Genetic Transformation And Expression Analysis Of A Vascular Tissue-Specific Promoter From Poplar Clones

Poplars belong to Salicaceae Populus, which is very popular throughout in the china. It not only is an important sylvicultural and commercial tree, but also has important use in maintenance balance of ecosystem and the resolvent of wood shortage.Popla has many advantages,such as fast growth, good adaptability, wide adaptation. Selecting Triploid of PopulusTomentosa,741poplar,Populus×euramericana'Neva',Populus×euramericana'Guariento'and tobacco the experiment materials,the leaf regeneration systems were established and optimized the Agrobacterium tumefaciens mediated transformation system. Mediated by Agrobacterium tumefaciens, transfer pProNAC068::GUS were integrated into tobacco,pProNAC157::GUS were integrated into Poplars and tobacco genome.Some transgenic plants were tested on DNA levels and GUS gene activity.The main results were as follows:Kanamycin (Km) critical concentration was 50 mg·L-1 and Cefotaxime was 300 mg·L-1for the tobacco transformation. pProNAC068::GUS and pProNAC157::GUS were transformed into mode plant with leaf disc transformation respectively. At last we obtained 17 and 3 plants of Km resistant.PCR detections indicated that genes were integrated into the tobacco genome.A GUS histochemical assay showed that pProNAC068::GUS of GUS activity was found in root, stem and leave, but the pProNAC157::GUS of GUS activity was not found in root, stem and leave.The leaf high frequency transformation system of Triploid of PopulusTomentosa and 741 poplar was established.First, a high frequency regeneration system of Triploid of PopulusTomentosa has been established.The best shoot regeneration mediumwas MS+6-BA 1.0 mg·L-1+NAA 0.1 mg·L-1 and MS+TDZ 0.1 mg·L-1+NAA 0.1 mg·L-1, the root induction medium was 1/2 MS+6-BA 0.3~0.5 mg·L-1.Infected liquid concentration OD600 was 0.4~0.6, infection of leaf explants for 8~10min with Agrobacteria and cocultivation for 2 days after infection would befavorable for the transformation. Kanamycin critical concentration was 50 mg·L-1and Cefotaxime 400 mg·L-1 was a suitable concentration to control the propagation of Agrobacteria.16 and 2 plants of Km resistant with pProNAC157::GUS were gained via strictly selection by Km. The results of PCR showed that the foreign gene has been integrated into genome of Triploid of Populus Tomentosa and 741 poplars. A GUS histochemical assay showed that GUS activity of Triploid Populus Tomentosa was only found in some small amount of the stem and was not found in root and leave; the GUS activity of 741 poplars was not found.The regeneration system of Populus×euramericana'Guariento'was established. The shoots regeneration medium were MS+ 6-BA 0.6 mg·L-1+NAA 0.2 mg·L-1 .Then the regenerated plants were rooted on 1/2 MS+IBA0.3 mg·L-1.Kanamycin (Km) critical concentration was 30~35mg·L-1and Cefotaxime 400 mg·L-1 was a suitable concentration to control the propagation of Agrobacteria. Several crucial factors influencing the transformation efficiency were studied.It was found that OD600=0.4, infection of leaf explants for 8~10 min with Agrobacteria and cocultivation for 2 days after infection would befavorable for the transformation.Mediated by Agrobacterium tumefaciens pProNAC157 genes were transformed into Populus×euramericana'Neva'and Populus×euramericana'Guariento',there were 2 Kanamycin resistant buds of Populus×euramericana'Neva'.PCR detections indicated that genes were integrated into the Populus×euramericana'Neva'genome. A GUS histochemical assay showed that the GUS activity was not found in root, stem and leave.