobacteritunm in the phrase of loizarithlm Growth was deluted to 1/10 of original concentration, explants were mixed with cell suspension of Agrobacterium in 6-8 minutes, three days col-cultivated on the axenic filter paper.(3) General DNA of transgenic plants was taken as template, the DNA of plasmid as was taken masculine contrast, the DNA of common green tabacco plants was taken as negative contrast, then carried through PCR amplification. PCR analysis showed that 21 plants were masculine plants. The percent of transformation frequency is 56.7%. we elementarily confirmed that CaNZ gene existed in the DNA of plants.(4) A majority of plants analysed masculine were short and grow slowly,compare to the contrast.Certainly,there were morphological characters. We need more evidences if want to confirm the effect of gene transformed on calcineurin signalling mechanisms.