Construction And Analysis Of A Normalized CDNA Library From Metarhizium Anisopliae Var. Acridum Germinating And Differentiating On Locusta Migratoria Wings

Metarhizium anisopliae, an economically important entomopathogenic fungus widely applied at home and abroad, plays an important role in biological control of insects. Mycoinsecticides have many advantages over chemical insecticides, including low toxicity to non-targeted organisms, high insect specificity, and low environmental impact, but the slow killing speed has been considered a potential drawback which prevents the utilization of these fungi against insects. After germination on parasitical insect cuticle, M. anisopliae will differentiate and form an important infection structure called appressorium, which can not only secret abundant extracellular proteolytic enzymes to solubilize host cuticle, but also generate significant tip turgor pressure for penetration. The formation of appressorium is an important infection event that determines successful colonization of a susceptible host because an appressorium helps M. anisopliae establish a pathogenic relationship with its host. Thus researches on mechanisms of fungal pathogenesis may suggest strategies for the development of more efficient anti-insect mycoinsecticides.Many researchers have attempted to study the developmental and transcriptional events during M. anisopliae infection. The methodologies of their studies are that M. anisopliae were cultured to germinate and differentiate in liquid minimum medium supplemented with host cuticle or cuticle extract, followed by cDNA library construction and ESTs analysis. The fungus cultured in liquid medium is in saprophytic life, while the fungus infecting insect cuticle is under parasitical life cycle. Taking into account the different surrounding growing environment, submerged cultivation of the fungus may mask the complete gene expression profile of M. anisopliae during germination and differentiation in vivo. In order to more clearly and actually elucidate the mechanism underlying fungal pathogenesis, a method was established in this paper for constructing a normalized cDNA library of M. anisopliae var. acridum germinating and differentiating on locust wings whose nucleic acid had been eliminated before inoculation.For ensuring the constructed cDNA library is not contaminated with locust cDNA clones, a reagent called RNA fragmentation buffer was employed to degrade the locust wing nucleic acid before inoculation with M. anisopliae conidia. Besides, we designed locust specific primers with high sensitivity to monitor the existence of locust DNA or RNA in the cDNA used for library construction. Comparison of germination and differentiation rate of M. anisopliae grown on treated wings with that on non-treated wings indicated that the RNA fragmentation buffer treatment procedure does not influence the growing behavior of M. anisopliae. No locust cDNA clones were identified from 221 ESTs obtained from the cDNA library, and similarity searching and analysis of the ESTs showed the presence of some representative genes related to pathogenesis of M. anisopliae.The main results were as follows:1. The DNA and total RNA extracted from locust wings treated with RNA fragmentation buffer had been obviously degraded into small fragments.2. Based on the reported locust genes which are constitutively and highly expressed, we designed locust primer pairs with high sensitivity to detect whether the cDNA used for library construction contained locust sequence. And the results showed that the cDNA was not contaminated with locust sequence, indicating that RNA fragmentation buffer was effective for degrading locust nucleic acid.3. The growing process of M. anisopliae on locust wings was observed with a digital microscope. The results showed that conidia of M. anisopliae germinated after 8 h of inoculation, appressorium formed after 16 h, most of the conidia had germinated on wings after 20 h, and plenty of appressoria formed after 30 h, with few penetration peg forming. There were no difference between the germination and appressorium formation rate of the M. anisopliae grown on treated wings with that on non-treated wings.4. Some of the M. anisopliae virulent genes, viz Mad1,Mpl1和Pr1A, were successfully amplified from the cDNA used for library construction, suggestion that the cDNA was representative of the gene expression of M. anisopliae infecting insects.5. A normalized cDNA library of M. anisopliae germinating and differentiating on locust wings was successfully constructed utilizing DSN (duplex-specific nuclease) normalization method combined with SMART (switching mechanism at 5′end of RNA transcript) library construction method. A total of 221 ESTs of high quality was obtained from the normalized cDNA library. These ESTs represented 162 unigenes (73.3%), indicating good normalization efficiency. Similarity searching of these ESTs against the locust EST database revealed that the cDNA did not contained locust cDNA clones.6. ESTs similarity analysis via BlastX software against NCBI GeneBank showed that 3 ESTs had no homology to any existed sequences, 66 ESTs encoded predicted proteins and the rest 93 ESTs represented a broad spectrum of genes of different biological functions, including protein metabolism proteins, RNA metabolism proteins, cell metabolism proteins, cell cycle related proteins and stress response proteins, etc.