Construction Of A CDNA Library Of Paclitaxel Producing Bacteria

Taxol is one kind of the highest effective anticancer drugs,however,the supply is seriously limited.Taxol-producing fungi may be a good way to solve this problem.This study focused on taxol-producing fungi HDFS4-26.In the process of taxol,total RNA respectively was extracted by Trizol Reagent,cDNA library was constructed and plasmids was excreted from this library.On the basis of twenty key genes coding in the biosynthesis pathway of taxol,analysised the best genes coding as biotin labeled probes,with DNA capture dynal beads constructed a system,which blotted with plasmids extracted from library,then obtained another library.Sequencing 580 randomly clones from this library,and finally 533 ESTs were obtained.This study provided a foundation for genetically engineering taxol-producing,fungi which could improve taxol yield in the future,and will be helpful to know about taxol biosynthesis at molecular level.The main results were follows:1.Taxol-producing fungi HDFS4-26 strains were fermented in S-7 culture for 9d.2.cDNA library has been constructed which capacity reached 2.3×10,the percentage of recombination was as high as 95%,the average size of inserts was larger than lkb.Sequencing 32 randomly clones from the cDNA library,and got 28 valid sequences which average size of sequences was 1260bp.The sequences were blasted against the non-redundant nucleotide and protein database of NCBI.The results showed 5 clones protein size of sequences larger than 300aa and 14 clones between 200?300aa.Therefore,the cDNA library constructed excellent.3.cDNA library has been selected which capacity reaches 1.75×106,the percentage of recombination was as high as 94%,the average size of inserts was larger than lkb.4.580 randomly clones from the cDNA library were sequenced at 5' end,obtained 533 high quality unigenes after trimming the vector sequence and eliminating low quality sequenees.The cDNA library which is included 39 contigs and 494 singletons with an average length 807bp.5.Those 533 ESTs were blasted agaist the non-redundant nucleotide and protein database of NCBI,240 sequences obvious similarily(E<e-5)with the databases(45%);261 sequences were unknown or describe ominous(49%);32 sequences can be considered a new genes(6%).6.240 unigenes have been classified based on Gene Ontology,142 unigenes have been classified in molecular function;52 unigenes have been classified in biological process;46 unigenes have been classified in cellular component.In taxol biosynthetic pathways we predicted 12 unigenes related of enzyme gene.