Study On Overexpression Of Candidate Genes A11450 And A02725 In Cladosporium Cladosporioides MD2

Paclitaxel is a broad-spectrum,high-efficiency and low-toxic anticancer drug.Paclitaxel-producing endophytes are one of important natural resources for paclitaxel production.However,the taxol yield of these reported paclitaxel-producing endophytic fungi are low and not suitable for industrial application.Overexpressing of rate-limiting genes and regulatory genes involved in taxol biosynthesis is one effective way to construct a stable and high taxol-yield engineeried strains for industrial application.However,there is little knowledge about the fungal taxol biosynthesis pathway and related genes.Therefore,cloning and function analysis of fungal taxol-related genes are keys for improving the taxol yield of endophytic fungi by genetic engineering.In this study,2 candidate genes A11450 and A02725 were selected from the paclitaxel-producing fungus Cladosporium cladosporioides MD2,and A02725 was cloned and the construction and function of A02725 was predicted by bioinformatics methods.Overexpression vectors of these two candidate genes were constructed and transferred into C.cladosporioides MD2 to produce the corresponding transgenic strains.The influence of overexpression of candidate genes A11450 and A02725 on taxane biosynthesis of C.cladosporioides MD2 was analyzed.These results laid a foundation for further function study of the two genes and provided candidate genes for engineered strains with high taxol yield.The main results of this study were as follows:(1)The full-length DNA sequences of candidate gene A02725 were cloned.The DNA sequence of candidate gene A02725 contained 1524 bp and did not have intron.The coding protein of candidate gene A02725 had 62% identical to Geranylgeranyl pyrophosphate synthase(GGPPS)from Cercospora Zeina.The coding protein of candidate gene A02725 did not contain transmembrane structure and signal peptide,and harbored a polyprenyl synthetic domain.(2)Overexpression vector of candidate gene A11450 was constructed base on pTFCM and transfferd into C.cladosporioides MD2 with an Agrobacterium tumefaciens-mediated transformation method to generate 25 A11450-transgenic strains.The copy number of the exogenous genes in the A11450-transgenic strains were detected by Southern blot technology.Three A11450-transgenic strains(4-6,4-19 and 4-20)with single copy of exogenous genes were randomly selected for QRT-PCR analysis of the expression levels of candidate gene A11450.Then,three taxanes were extracted from the transgenic strain 4-20 with the highest relative expression level of candidate gene A11450 and detected by HPLC method.The yield of 10-deacetylbaccatin III,bacatin III,and paclitaxel of the A11450-transgenic strain 4-20 was about 1.24-1.36 folds of that of the control(non-transgenic)strain,suggesting that overexpression of candidate gene A11450 could increase the yield of 10-deacetylbaccatin III,baccatin III and paclitaxel in C.cladosporioides MD2.(3)Overexpression vector of candidate gene A02725 was constructed and transferred into C.cladosporioides MD2 to produce 18 A02725-transgenic strains.Southern blot was used to detect the copy number of the exogenous genes in the A11450-transgenic strains.Three of A02725-transgenic strains(7-10,7-14 and 7-18)with single copy integration were randomly selected for detection of the expression levels of candidate gene A11450 by QRT-PCR.Then,three taxanes were extracted from the A11450-transgenic strain 7-18 with the highest relative expression level of candidate gene A11450 and detected by HPLC method.The yield of 10-deacetylbaccatin III,bacatin III,and paclitaxel of the A11450-transgenic strain 7-18 was about 1.34-1.44 folds of that of the control strain,indicating that overexpression of candidate gene A02725 could increase the yield of 10-deacetylbaccatin III,baccatin III and paclitaxel in C.cladosporioides MD2.The above results preliminarily revealed that overexpression of candidate genes A11450 and A02725 could promote the paclitaxel biosynthesis of C.cladosporioides MD2.These results laid the foundation for the further function study of the two candidate genes by gene knockout technology and heterogenous expression technology.