Biosynthesis Regulation Of Paclitaxel In Cephalotaxus Mannii

Cephalotaxus mannii distribute at the most southren part among Cephaltaxaceae species, which is an endangered, rare and national key protected plant. Its bark, stems, leave and seeds contain abundant alkaloid substance harringtonines with anticancer activities. Its potential commertial value has attract great attentions.Besides, paclitaxel (taxol), an anticancer agent, was first found by us in Cephalotaxus mannii. With2-year-old seedling as material, we dissect gene express and regulation involved in paclitaxel biosynthesis after challenged by elicitors. These results could provide some theoretical references for Cephalotaxus mannii genetic resources exploring and artificial induction of biosynthesis of taxol. The main results are as follows:(1) By using chlorophyll fluorescence techniques, the two fluorescence kinetic parameters Y(NPQ) and Y(NO) are affected after challenged by elicitors, which indicated that we can use elicitor to micic the stress Cephalotaxus mannii exposures.(2) Firstly, taxol was detected in Cephalotaxus mannii by HPLC. With the established methods, bacctin III, the precursor of taxol biosynthesis, was found accordingly. Moreover, contents of taxol and bacctin III both increased after elicited by MeJA (100μM).(3) By homology cloning technology, the GGPPs and TAT gene fragments were obtained in Cephalotaxus mannii. Base on it, GGPPs cDNA gene sequence was achieved by RACE technology. The sequence analysis showed that the cDNA sequence is1694bp in length with a poly A tail. The gene was deposited in Genbank with Accession number JX971119. And bioinformatics of the genes was analyzed accordingly.(4) The Real Time PCR results show that, MeJA (100μM), Flg22(1μM), Harpin (50mg/mL), three different elicitors all can upregulate GGPPs and TAT expression, two taxol biosynthesis related genes. However, the expressions varied in speed and amplitude.(5) After establishment of the genomic DNA methylation sensitive amplified polymorphism (MSAP) method in Cephalotaxus mannii, genomic DNA demethylation was found when challenged by elicitor.