| The filamentous fungus Neurospora crassa is an extremely useful organism for studying basic biological mechanisms. In this study, by BLAST analysis, we identified a homologous gene of Arhrobotrys oligospora AoMadl in Neurospora crassa genome, NCU10687(NcMadl). And the protein sequence was analyzed based on its amino acid and nucleotide sequences. Then, the NcMadl gene were knocked out by homologous recombination techniques and complemented to study its biological function in N. crassa.In additional, to study the roles in different fungal species, we expressed the AoMadl gene in the wild type and NcMadl deleted strain of N. crassa. The results of this paper for further study N. crassa cell wall protein function.The main results of this study were described as follows:1 NcMadl knockoutThe upstream and downstream homologous fragment were inserted into the plasmid pAgl-H3, on each sides of the hygromycin resistance gene (hph), the resulted knockout plasmid were transformed into N. crassa (ku70RIP) using agrobacterium-mediated strategy, transformants were verified by PCR and Southern blot methods.2 Strain crossingTo obtain an auxotrophy strain (his-) of N. crassa of the NcMadl deleted strain, the 301-6 (His-) was crossed with ΔNcMadl strain to obtain a No.66 strains (ΔNcMadl, His-), as a recipient strain for gene completion and heterologous expression.3 Construction of Neurospora crassa NcMadl gene complementation strains, AoMadl heterologous expression strains and AoMadl heterologous complement strainsThe whole genes of AoMadl and NcMadl were inserted into pEASY-T5 Zero plasmid to obtain pT5-AoMadl and pT5-NcMadl, followed by insertion of a his3 gene fragment to each plasmid to make the expression vectors pT5-AoMadl-His and pT5-NcMadl-His. The two plasmids were introduced into N. crassa wild-type 301-6 and ANcMadl strains to obtain NcMadl complement strains, AoMadl overexpression strain and heterologous expression strains.4 Phenotype comparisonThe phenotypes were compared between the N. crassa wild type and the Genetic engineering strain to identify the role of NcMadl. Obtained and cell wall protein production, comparative analysis of mycelium growth rate, morphology of colonies, sporulation capacity and stress resistance. It was found that, ΔNcMadl strains outside the cell wall glycoprotein decreased after covering NcMadl gene, glycoprotein recovery. AoMadl overexpression strain glycoprotein cell wall surrounding dense than the wild type, and the growth rate is slower than the wild-type strain, fluorescent whitening agents than wild-type factor sensitive to inhibition of cell wall synthesis. Description NcMadl genes involved in the synthesis of cell wall glycoproteins, AoMadl protein ofNeurospora crassa growing influence.The main innovations of this paper:1 A method using Agrobacterium mediated successfully and effectively obtained the knockout strains of Neurospora crassa NcMadl gene, and covering NcMadl gene to knockout strains, this is done in order to better study the function of the protein NcMadl. While AoMadl homologous genes from Arthrobotrys oligospora was expressed in Neurospora crassa, but also further investigated AoMadl protein.2 Transformants were compared with the wild-type strain in surrounding cell wall polysaccharide, growth rate, colony morphology, sporulation capacity, resistance and other aspects. The role of the Madl protein in non-pathogenic filamentous fungi Neurospora crassa was preliminarily expounded, and to lay a foundation of further study on this kind of protein... |
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