Identification Of Genes Expressed By Metarhizium Anisopliae On Locusta Migratoria Wing And Haemolymph

Metarhizium anisopliae, an economically important entomopathogenic fungus widely applied at home and abroad, plays an important role in biological control of pest. 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 pest. After germination on host 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.In order to escape the host immune reaction after invading in vivo, M. anisopliae will require not only morphological alterations but also adaption to the hyperosmosis, consuming host nutrition, and killing host finally.The formation of appressorium and colonization in vivo are important infection events that determine successful infection of a susceptible 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, and were transferred to cell-free hemolymph obtained from host.cultured, 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 identify the differentially expressed genes during infection process of the locust wings vs. in locust haemocoel hyphae, a SSH library was established in this paper. In addition, a cDNA library of M. anisopliae var. acridum colonization in vivo was established. After sequencing and bioinformation analysis, the two librarys can supply some candidate genes for identifation of virulence genes.The main results were as follows:1. A subtracted cDNA library was constructed by suppression subtractive hybridization based on M. anisopliae CQMa102 germinating and differentiating on locust wings as a tester and the locust haemocoel hyphae as a driver.2. A total of 1890 clones from the subtracted were screened using dot blot method, and 460 positive clones were verified. The sequenced 460 clones resulted in 329 high quality ESTs which represented 78 uniESTs.3. ESTs similarity analysis via BLASTx software showed that 32 ESTs had no homology to sequences with known ESTs, 22 ESTs encoded predicted proteins and the rest 24 ESTs represented a broad spectrum of biological functions, including protein metabolism proteins, cell metabolism proteins, cell sturctrue and function proteins and stress response proteins, etc.4. A total of 12 clones predicted to be differentially expressed by SSH analysis were tested by semi-quantitative reverse transcription PCR. The results showed that those genes were differentially expressed on infection process of locust wings.5. A cDNA library of CQMa102 in locust haemocoel hyphae was constructed successfully utilizing SMARTTM (switching mechanism at 5'end of RNA transcript) library construction method.6. A total of 396 clones were screened using dot blot method. The sequenced 396 clones resulted in 318 high quality ESTs which represented 187 uniESTs. ESTs similarity analysis via BLASTx software showed that 22 ESTs represented a broad spectrum of biological functions, including protein metabolism proteins, cell metabolism proteins, cell sturctrue and function proteins and stress response proteins, etc.