Identification And Functional Expression Of Three Chitinases From Fungus-Growing Termite Of Macrotermes Barneyi

Termites,social insect,which have strong ability of degrading lignocellulose,play an important role in the carbon cycle of the ecosystem.According to whether including protozoa,termites can be divided into lower termites and higher termites.Among the higher termites,there are fungus-cultivating termites symbiotic with fungi.Fungus-cultivating termite food contains fungal spores,and the main component of the cell wall of the spores is chitin,so there may be chitinase in the gut of termite for degradetion of fungal spores.At present,the study on termites chitinase is very little.The Macrotermes barneyi is one of the species of fungus-cultivating termites,and the transcripts of the whole intestine of the M.barneyi have been sequenced.In this study,three chitinase genes may be involved in chitin metabolism,which were genetically cloned and expressed,as follows:1.Firstly,we analyzed the sequencing results of the M.barneyi,finding that 40 unigenes of chitinase by searching the keyword "chitinase".The 40 chitinase genes were subjected to a series of bioinformatics analysis,then three genes were screened for further exploration.2.Furthermore,the expression of unigene comp133624_c0 was the highest in the foregut,277.21.According to the bioinformatics analysis of the fragment,the primers were designed and the full length of the cDNA was obtained by PCR and then expressed in E.coli JM109;The expression of unigene compl74658_c0 was the highest in the midgut,523.34.According to the bioinformatics analysis of the fragment,the primers were designed and the full length of the cDNA was obtained by PCR.The gene was expressed in E.coli JM109,BL21(DE3)and Pichia pastoris;The expression of unigene comp182537_c0 in midgut and hindgut was relatively high,which was 750.43 and 1638.2 respectively.It was found that the fragment encodes a chitinase containing CBM-14(binding domain),then the primers were designed and the full length of the cDNA was obtained by PCR.And then it was expressed in E.coli JM109 and BL21(DE3).3.Lastly,we performed qPCR and RT-PCR experiments on the above genes to confirm whether they were consistent with the dominant expression in intestinal parts of the transcriptional sequencing results.In conclusion,three chitinase genes were successfully cloned and expressed in E.coli.Their recombinant proteins were able to detect chitinase activity,but the activity was not high.It may be due to the expression of the insect-derived protein are not fitin E.coli,the recombinant protein is not well modified and folded,resulting in the majority of recombinant proteins are formed inclusion bodies.Besides,unigene comp174658_c0 was successfully cloned and expressed in P.pastoris.In addition,the results of qPCR and RT-PCR showed that the expression level of two genes in intestinal parts were consistent with that of transcriptome sequencing,except for comp174658_c0.