Studies On Tissue Culture Of Floral Organs Of Saffron (Crocus Sativus L.) And Expression Of CsZCD Gene In E.coli

Saffron {Crocus sativus L), a perennial herbage of Iridaceae, is a precious herbal medicine and widely used as a colorant and flavoring agent. To explore the resources of saffron and officinal ingredient, tissue culture and molecular biological research of saffron were studied in this thesis. By using saffron's perianth and style as material, we set up a system of the induction and screening marker of callus, the induction, subculture and optimization of stigma-like structures, and in vitro flowering. At the same time, the Crocus zeaxanthin 7,8(7',8')-cleavage dioxygenase gene (CsZCD) was expressed in E.coli, and the product was purified. The following results were obtained.1. The callus and stigma-like structures were induced from Saffron's stigma, stamen, ovary, perianth, style, side bud and young leaf. The induction rate of callus and stigma-like structures from perianth and style were high in the medium with MS + NAA5 mg·L^-1+ 6-BA5+ 4%sucrose +agar 0.8% and B₅ + KT 5 + IAA4 + 2,4-D 1 + Pro 0.5+3%sucrose+agar 0.8%. Induced in medium of B₅ + KT 5 + IAA 4 + 2,4-D 1 + Pro 0.5+3%sucrose, loose style callus were easy to be subcultured.2. In the induction of style-stigma-like structure from saffron's perianth and style, we used medium with MS + NAA 5 + 6-BA 5+sucrose 4%+agar 0.8% and B₅ + KT 5 + IAA 4 + 2,4-D 1 + Pro 0.5+ sucrose 6%+agar 0.8%, and chose different length of top bud, in the different illumination and temperature, and sucroseconcentration gradient, optimized regeneration conditions of style-stigma-likestructure. The medium with MS + NAA 5 + 6-BA 5+sucrose 6%+agar 0.8%,18°C with dark is selected as the best medium for style-stigma-like structure. In this medium, the perianth style-stigma-like structure regeneration ratio can arrive 75.0%.3. Saffron's stigma, stamen, perianth, style were used as explants to induce in vitro flowering. The result showed that only perianth and style can be induced in vitro flowering in 20+2°C with dark. The induction ratio of perianth in MS + NAA 5 mg ? L':+ 6-BA5+ sucrose6%+agar 0.8% is 60%, and this ratio of style in B5 + KT 5 + IAA 4 + 2,4-D 1 + Pro 0.5+sucrose 4%+agar 0.8% is 25%.4. The present study explored the effects of antibiotics on growth of loose callus from saffron's style, including kanamycin(Kan), Hygromycin(Hyg), Ampicillin(Amp), cefotaxime(Cef) and Timentin(TMT), and initially determined the strategy of GMOs(genetically modified organism) screening of saffron. When the concentration of Kan is 500 mg ? L"1, callus growth would be inhibited significantly. As the bacteria inhibition, Cef had little effect on callus growth, Amp would induce the callus growth, its growth rate was increased obviously when high concentration of Amp was added. However callus growth was inhibited when TMT concentration at 750 mg ? L"1.5. For prokaryotic expression, the CsZCD which codes for a key enzyme in biogenesis of crcrcetin glycosides was cloned in saffron. The CsZCX>-NEW gene was obtained by PCR. The two gene were inserted into a expression vector pQE31 respectively. The recombinant plasmid CsZCD -pQE-31,CsZCD-NEW-pQE-31were constructed. In response to the IPTG induction, CsZCD, CsZCD-NEW gene was expressed in E.coli M15 and gave rise to the fusion proteins consisting of 6XHis fused to the N-terminal. Their contents were respectively about 13.7% and 14.4% among total bacterial cell protein. SDS-PAGE and Western blot analysis indicated that the fusion proteins with 49kD and 52KD were expressed stably in the form of insoluble inclusion bodies after IPTG induction. After denaturation, the purified recombinant proteins were purified by His Trap? HP chromatography column, and prepared for further research.