Effect Of SP Promoter And Intron On The Expression Of Foreign Gene In The Transgenic Poplar

Popular is an important sylvicultural and commercial tree, which is very popular throughout china and other places of the world. Triploid is the star among the Poplar and has become one of the most important sylvicultural trees for its high values such as growing fast, high resistance, long fibre etc. In addition, because the high sterility character makes Triploid Poplar have to reproduce by clonal propagation, there is little biosafety problem when the transgenic plants are put into the field. So we select the BT-18 Triploid Populus Tomentosa as the research material. Several factors such as hormone concentration were investigated to optimize the regeneration system in vitro. A high frequency regeneration system in culture has been established.SP promoter was achieved by artificially splicing the sequences derived from the octopine synthase (ocs) and mannopine (mas) genes. The SP promoter is approximately 156-fold and 26-fold stronger than are the CaMV35S and double CaMV35S promoters in the leaf tissue. It is also observed a root-preferential pattern of expression. In our study, four expression vectors were constructed by spliced SP and CaMV35S promoters to GUS and intron-GUS reporter genes respectively. Basing on the high frequency regeneration system of BT-18 Triploid Populus Tomentosa, we obtained lots of transgenic plants through the developed Agrobacterium-mediated transformation method. Then we assayed the expression level between SP promoter and CaMV35Spromoter in different tissues of BT-18 Triploid Populus Tomentosa and analyzed the effect of intron on the expression of the foreign gene. This study will provide some basic and applied information for the future task of transgenic poplar. Conclusions are drawn and shown as following: First, A high frequency regeneration system has been established. The best shoot regeneration medium is MS supplemented with 0.5mg/L 6-BA, 0.3mg/L IBA, 0.15mg/L KT and 30mg/L Ad; The best shoot elongation medium is MS supplemented with 0.3mg/L 6-BA, 0.1mg/L NAA, 0.1mg/L KT and 20mg/L Ad; The best root regeneration medium is 1/2MS supplemented with 0.1mg/L NAA and 0.05mg/l IBA. The survival pressure of Kan for the regeneration of transgenic shoot was confirmed as 40mg/l by the gradient antibiotic resistance experiment. The browning problem was prevented by adjusting the concentration of hormone, reducing the time of sterilization, continually refreshing the medium, adding sorbent such as active carbon etc. The problem of vitrification is resolved by reducing the concentration of Cytokinin, healing the culture bottle by the breathing freely lambskin, increasing the concentration of agar, using the 1/2MS as the root regeneration medium etc. Second, we constructed two clone vectors (pUCGI, pUCG) and two expression vectors (pBI2, pBISN2). The construction method has been carefully studied.Third, we developed the traditional Agrobacrium-mediated transfomation method and make it more efficient. Lots of transgenic plants have been obtained through the Kan screen. Then the transformants were identified by the PCR analysis. Contaminations in PCR are analyzed and resolved.Forth, the histochemical analysis of immediate GUS activity shows that the level of GUS activity expressed by the SP promoteris higher than those expressed by CaMV35S promoter. In addition, we observed the GUS activity obtained with the CaMV35S－GUS fusion gene in agrobacterium. But no GUS activity was detected with the CaMV35S-intron- GUS fusion gene in agrobacterium due to the lack of a eukaryotic splicing apparatus. So the intron containing GUS gene is better than GUS gene as a reporter gene in transient transformation experiments.Fifth, the histochemical analysis of stable GUS activity and fluorescence quantity assay showed that:(1) SP promoter drives the basipetal GUS expression in the transgenic poplar and the older the leaf, the higher activity of GUS;(2) Comparisons have been made based on the range of expression levels found for each promoters. The SP prom...