Screening Of Taxol-producing Endophytes,and Clone And Analysis Of Candidate Genes Related To Taxol Biosynthesis

Taxol is a highly effective anticancer drug,but its source of yew trees are extremely scarce,which severely limits its widely used in the clinic.Using endophytic fungi fermentation to produce taxol is considered to be one of the most promising ways to solve the problem.However,due to low yield and instability of taxolproducting fungi,so far,no one endophytic fungus has been used in industrial production.Therefore,screening of taxol-producing fungi and then building stable high-yield genetic engineering strains by molecular breeding technology is the main way to achieve its industrial application,and meanwhile,clone and functional analysis of genes related to taxol biosynthesis is key for performent of genetical modification of taxol-producing fungi.In this study,endophytes from the barks of yew tree were isolated,and the endophytes producing taxol and its precursors were screened by HPLC-MS method,which provided new resources for the study of taxol-producing endophytes.Additionally,a taxol-producing fungus,Cladosporium cladosporioides MD2,was used as material to screen candidate genes related to taxol biosynthesis,after sequencing of genome and transcriptome of C.cladosporioides MD2.The DNA and cDNA full-length sequences of candidate genes were cloned.Expression characteristics of candidate genes responsing to methyl jasmonate(MeJA)were analyzed by qRT-PCR and copy numbers of candidate genes in the chromosome were analyzed by southern blot.Then,prokaryotic expression vectors of two candidate genes were constructed and prokaryotic expression conditions of them were optimized.Fungal expression vectors of two candidate genes were also constructed,and spores of Cladosporium cladosporioides MD2 were transformated by Agrobacterium tumefaciens to obtain transgenic strains.Results of this study were as follows:(1)A total of 90 endophytic fungal strains and 200 endophytic bacterial strains were isolated from the inner barks.The results showed that there were two fungal strains producing taxol,baccatin III and 10-deacetyl baccatin III,one producing taxol and baccatin III,two producing baccatin III,and two bacterial strains producing taxol.The fungus MHZ-32 was preliminarily identified to belong to Phomopsis sp.(2)16 candidate unigenes were found out by phylogenetic analysis.DNA full-length sequences for 16 candidate unigenes were obtained and cDNA full-length sequences for 12 candidate unigenes were obtained.The result of southern blot showed that these candidate genes exited in chromosome in single copy number.The result of qRT-PCR showed that,in the range of 30 hours inducted with 100 ?M methyl jasmonate,the relative expression amount of one candidate gene increased,four dropped significantly,six dropped slightly,and one dropped firstly,increased subsequently and then decreased.(3)Bioinformatics analysis were carried out for unigeneA09801 and A03231.The full-length sequences(1674 bp)of DNA and cDNA of UnigeneA09801 could encode 557 amino acids,and the hydrophilic encoding protein possessed 61291.8 Da molecular weight and 5.47 isoelectric point value,C2717H4201N743O840S18 molecular formula,and had no trans-membrane structure and signal peptide.At the N end of A09801 encoding protein,GMC and FAD domains were contained,and at the C end,only GMC domain was contained.The full length sequences(918 bp)of DNA and cDNA of UnigeneA03231 could encode 305 amino acids,and the hydrophilic encoding protein possessed 33071.5 Da molecular weight and 5.69 isoelectric point value,C1457H2314N406O448S12 molecular formula,and had two trans-membrane structures but no signal peptide.At the N end of A03231 encoding protein,ADH short C2 and ADH short domains were contained.(4)The prokaryotic expression vectors of UnigeneA09801 and A03231 were constructed and prokaryotic expression conditions of UnigeneA09801 and A03231 were optimizated.The prokaryotic expression conditions of UnigeneA09801 in E.coli BL21 were: 32 ? induction temperature,1.2 mM TPTG dose,4 h induction time.The prokaryotic expression conditions of UnigeneA03231 in E.coli BL21 were: 28 ? induction temperature,0.6 mM TPTG dose,10 h induction time.Both of the two expressed proteins were soluble.The results laid a foundation for the purification and catalytic function research in vitro of UnigeneA09801 and A03231 expressed products.(5)Firstly,fungal expression vector pOrigin was constructed,then fungal recombinant expression vector pO-A09801 and pO-A03231 were also constructed.These laid a foundation for overexpression analysis of UnigeneA09801 and A03231 in the future.