Cloning And Functional Analysis Of Adenylyl Cyclase Gene In Setosphaeria Turcia

Setosphaeria turcica is an important phytopathogenic fungus, causes Northern Corn Leaf Blight which is a major leaf disease of maize, and always results in significant economic loss. The research showed that cAMP signal transduction pathway was extracellular signal transduction pathway in fungi widespread, and played an important role in regulating the growth, development and pathogenicity of phytopathogenic fungi. This research cloned the adenylyl cyclase (AC) gene from S. turcica. In this study, we cloned the sequence of adenylyl cyclase gene from genomic DNA of S. turcica strain 01-23 by candidate gene approach and Genome Walking technology. Then we took the functional analysis of STAC through the experimentation of the gene-disruption and STAC gene-specific inhibitor. As a result, this gene was involved in conidiation, appressorium formation, pathogenicity and secondary metabolism, respectively. This work would lay a foundation for investigating the role of cAMP pathway between pathogenic fungi and host.A specific inhibitor (MDL-12,330A, Hydrochloride) of AC could inhibit the conidium germination and appressorium formation of S. turcica. As the concentration of MDL-12,330A Hydrochloride was increasing, the inhibition rate became stronger.Degenerate oligonucleotide primers were designed based upon conserved nucleotide sequences of the known STAC gene in plant pathogenic fungi. We amplified their homologous fragments from genomic DNA of S. turcica strain 01-23. Then its flanking regions were obtained by Genome Walking technology. As a result, we got the 4465 bp DNA fragment. The specific probes of STAC were prepared to apply for Southern blotting. The results showed that STAC gene only had single copy in genomic DNA of S. turcica.The homology sequence alignment of STAC gene was carried out through Position-Specific Iterated BLAST software of NCBI website. As a result, the identity of AC gene was about 58%～91% in many fungi such as Pyrenophora tritici-repentis,Aspergillus fumifatus,Neurospora crassa,Cyptococcus neoformans.The STAC gene replacement vector was constructed with the gene homologous combination theory and PEG-mediated gene transformation system. Transformants were screened by hygromycin B and PCR (polymerase chain reaction) with specific primers corresponding to hygromycin phosphotransferase gene. One STAC gene-disruption mutant, named asΔstac, was obtained by Southern blot analysis performed with the DIG-labeled hph gene. The study of phenotypic analysis showed that the growth rate of mutant was faster than the wild strain, the color of aerial hyphae was gray, it failed to sporulate, toxin activity was significantly reduced and resistibility of the muntant strain was enhanced in osmotic stress conditions. The conclusion was that STAC gene of S. turcica regulated generation of the spore, formation of the appressorium, the growth of the mycelial and pathogenicity.