Abstract:

According to the published DNA sequence (GenBank Z00044.1) of tobacco chloroplast high-frequency homologous recombination fragment, primers were designed to amplify a 3.663 kb DNA fragment named ctDNA from chloroplast genome of tomato by PCR. The fragment was not only 96.7% homology compared with the tobacco fragment reported in GenBank, but also 95.8% homology compared with the tobacco fragment used in this paper. The tomato chloroplast multicistron expression vector pLM2（Fig.1） (-psbD-Prrn-RBS-man-RBS-gfp-RBS-aadA -psbA3’-trnG-) was constructed with ctDNA, Prrn, man, gfp, aadA and psbA3’. And the tobacco leaves were bombarded with golden particles coated with the vector pLM2. Three transgenic tobacco shoots were obtained on the screening medium. The function of aadA gene was identified by screening medium（Fig.3）, and the function of gfp gene was confirmed by laser scanner（Fig.4）. The expression of man gene was identified by Western blot（Fig.5）. The genes man, gfp and aadA being in the chloroplast of tobacco were confirmed by PCR（Fig.6）. And the tobacco chloroplast multicistron expression cassette integrating in tobacco chloroplast genome DNA was confirmed by RFLP（Fig.7）. The results were showed that the three genes( man, gfp, aadA ) which were in the tomato chloroplast multicistron expression vector pLM2 were expressed in tobacco chloroplasts.

Key words: Multicistron, Construction of Tomato chloroplast expression vector, Tobacco plastid genetic transformation, Bombardment microprojectiles