The Research On Regulable Platid Expression System In Tabacco

Foreign proteins could be highly accumulated in the plastids of higher plants with plastid transformation. Comparing with nuclear transformation, plastid tranformation has many advantages, such as higher expression effeciency, more bio-safety, and expressing prokaryotic genes without modification. But the same as nuclear transformation, plastid transformation also has the problems of bio-safety of antibiotic markers and development obstacles due to the overexpression of foreign gene. The present research has studied the co-operation system of plastid and nucleus in tabacco, in which the nuclear and plastid genomes were both transformed. The nucleus was transfomed with T7 RNA polymerase gene under the control of the promoter of Ale, which could be induced by alcohol; and the plastid was transformed with foreign genes (such as GUS or NK) under the control of T7 promoter. In this system, high level expression of the transgenes in plastids can be controled, and the development of the plant can not be influenced.In this study, T7 RNA polymerase expression cassette controled by Ale promoter was firstly transferred into the nuclear genome of tobacco and transformants were obtained which were detected with PCR, Southern-blot and RT-PCR analysis.It's proved that the T7 RNA polymerase gene has already been integrated into the nuclear genome and expresssed. Then, two tobacco plastid expression vectors and two lettuce plastid expression vectors were constructed. They contained the homologous fragments of the plastid genomes respectively, the foreign gene (GUS or NK) expression cassette controled by T7 promoter and the expression cassette of the marker gene of BADH. At last, wild type tobacco and tobacco, whose nucleus has already been transformed by T7 RNA polymerase, were transformed with the vectors using aadA or BADH as marker gene, respectively. Spectinomycin(Spe) and streptomycin(Str) resistant transformants wereobtained. And the transformation results were discussed. The application of antibiotic marker free selection with BADH gene was also tried in the plastid transformation of tobacco.Betaine aldehyde resistant and PCR positive plants were obtained.In the end,a limitation result of this system was discussed.In the second part of the thesis, a chemical, Betaine aldehyde chloride (BAC), was found to have potential to significantly enhance the regeneration frequency of plant tissue by using Lactuca sativa as a model. Lettuce leaves were used as the explants and the calli were induced and the shoots were regenerated on the medium containing BAC (5mM). Compared with the control, in which the explants were cultured on the same medium but without BAC, the frequencies of callus induction and shoot differentiation were increased 29.3% and 42.0%, respectively. The reason that BAC could increase the frequency of plant regeneration was discussed.