Cloning Of Mapmi Gene And Its Functional Analysis In Entomopathogenic Fungus, Metarhizium Acridum

The pathogenesis of Metarhizium acridum has been mostly studied at home and abroad, which mainly focused on infection, injection and sporulation. Key period of M. acridum is that the period of invading the hemolymph, colonizing and accommodating the insect,s hemolymph. It is also important for fungal pathogenicity. A normal subtracted cDNA library was constructed using M. Acridum in the haemolymph of Locusta migratoria. Based on this, a 320bp-length EST fragment was chosen for further analysis. This protein was homologous with phosphomannose isomerase in other fungi. The Mapmi ORF was obtained by screening full length cDNA library. We analyzed its function by suppressing its transcript level using RNA interference (RNAi) technique.The main achievements are as follows:①A 1326bp ORF was cloned and sequenced, Its Genbank accession number is HQ123439. This gene encodes a protein of 431 amino acids. We got the DNA sequence by using comparison between ORF sequence and genomic sequence of M. acridum with DNAMAN tool, Which displayed that the full DNA length of Mapmi was 1386bp and contained two exons and one intron at position 145bp from the start codon, The intron had the typical border of GT…AG. The Mapmi gene Molecular Weight was 47.65 kDa, with a predicted pI of 5.31. No signal peptide was found at the N-terminal sequence when analyzed by using SignalP②In order to analyze the function of Mapmi, a dual promoter cassette containing the pmi fragment was constructed by using pDPB vector. The pmi-RNAi cassette was cut off and cloned into pK2 binary vector containing a T-DNA harboring a bar cassette (pK2-Mapmi-RNAi). Transformation of M. acridum was carried out mediated by Agrobacterium tumefaciens. Ten transformants were confirmed to carry Mapmi-RNAi cassette by PCR analysis, The Mapmi expression of RNAi transformants was analyzed by real-time RT-PCR. The result showed that The transcription levels of Mapmi-RNAi transformants were reduced significantly compared with the wild-type. However, RNAi efficiency in Mapmi transcription varied from 42.5-89.8% among three Mapmi-RNAi isolates③We analyzed the growth of Mapmi-RNAi transformants on PDA medium and PDA containing cell stress chemicals. The transformants were underwent the growth characterization on PDA medium and PDA containg CR, KCl and H2O2. Growth of RNAi mutant was slightly slower than the wild-type isolate on PDA, while other three media significantly inhibited the growth of RNAi mutant, with PDA amended with KCl resulting in the most inhibition.④We analyzed the growth of Mapmi-RNAi transformants on Czapek-dox medium in which the sucrose was replaced with glucose, fructose, and mannose at different concentration respectively. The result revealed that the growth of Mapmi-RNAi mutant was greatly influenced by carbon source. Glucose could be better utilized than fructose by the wild type and mutant strains when either of them were the sole carbon source, but RNAi mutant had an inhibited growth with either of them as carbon source compared to the wild strain. Trace mannose or grate concentration mannose could not well support the growth of the wild type and mutant strains, moreover, RNAi mutant was more sensitive to the nutrient deficiency.⑤The dry weight of hyphae between Mapmi-RNAi strains and wild type was significantly different in 1/4SDAY liquid medium amended with CR, KCl and H2O2 respectively.⑥We analyzed germination and conidiation under stress condition of Mapmi-RNAi mutant on PDA medium and with cell stress chemicals. We got that compared with the wild type strain, Mapmi-RNAi strains were not significantly different between the RNAi and wild type strains on PDA. On PDA with CR, KCl and H2O2, the RNAi stain had significantly lower germination and conidial production than the wild-type strain.⑦The virulence of Mapmi-RNAi mutant was significantly reduced in comparison with the wild strain, sugggesting that the Mapmi gene was related to the virulence of M. acridum.