Studies On The Differential Expression Of CDNAs Induced By Methyl-jasmonate In Taxus Chinensis Var. Mairei

Taxol, a diterpene amide, has been approved for the treatment of cancers by FDA in 1992. Taxol can promote microtubule's aggregation and restrain its decompose. It can restrain cell's mitosis and make cell's division block in G2/M stage. It is widely distributed at a relatively low content in the yew trees (Taxus spp.), so it is very important for all of us to know the synthesis mechanism and enhance the yield of taxol.Differential display reverse transcription polymerase chain reaction (DDRT-PCR), one of the most widely employed techniques to identify differential gene expression, has become a powerful tool in the area of molecular genetics since 1992 when the technique was invented by Liang Peng and Dr. Pardee. The technique is simple, time-saving, sensitive and reproducible.In present research, we adopted both mRNA differential display and silver staining techniques to isolate cDNA induced by MJ(200 p M), total 69 differential expression cDNA fragments were obtained. Suspending cells induced by MJ(200uM) for 6h 16h 24h was used as treatment sample, and suspending cells without induced as control sample. Differential expression of mRNA between treatment sample and control sample was analyzed by DDRT-PCR. The PCR amplification products were separated by 6% denaturing polyacrylamide gels. Distinct bands were visualized by silver staining. The length range of differential displayed PCR products mostly extended from 150 bases to 750 bases. Among the total 69 cDNA fragments, only 10 positive cDNA fragments have been cloned and sequenced after being testified by Reverse Northern, seven up-regulated and three down-regulated cDNA fragments included. These fragments were cloned in the pGEM?T Easy Vector. Through sequencing and then querying EST database of GeneBank, we can find that four of them have high homologous sequences while the other six have poor. Of these ten fragments, W-10 was 93% homologous to Taxus chinensis taxadiene synthase cDNA clone. Especially, it strongly expressed after being treated 6 hours later with MJ. This is correspondent to the expectation. In order to know its function, we must get its full length. Put under a strong promotor it will improve synthesis of taxol, there is still much work to do. Three fragments were highly homologous to other plants cDNAs, but not related to taxol synthesis. The other six were low homologous to EST library and, therefor, it most probably be a novel gene, and there is still much work to do for its full length and its function identification.