Cloning And Functional Analysis Of MaChy Gene In Metarhizium Acridum

The ascomycetes Metarhizium acridum was well characterized entomopathogenic fungi and widely used in biological control programs. The overall host range of M. acridum is broad. During the invasion steps , the formation of appressorium was very important.So cloning and identification of the Characteristics of appressorium formation related gene are necessary to clarify the characteristics of appressorium formation and other important traits related to the molecular mechanism.Based on the Metarhizium acridum CQMa102 subtracted DNA library which constructed by suppression subtractive hybridization (SSH) before penetration.we chose one EST which was highly expressed before penetration. Using the Metarhizium acridum CQMa102 normalized full-length cDNA library during the conidiation stage,we have got the full-length cDNA of MaChy .At the same time,we got the DNA of MaChy through the DNA of Metarhizium acridum CQMa102.In addition, we use RNAi to analyse the function of MaChy gene.①A 1202bp DNA fragment which contained the full-length MaChy gene as well as upstream and downstream regulatory sequences was cloned and sequenced. The putative coding sequence which contained a 1074 bp ORF, a 80 bp 5'UTS and 50 bp 3'UTS was identified using ORF finder analysis and BLASTX. A protein of 357 aa with a predicted molecular mass of 39.78kDa and a pI of 5.53 was deduced. Without introns in the full-length DNA , and the deduced protein was predicted with an online program. (http://www.predictprotein.org/).②The polypeptide contains four potential site of phosphorylation by casein kinase 2 (amino acids SELD , THVE , SKHE and STLE at residue 104,134,179 and 341, respectively), four potential site of Protein kinase C phosphorylation (amino acids TIR, SMR, TKK and TPK at residue 4,77,300 and 323, respectively), three potential N-glycosylation sites (amino acids NASK , NASE and NSTD at residues 177,206 and 322, respectively) and four potential myristylation sites (amino acids GTVINH, GSGDSL, GARLGL and GNGYSR at residues 122, 144, 238 and 262,respectively)③The LT50 against locust was measured with the conidia of the wild-type and the RNAi mutant, respectively. The results showed there were no significant differences between the wild-type and the RNAi mutant. It indicated that knockdown transcripts of MaChy gene had no effect on the virulence of M. acridum. ④Spore germination and appressorium formation on locust L. migratoria wings was observed by electron microscopy.But there was not significantly different between the wild-type and RNAi mutant. This demonstrate that the MaChy gene has no effect on spore germination and appressorium formation.⑤Relative expression of MaChy in 3 different stages were analyzed by real-time RT-PCR. The greatest expression of MaChy gene was occurred during the appressorium formation stage.it was 2.5 times to the expression in the haemolymph. And the expression in the haemolymph was nearly equivalent with the expression in the1/4SDA medium.