Cloning And Transgenic Tobacco Identification Of Cbr From Dunaliella Salina

Dunaliella. Salina, enkaryotic unicellular green algae, able to proliferate under saline conditions ranging from 0.1 to 5. 5 mol/L NaCl. The reason of the salt tolerance was that there was a novel osmoregulation mechanism an a novel carotenoid biosynthetic pathway in D. Salina. In order to study the salt tolerance mechanism at the gene level, our laboratory successfully cloned the gene cbr (.carotene biosynthesis related} from D. salina by the traditional homogene cloning method. Then, the ORF of the cbr was obtained by RT-PCR and was introduced into tobacco for studying the gene function. By the RACE technique, the full-length cDNA of cbr was successfully constructed in D. salina. We analyzed the effects of osmotic shock on the gene expression of cbr by using sensitive semi-quantitative RT-PCR.1. Using the traditional homogene cloning method, we cloned a cDNA fragment of the cbr from D. Salina. The result of DNA sequencing indicated that 67.0% of the sequence of the fragment was homologous to the cbr in D. bardwail. Then, the full-length cDNA of cbr was constructed by the RACE technique based on this sequence.2. D. Salina was treated with hypoosmotic shock (NaCl 1.5mol/L→0. 5mol/L) and hyperosmotic shock (NaCl 1.5mol/L→3mol/L) for 3h, 6h, 12h, 24h, 48h. In order to analyze the effects of various osmotic shocks on the gene expression of cbr, serai-quantitative RT-PCR was performed with an inner standard of 23S RNA from D. Salina. The result showed that hypoosmotic shock and hyperosmotic shock had obviously different effects on the gene expression of cbr, respectively.3. Firstly, the vector pCAMBIA2301G with two gus reporter genes was constructed. Then one gus was substituted with the coding sequence of cbr obtained by RT-PCR, and pCAMBIA2301G-cbr was constructed. Finally the pCAMBIA2301G-cbr was introduced into Agrobacterium tumefaciens using the freeze-thaw method.4. After infected by A. tumefaciens, the leaf discs of tobacco were selected on plates containing MS inducement medium supplemented with 75mg/L kanamycin. Regenerated plants were transplanted into vermiculite:sand(1:1) medium.5. The regenerated plants were identified by PCR, Southern blot analysis and RT-PCR. PCR analysis showed that approximate 27% tested plants produced the target band. A few of the plants produced the band in the Southern blot analysis. Three plants were selected randomly for RT-PCR analysis. The target band was amplified in all the three plants.