Cloning And Functional Analysis Of The Mamrd1 Gene In Metarhizium Acridum

Metarhizium acridum is an important entomopathogenic fungus. We have successfully constructed subtracted cDNA library with M. acridum genes differentially expressed in vivo using suppression-subtractive hybridization. Following bioinformatic analysis and access of related literature to these EST sequences, one multiple RNA-binding domain protein gene (Mamrd1) was selected in this study.The full-length cDNA of Mamrd1 was obtained (Acc No. HQ268501). Bioinformatics analysis revealed that, Mamrd1 included three exons, 2 introns, and translated into 841 amino acid polypeptide (Acc No. ADZ99447) with a predicted molecular weight of 92.7 KD and isoelectric point of 6.12. Secondary structure revealed that, similar as the yeast Mrd1p, Mamrd1 contained five RBD domains.In this study, an engineered strain with down-regulating Mamrd1 using RNA interference (RNAi) was constructed,The growth characteristics of the RNAi strains on artificial media and inside host insect, gene expression and virulence were analyzed.On artificial medium, quantitative PCR analysis showed that the expression of Mamrd1 in Mamrd1-RNAi strains reduced 49%. Study on quantitative analysis before and after the rRNA processing in Mamrd1-RNAi strains showed similar as yeast Mrd1p, Mamrd1 participated processing of pre-rRNA. Compared with wild-type, growth rate of Mamrd1-RNAi strain was not significant, but the colony was fluffy, and mycelial layer was thicken. Microscope analysis found, RNAi strains had significantly reduced hyphal branching, increased hyphal branching interval and fewer sporulation structure and biomass .The percente germination didn't significantly affected when down-regulating the Mamrd1,.For the test of M. acridum growth inside insect, Mamrd1 gene was tup-regulated 13 times after the fungi infested locusts. In the host, the expression of Mamrd1 in Mamrd1-RNAi strains was inhibited 90%. Bioassay test showed that RNAi strain had a significant lower virulence than the wild type. Growth analysis showed that the growth of RNAi strain inside insect was significantly inhibited.In summary, Mamrd1 participated in ribosome processing in M. acridum. The Mamrd1 was upregulated during the infection process and affected the growth, sporulation and virulence in M. acridum.