Cloning And Expression Of Genes Encoding Laccases In Termitomyces Albuminosus, Culex Pipiens Pallens And Musca Domestica

Laccase(EC 1.10.3.2) is one of copper-contained polyphenol oxidases,and it exits in fungi(especially white-rot fungi),plants,insects,and bacteria.Laccase has broad substrates(about 250 kinds),which mainly are phenolic and aromatic compounds.Laccase has provided great application potential in ligin degradation, biobleaching in pulps,environment protection,processing of foodstuffs and beverages. In this paper,we analyzed the characteristics of laccase from the symbiotic fungi (Termitomyces albuminosus) of Odontotermes formosanus,induced laccase production,cloned a laccase gene from the fungi and expressed it in Escherichia coli. Meanwhile,we also cloned laccase genes from Culex pipiens pallens and Musca domeslica,and analyzed the relation between gene expression of laccase and resistance of fenvalerate in Culex pipiens pallens population.The main results are as follows:1.Analysis of the distribution of laccase in alimentary tract of workers of O. formosanus,nymph,T.albuminosus,and fungus comb,as well as effect of temperature,pH value and inhibitors to the activity of laccase.The results showed that T.albuminosus exhibited the highest laccase activity,reached 13.36 U/g;followed by the fungus comb,its laccase activity was 4.24 U/g;in alimentary tract of workers,its laccase activity only was 1.05 U/g;in nymph,the laccase activity could not be detected.These laccases from different materials exhibited high activity in acid condition(pH 3.0-3.5) and thermal stability.Also,these laccases could be inhibited strongly by L-cysteine,DTT and fluoride ions.The results suggest that T. albuminosus takes part in lignin degradation of plant litter through secreting laccase into fungus comb in colonies of O.formosanus.2.Induction of laccase production in environment with different cultivation media and CO₂ concentrations.The results showed that T.albuminosus could be cultured in PDA,CDA,PD,and KB(with LN/HN) media,but the mycelium of these cultured fungi was different from the original fungi.The fungi cultured in PDA and KB(LN) expressed low laccase activity.The T.albuminosus could grow normally in the environments with 4%or 10%and 15%of CO₂ concentration,and expressed low laccase activity,but it could not grow normally in the environments with 25%or 50% and 75%of CO₂ concentration.The results suggest that the laccase activity of T. albuminosus can be induced with cultivation media and CO₂ in laboratory.3.Cloning of a laccase gene with RACE method from T.albuminosus and expression of this laccase gene in E.coli.The full-length of laccase gene(talcc) from the symbiotic fungi is 2295 bp,contained an open reading frame(ORF) of 1551 bp. This cDNAs was composed of 15 exons,and encoded a putative 517 amino acid protein.The predicted molecular weight(MW) and isoelectric point(pI) for the mature protein of talcc are 55.8 kDa and pH 5.92,respectively.After alignment with other fungus-originated laccases,the TaLCC presented the highest similarity(about 67%) to CbLCC from Cyathus bulleri,but had only 47%similarity to laccase from the same genus fungi.Meanwhile,the cDNAs of talcc with native signal peptide was transformed into the expression vector of pET-28a,and the recombinant vector of pET-28a/talcc was transformed into E.coli of BL21.After induced 1-3 hours with IPTG,the recombinant strain secreted a 60 kDa protein.This protein was the recombinant laccase through analysis of Western blot.Unfortunately,no laccase activity was detected in this purified protein.4.Cloning of laccase genes from C.pipiens pollens and M.domestica with RACE method.The laccase gene CpLac2 of C.pipiens pollens contained an ORF of 2289 bp that encoded a putative 762 amino acid protein.The predicted MW and pI for the mature protein of CpLac2 were 81.2 kDa and pH 6.21,respectively.The putative protein had a signal peptide of 31 amino acids.After blasted with other insect laccases, CpLac2 had 78-90%identity with the insect laccase 2 family.This means the CpLac2 belongs to the laccase 2 family.A laccase fragment of 1482 bp was cloned from M. domestica.This sequence also presented >90%identity with insect laccase 2 family, and it also means this sequence belongs to the laccase 2 family.5.The developmental expression model of CpLac2 in C.pipiens pollens was measured by RT-PCR.The result showed the CpLac2 was abundantly expressed in egg,the 4^th instar larva and pupa,which suggested the role of CpLac2 for egg chorion tanning and cuticular sclerotization.Meanwhile,the expression of CpLac2 in fenvalerat-susceptible and -resistant strains of C.pipiens pollens was measured by real-time PCR.The result revealed the CpLac2 was significant higher expressed in resistant strain than in susceptible strain.The overexpression of CpLac2 in resistant strain suggested that resistance could derive from reinforcement of the cuticle,which decreased the penetration of insecticide in cuticle.