| Taxol,an effective anticancer drug,is mainly extracted from Taxus,but the natural resource of Taxus is scarce.Fermentation of taxol-producing endophytic fungi is one promising and alternative way to produce taxol.However,the taxol yield of these reported taxol-producing endophytic fungi is low and unsuitable for industrial application.Overexpression of rate-limiting enzyme and regulatory genes related taxol synthesis is one way to obtain high taxol-yield engineered strains.However,little is known about the biosynthetic pathway and function genes of fungal taxol.Therefore,cloning and function research of the genes related to fungal taxol biosynthesis is a key to reveal the biosynthetic pathway of fungal taxol and improve fungal taxol yield by genetic engineering technology.In this study,candidate genes A10182 and A10251 were cloned from one taxol-producing endophytic fungus Cladosporium cladosporioides MD2,and the influence of overexpression of the two candidate genes on the yields of taxol and other 5 taxanes in Cladosporium cladosporioides MD2 were analyzed.These results laid a foundation for further function research of candidate genes A10182 and A10251.The main results in this study were as follows:(1)The full-length DNA sequences of candidate genes A10182 and A10251 were cloned and analyzed by bioinformatics.The sequence length of A10182 and A10251 was 678 bp and 906 bp,and the encoding products of them had high homology with one hypothetical protein(GenBank no.XP007808865.1)of Endocarpon pusillum and one hypothetical protein(GenBank no.KXT16021.1)of Pseudocercospora muase,respectively.The encoding proteins of A10182 and A10251 were belonging to hydrophobic protein family,and did not have trans-membrane structures and signal peptides.The encoding protein of A10182 contained 225 amino acids,possessed 24.984 kDa,and harbored 4 alpha helices and 10 extend strands.The encoding protein of A10251 contained 301 amino acids,possessed 33.320 kDa,and had 3 alpha helices and 1 extend strand.(2)Overexpression vector of candidate gene A10182 was constructed and transformed into C.cladosporioides MD2 with Agrobacterium-mediated transformation method.A total of 23 A10182-transgenic strains were obtained after plate resistance screening and amplification verification of resistance genes hph.Southern blot analysis showed that the exogenous genes of 13 transgenic strains were integrated into host chromosome with single copy.Three transgenic strains were randomly selected for qRT-PCR analysis of the expression levels of candidate genes,and the results showed that the expression levels of A10182 in transgenic strains were increased in comparison to the control strain(untransgenic strain).The expression level of A10182 in transgenic strain AC was highest and about 2.0 folds of that in the control strain.HPLC analysis result showed that the yields of taxol(5.187±0.016 ?g/g),10-deacetylbaccatinIII(20.411±0.335 ?g/g),10-deacetylpaclitaxel(4.518±0.091 ?g/g),7-xylosyl-10-deacetylpaclitaxel(3.434 ± 0.061 ?g/g)in the A10182-transgenic strain AC were higher than that of taxol(5.033±0.132 ?g/g),10-deacetylbaccatin III(15.062±0.304 ?g/g),10-deacetylpaclitaxel(4.044±0.089 ?g/g),7-xylosyl-10-deacetylpaclitaxel(2.387 ± 0.059 ?g/g)in the control strain,respectively.In addition,docetaxel was detected in the transgenic strain AC(about 2.709±0.106 ?g/g)and undetected in the control strain.These results indicated that overexpression of candidate gene A10182 could promote the synthesis of these above taxanes in C.cladosporioides MD2.(3)Overexpression vector of candidate gene A10251 was constructed and transformed into C.cladosporioides MD2 to produce 30 of A10251-transgenic strains.Southern blot analysis showed that the exogenous genes of 4 of 14 transgenic strains were integrated into host chromosome with single copy.QRT-PCR analysis showed that the expression levels of A10251 in transgenic strains were increased in comparison to the control strain,and the A10251 expression level in transgenic strain AC was highest and about 2.0 folds of that in the control strain.HPLC analysis result showed that the yields of taxol(6.022±0.116 ?g/g),10-deacetylbaccatin III(22.304±0.442 ?g/g),10-deacetylpaclitaxel(4.209±0.097 ?g/g)and 7-xylosyl-10-deacetyl paclitaxel(3.378±0.074 ?g/g)in the transgenic strain AC were higher than that of taxol(5.033±0.132 ?g/g),10-deacetylbaccatinIII(15.062±0.304 ?g/g),10-deacetyl paclitaxel(4.044±0.089 ?g/g)and 7-xylosyl-10-deacetylpaclitaxel(2.387±0.059 ?g/g)in the control strain,respectively.Docetaxel was detected in the transgenic strain AC(about 1.983±0.094 ?g/g)and undetected in the control strain.These results indicated overexpression of candidate gene A10182 could increase the synthesis of these taxanes in C.cladosporioides MD2. |
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