Preliminary Study On The Constitutive Toxins Synthetic Gene Cluster Of Neurospora Crassa

Neurospora crassa is a kind of multicellular filamentous fungus belonging to neurospora.Mycelium is transparent and has partition and branching,aerial hyphae can produce orange conidia.Neurospora crassa is widely used in laboratory because of fast growing,easy to cultivate,and clear genetic background.Neurospora crassa synthesized synthetic toxin to resist insects.To avoid damage to their cells,Neurospora crassa forms a specific subcellular structure to store the toxins.Polyketide synthase 5(PKS5)and genes coding short chain dehydrogenase reductase(DH),cytochrome pigment 450(CYP450-1,CYP450-2),major facilitator superfamily(MFS-1,MFS-2),sulfate transporter(SULT),transcription factor(TF)form biosynthetic gene cluster of constitutive toxin by Neurospora crassa(CTNC).CYP450 is a kind of active protease with high content and function in fungi,which can degrade the toxic substances.MFS is able to take the toxin produced by fungi to the outside of the cell.SULT transported sulfate radical into the cell to synthesis cysteine.Transcription factor play a dual role in the regulation of gene expression: 1.regulate the secondary metabolites of filamentous fungi;2.react to the signal transduction of reactive oxygen free radicals which are harmful to their own.In this paper,the following studies are carried out:1.The 10 mutants were obtained by using the ku70 mutant as the starting strain,and the changes of the metabolites were detected by thin layer chromatography(TLC).2.PKS5 was linked to pYH-WA-pyr G-KI vector and was integrated into Aspergillus nidulans(6450,1898).The secondary metabolites were detected.3.TF was linked to pETMALc-H vector and transformed into Escherichia coli BL21.The obtained protein was used as antigen to inject rabbit,and the corresponding antibody was obtained from rabbit serum.4.The ku70 mutant was used as the starting strain,TF was over expressed in situ.The protein was extracted and the expression level of TF was detected by Western Blot.The results showed that 4 of the mutants of Neurospora crassa were transformed into white and did not produce spores,which indicated that the genes knocked out were closely related to the formation of spores.By TLC detection,the number of secondary metabolites are decreased.PKS5 was successfully expressed in Aspergillus nidulans(6450,1898).The results of TLC and HPLC showed that some new substances were produced in the secondary metabolites of Aspergillus nidulans(6450,1898)mutants.The transcription factor was successfully expressed in BL21,and the corresponding antibody was obtained by injecting rabbits.Extraction of TF over expression strain protein and found that TF was up-regulated by Western Blot.