Cloning And Prokaryotic Expression Of Genes Encoding OsCAS Or OsLIMS And Their Genetic Transformation

Previous studies have shown that rice emits more volatile terpenoids,β-caryophyllene and limonene when rice plants were treated with MeJA/JA or infested by the rice stripped stem borer, Chilo suppressalia (Walker). Moreover, it has been demonstrated thatβ-caryophyllene has a strong attraction of Anagrus nilaparvata Pang et Wang, an egg parasitoid of rice planthoppers. However, the role ofβ-caryophyllene and limonene in the whole induced rice defense against herbivores remains unknown. In order to determine the biological functions ofβ-caryophyllene and limonene in defense response of rice plants, the full length sequences of both OsCAS and OsLIMS genes were cloned, and their prokaryotic expression, mutant (overexpression or RNAi) lines, and induced expression profile of the OsCAS gene were studied. The results are as follows:1,According to the published sequence, a full length sequence of the rice OsCAS gene was PCR amplified and cloned in pMD19-T vector. Phylogenetic trees analysis of the rice OsCAS protein with other characterized plantβ-caryophyllene synthase revealed that rice OsCAS gene had a high identity to Zea mays (homologous 99%), whereas a low identity to Arabidopsis thaLiana, Medicago truncatula, Cucumis sativas and Artemisa annua (homologous only 51%). Infestation by rice brown planthopper Nilaparvata lugens (St(a|°)l) (at 1 and 12 h) or MeJA/JA treatment (at 1 and 24 h) enhanced the expression levels of the rice OsCAS gene, but H₂O₂ treatment didn't express. A prokaryotic expression vector pET32a(+)-OsCAS for OsCAS gene was construced and expressed in E.coli BL21(DE3). SDS-PAGE analysis showed that the recombinant protein was obviously expressed at 3, 4 and 5 h after induced by IPTG. By using Agrobacterium-mediated transformation system, rice mutant lines with overexpression or RNAi of the OsCAS gene were set up, which will obtain the foundation for elucidating the function of the gene in the defense responses of rice plants.2,According to the published sequence, a full length sequence of the rice OsLIMS gene was PCR amplified and cloned in pMD19-T vector. Phylogenetic trees analysis of the OsLIMS protein with other characterized plant limonene synthase revealed that rice OsLIMS gene had a low identity to other plants (homologous 41%), which is obviously lower than other plants each other (homologous 45%-99%). A prokaryotic expression vector pET32a(+)-OsLIMS for OsLIMS gene was construced and expressed in E.coli BL21(DE3). SDS-PAGE analysis showed that the recombinant protein was obviously expressed at 4,5,6,7 and 8h after induced by IPTG. By using Agrobacterium-mediated transformation system, rice mutant lines with overexpression or RNAi of the OsLIMS gene were set up, which will obtain the foundation for elucidating the function of the gene in the defense responses of rice plants.