| Potato(So]anuni tuberosum L) plants were firstly transformed with the gene(h5nla) encoding an Avian Influena A (H5NI)Virus, and the gene was expressed in transgenic potato. Meanwhile, the transfer system was optimized in order to realize the aim of transgenic plant as low-cost vaccine production system. The results were as follows. 1. To optimize the regeneration system of potato using tuber disc as explants, it is necessary for the chosen media to have the high efficient regeneration shoots of tuber disc. Using MS as the basic medium and adjusting the hormone content and rate, four media were obtained which were Ml, M2, M3, M4. By comparing the regeneration rate of tuber discs on the four media, M4(MS-f-2.Omg/L BA+3.OmgIL ZT+0.5mgIL NAA+0.Smg/L GA3) was selected as the highest efficient one for shoots regeneration. And on M4 medium, ahead of time 20 days or so, shoots could be regeneraed from 89.4 percent tuber discs. Meanwhile care was taken to select more efficient media combination for making plants stronger and producing more tubers. The media were M7( I /2MS+0. 5mgIL BA+0. 5mg/L GA3+3%Suc) combinating with M14(MS?.Omg/L BA+3.Omg/L ZT+l0%Suc) , by which the number of tubers produced was twice as much as that on other media. The high efficient regeneration system is essential for high efficient transforma- tion system. 2. Four plant expression plasmids were constructed for transformation. P13O1BG was constructed for expressing green fluorescent protein gene(egfr); pl3OlB Q G was for enhanced 4 expression of eg4~, by inserting a 65bp enhancer( Q) between 35s promoter and gene egfp; p13OIH5NI was for expression of Avian Influena A (H5N1)Virus gene h5nla; pl3OlHBs was for expression a small subunit protein of Hepatitis B Surface Antigen(HBsAg). In the four plasmids, the CaMV35s promoter and nos terminator fragments were syncretized with reporter gene and targeted gene. The selectable maker gene terminated by polyA fragment for transformation of plants was hygromycin phosphotransferase gene which allows selection with hygromycin. And the selectable maker gene for plasmids was neomycin phosphotransferae gene, which allows selection with kanamycin. Agrobacterium strains LBA4404, EHAIO5 and AGL1 cells were transformed by the direct method with plasmids mentioned above prepared from Escherichia coil clones. 3. The transformation results mediated by gunpowder(ZHFQ-2), demonstrated?that the explants were seriously damaged during the transfer process, and no transfer plants were selected; By contrast, the results mediated by Agrobacterium were stable. 4. By comparing several conditions of transformation mediated by Agrobacterium, we found that the transfer efficiency was highly effected by strains, content of strain cells, hormone, and AS etc. Among the three strains (LBA4404, EH1O5 and AGL1), the invasiveness of the strain EHA 105 was the highest. The GUS transient activity under different transfer conditions showed that the following conditions selected were much more efficient for transformation: Agrobacteria that were resuspended and diluted with solution(MS+0.Smg/L GA3+AS I Oumol/L) four times, invaded potato tuber discs swirring at lOOrpmI28 0C for 20-30mm. Following co-cultivating, the explants were transfered to M4 medium with the addition of 30 u mo]IL AS...
|
PDF Full Text Request