| Metarhizium anisopliae, a kind of entomopathogenic fungus applied worldwide, plays an important role in biological control of insects. The process of Metarhizium anisopliae infected host is dynamic and complex,but currently only sets out the mechanism that Metarhizium anisopliae penetrate the host epidermis by secreting protease and chitinase as well as adopting host nutrition by scerting acid phosphatase and trehalase after invading the host hemolymph. Critical period of pathogentic fungi is that the period from invading the hemolymph to the death of host and this research stage is not clear, but it is also very important for fungal pathogenicity. Based on the Metarhizium anisopliae CQMa102 subtracted cDNA library which constructed by suppression subtractive hybridization in vivo by Metarhizium anisopliae in the haemolymph of Locusta migratoria, we chose the EST which was highly expressed during this stage. The number of the ESTs are GO004730 and GO004735 respectively.The two predict proteins have very homology with prenyltransferase and alpha-1,2-mannosyltransferase in other fungi respectively.Using the SMART method, the full-length cDNA were obtained respectively.We analysis gene expression profile of Mpt by quantitative PCR, meanwhile we use RNAi to analyse the function of Mmmnt1 gene.The main achievements are as follows:①A 1557 bp DNA fragment which contained the full-length Mmnt1 gene as well as upstream and downstream regulatory sequences was cloned and sequenced. The putative coding sequence which contained a 1296 bp ORF, a 125 bp 5'UTR and 136 bp 3'UTR was identified using ORF finder analysis and BLASTx.Genbank accession number is GU990223 and encodes a protein of 431aa with a signal peptide MVAGNARYVRYIIIAVFTLAVFYFVSNSKY. The full cDNA length of Mpt gene is 1194bp,which includes 1026bp ORF,63bp 5'UTR and 105 bp 3'UTR.Genbank accession number is GU271134 and encodes a protein of 341aa with a signal peptide MAWFTGRFMAGEVAAILSAFALGYL.②The DNA sequence and cDNA sequence comparison with bl2seq sorftware showed that the full DNA length of Mmnt1 was 1711bp and contained two introns (177bp-291bp),(1469bp-1525bp). The full DNA length of Mpt was 1380bp, included one intron (862bp-937bp). The introns of two genes had the typical borders of GT…AG. ③Quantitative PCR detection the expression level of Mpt gene in different infection stages demonstrated that the expression was very slow in appressorium , initial infection and 1/4SDA medium conditions and the relative expression levels were 17.68%,17.70% and 4.85% respectively. However, the relative expression levels were 81.23% and 100% in the stage of I7 and LC and had a significant increase compared with former infection stages.④We obtained five RNAi mutants by PCR screeing.The Mmnt1 transcript of the wild-type and RNAi transformants were analyzed by real-time RT-PCR. In comparison with the wild-type, the Mmnt1transcript was inhibited in some degree in the RNAi mutants at different infection stages, with the efficiency of RNAi ranging from 40%-80%.⑤RNAi strains and wild type strains were inoculated to 1/4SDA liquid medium or 1/4SDA amended with various cell wall damage elements. The liquid biomass results showed that mycelium dry weight of RNAi strains was significantly lower than the wild type strains in 1/4SDA amended with KCl or H2O2. However, the difference was not obvious between the RNAi trains and the wild type strains in 1/4SDA or added Congo red, Calcoflour white.⑥The results suggested that the growth of RNAi mutants was significantly inhibited in solid PDA amended with KCl or H2O2 compared to the wild type. However, the difference was little between the RNAi mutants and the wild type in other culture conditions. This result was consistent with the liquid biomass.⑦Virulence tests using Locusta migratoria revealed a significant reduction in mo rtality and speed of kill by the RNAi mutants compared to the wild type. Topic infection and injection bioassays showed that lethal time was about one day longer than the wild type . Interference the Mmnt1 gene affected the virulence of spores, thus we speculatedthat the gene was related to the virulence of Metarhizium anisopliae. |
