Clone And Expression Of The Chitinase Chit1 Gene From Entomopathogenic Fungi Nomuraea Rileyi Cq Strain

Nomuraea rileyi is an important entomopathogenic fungus that is able to kill more than 40 noctuid insect species such as Helicoverpa arnigera,Spodoptera exigua,Trichoalusia ni and often causes epidemic of the insect in the field.It has been extensively used to control agricultural and forestry pests and shows a great potential application in IPM(Integrated Pest Management) system. The researches about Nomuraea rileyi are focused on Biology,Biological control,Genetic variability from the 1970's to now,but no any research papers about Molecular Biology of infection mechanism.During infection,the fungal pathogens produce a series of cuticle-degrading enzyme such as chitinase,proteases and lipases which facilitate penetration through cuticle.Under in vitro conditions,fungi may be induced to produce exocellular chitinases that digest available chitin substrates.However,the cuticle of insect is composed of about 30%chitin,which is major barrier during infection of entomopathogenic fungus,such chitinases play a role in infection process to disrupt the cuticle barrier.Nomuraea rileyi Cq strain is isolated from Southwest University in china. We report the expressing and cloning chitinase from the dimorphic entomopathogen Nomuraea rileyi Cq strain,compare its sequence to other fungal chitinases.The chitinase were expressed in Pichia pastoris and tested for enzymatic properties.The results are as followings:1.Genomic DNA was extracted from the mycelial preparations using a modified CTAB method,Using Primer5.0 designed different special primers for different PCR amplifications.We analyzed cloned sequence by DNAMAN software.Amplification of the genomic DNA produced 2756 bp sequence that is available from the NCBI GenBank database with the Accession number EU795711.The longer fragment contains a 76bp untranslated sequence at the 5' end,a 1511bp open reading frame(ORF) encoding 424 amino acids,and a l169bp untranslated sequence at 3' end terminating.Analysis of the ORF predicted a 20 aa signal sequence,the 404 aa mature chitinase had a molecular mass of 44 kDa with a calculated pI of 5.27.This sequence contained two highly conserved regions of the active domain of the family 18 glycosyl hydrolases including a presumed enzymatic actice site and a potential chitin-binding domain.A BLASTX search of the deduced amino acid sequence demonstrated similarities to a number of fungal chitinases classified in family 18 of the glycosyl hydrolases such as Nomuraea rileyi MJ(AAP04616),Metarhizium flavoviride(CAB44709),Hypocrea lixii(BAB40589),Lecanicillium lecanii(ABD77096),Isaria farinose(ABD64606),Stachybotrys elegans(AAM70478),Beauveria bassiana(AAN41260), respectively,identify with 99%,79%,74%,74%,74%,69%and 64%,respectively.2.The chit1 gene was cloned into the expression vector pPIC9K to construct the recombinant plasmid pPIC9K-chit1 and was expressed in P.pastoris expression system and chitinase chit1 was secreted into the culture medium under the induction of methanol.The time course of chitinase activity showed the accumulation of chit1 peaked at 72h and one band of proteins with molec- ular mass of around 41 kDa were clearly appeared in the transformats in SDS-PAGE analysis and digestion of chitin showed the transparent parts was most obvious in 72h.