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Construction and preliminary analysis of a cDNA library from Locustainfected with Metarhizium.pdf

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Construction and preliminary analysis of a cDNA library from Locustainfected with Metarhizium

Journal of Insect Physiology 56 (2010) 998–1002Construction and preliminary analysis of a normalized cDNA library from Locusta migratoria manilensis topically infected with Metarhizium anisopliae var. acridum Jie Wang, Yuxian Xia * Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Genetic Engineering Research Center, College of Bioengineering, Chongqing University, Chongqing Engineering Research Center for Fungal Insecticides and Key Lab of Functional Gene and Regulation Technologies Under Chongqing Municipal Education Commission, Chongqing 400044, PR China A R T I C L E I N F O Article history: Received 22 January 2010 Received in revised form 5 May 2010 Accepted 6 May 2010 Keywords: Locust Entomopathogenic fungus Pathogenesis cDNA library ESTs Immune-related genes A B S T R A C T The insect immune response to fungal infection is poorly understood at the molecular level. To explore the molecular basis of this process, a novel method to analyze the gene transcripts of insects in response to pathogenic fungus was established. A normalized cDNA library based on the SMART method combined with DSN (duplex-specific nuclease) treatment was constructed using mRNA extracted from the fat body and hemocytes of Locusta migratoria manilensis 6–24 h after being topically infected with Metarhizium anisopliae var. acridum. Analysis of 259 unigenes out of 303 sequenced inserts from the cDNA library revealed that the cDNA library was not contaminated with M. anisopliae transcripts and validated the presence of the immune-related genes characterized here. These results suggest that this method overcame the difficulties of contamination from a fungal source in constructing the host cDNA library frommycosed insects and proved that thismethod is reliable and feasible for investigation of host genes in response to fungal infection. Further studies of the expressed sequence tags from this library will provide insights into the molec