Analysis Of Microcycle Conidiantion In Metarhizium And Its Related Genes

Metarhizium anisopliae, a kind of entomopathogenic fungus applied worldwide, plays an important role in biological control of insects. The spore or conidium is the primary means for biology control. Thus, the ability of sporulation is an important factor in selecting Metarhizium anisopliae strain. However, little is known about the genetic mechanisms controlling the process of sporulation until now.It is well known that most filamentous fungi including Metarhizium anisopliae form conidia upon differentiated stalk cells separated from mycelia (normal conidiation). In this study, we successfully induced the ascomycete Metarhizium anisopliae strain CQMa102 into a microcycle conidiation (sporulation occurs from the spores immediately after germination without, or with greatly reduced mycelial growth) on a SYA solid medium. Such microcycle conidiation of Metarhizium anisopliae resulted in a mass of yeast-like slime spores.In order to understand the molecular mechanism of microcycle conidiantion, a comparatively systematic study was carried out. Firstly, experiments were designed to select a solid medium fitting for microcylce conidiation. Secondly, a normalized full-length cDNA library of CQMa102 in micricycle conidiation stage and a subtracted library were constructed. Thirdly, a macroarray screen from the subtracted library for cDNAs up-regulated in the microcycle conidiation than in the normal conidiation was performed. Finally, the sequenced ESTs were analyzed using the bioinformatics methods.The main results were as follows:①The sporulation process of CQMa102 on different media was observed with a digital microscope. The spores grown on a SYA solid medium had a microcycle conidiation - asexual differentiation.②The conidia production was compared between the two different sporulation patterns. It showed that both the spore production and the sporulation speed of microcycle conidiation were greater than those of normal conidiation.③A normalized full-length cDNA library of CQMa102 in microcycle conidiation stage was constructed successfully utilizing DSN (duplex-specific nuclease) normalization method combined with SMARTTM (switching mechanism at 5′end of RNA transcript) library construction method. ④A subtracted cDNA library was constructed by suppression subtractive hybridization based on the microcycle conidiation cultures as a tester and the normal coniditiaon cultures as a driver.⑤A total of 1600 clones from the subtracted were screened using both dot blot and reverse Northern dot blot methods, and 400 positive clones were verified. The sequenced 400 clones resulted in 340 high quality ESTs which represented 285 uniESTs.⑥ESTs similarity analysis via BLASTx software showed that 109 ESTs had no homology to sequences with known ESTs, 119ESTs encoded predicted proteins and the rest 112 ESTs represented a broad spectrum of biological functions, including protein metabolism proteins, RNA metabolism proteins, cell metabolism proteins, cell cycle related proteins and stress response proteins, etc.