紫杉烯合成酶基因重组载体的构建及其在真菌中的表达

Series of fungal expression vectors were constructed on the basis of pIGF. A cDNA (TS) encoding taxadiene synthase was subcloned into the pIGF-derived expression vectors. The cDNA was fused in frame to part (GAM, encoding the upstream 498 amino acids) of the native Aspergillus niger gene encoding glucoamylase. The filamentous fungi (Gibberella fujikuroi and Aspergillus niger) were co-transformed respectively by the GAM-TS carrying vector and hph carrying vector pAN7-1 (hph : hygromycin B resistant gene, HmBr). Recombinants containing the whole GAM-TS fusion gene were obtained from the HmBr dominant transformants by PCR screening and Southern blot method.RT-PCR analysis of the recombinants showed that introns in the GAM were not processed by Gibberella fujikuroi. The GAM-TS carrying vector was modified by replacing the genomic DNA of the GAM with its cDNA fragment. The modified vector was introduced into G. fujikuroi and a number of recombinants harboring the whole heterologous fusion gene were obtained. Northern blot analysis showed that the fusion gene was expressed on the transcription level.The fusion gene was also proved to be expressed in Aspergillus niger recombinants. SDS-PAGE analysis of the recombinant Fa15 indicated that there was a characteristic band that corresponded to the taxadiene synthase. Secondary metabolites of the recombinant Ta1 were detected by hin-layer- chromatography (TLC) and a new spot was observed in comparison with the control (host strain). This component was isolated and analyzed by Fab-MS, showing that the possible molecular weight is 661.3.Further chemical analysis of the component and research on other recombinants are in progress..

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