| Termites(Insecta,Palaeodictyoptera),a kind of ancient insect mainly distributed in tropical and subtropical areas.Termites are generally divided into lower termites and higher termites according to whether possessing protozoa or not in their hindguts.They mainly live in the dark,damp environment,taking plant(wood,leaves,etc.)and plant degradation-humus as food.Termites' living environment are more vulnerable to be infected by foreign microbes,so in the process of cooperative coevolution termites may form a unique immune defense mechanism and have some unique genes.In addition,termites have strong degradation,destruction ability,and abundant digestive enzyme genes due to its complicated diets,which makes it a new important model to explore functional genes and new hydrolytic enzymes.Macrotermes barneyi a kind of fungus-growing higher termites,which widely distributed in the south of China.For specific culturing basidiomycete fungi branch in the nest and using it as food,it constitutes a symbiotic system including the termite itself,and fungi in vitro and gut microbes.The transcripts of the whole intestine of the M.barneyi have been sequenced,we found some cellulose genes,including endo-glucanase,?-glucanase,mannase etc.Besides,there are many high expression genes such as ?-1,3(4)-glucanase,chitinase,etc.MbEG and MbBG have been expressed in E.coli successfully,and their enzymology properties were studied,but researches on ?-1,3(4)-glucanase and chitinase from termites are less.In order toexplore these genes functions in termites,we conducted the following research:1.The transcriptome data analysis of M.barneyi was focus on looking for some high expression of functional genes.We found 141 glycoside hydrolase genes,belonging to 29 families,including 47 glycosyl transferase genes(GHs),112 polysaccharide lyase genes(GTs),6 carbon acetate esterase gene(CEs),8 supplementary active enzyme gene(AAs);206 chitin degradation synthesis related genes,24 nitrogenase gene,and some high expression of chitinase genes,?-1,3-glucanase genes.2.The cloning expression and enzymology properties analysis of?-1,3(4)-glucanase gene.Through the q-PCR,RT-PCR and bioinformatics analysis we found the gene was from M.barneyi itself.The full length of Mbbgl was obtained.The results showed that the cDNA was 1085 bp in the length and contained an open reading frame(ORF)of 1035 bp,which encoded 349 amino acids.Analysis of the ORF predicted a 24 aa signal sequence,the 325 aa mature chitinase had a molecular mass of 37.65 KDa with a calculated PI of 4.78.Blast analysis found that the sequence with Coptotermes formosamus ?-1,3(4)-glucanase sequence homology is as high as 83%,followed by the Zootermopsis nevadensis.The Mbbgl was transformed into E.coil BL21(DE3)to be expressed.For recombinant protein Mbbgl,the optimum temperature is 50?,the optimum pH of 5.5,against kelp polysaccharid.It was shown that the optimum substrate was laminarin and the Vmax is 21.83 U/mg,Km is 3.46 mM against it.Using TLC analysis to discover the degration products of laminarin by Mbbgl,we found that it not only has the enzyme activity,but also has a?-glucanase activity,even can degrade laminaribiose to monosaccharides.Due to its novelty,we analyzed its homologous protein structure modeling,and the codon optimization made it better expressed in prokaryotic expression system.3.The expression of chitinase genes from M.barneyi.We expressed the three chitinase genes which showed high expression in transcriptome in Piahia pastoris.The results showed that three MbCht genes have been successfully expressed in P.pastoris,Western blotting can detect signals.The restructuring protein MbChtl had weak activity against colloidal chitin qualitative,however,the activity of MbCht2 and MbCht3 have not been detected.In conclusion,the transcriptome data of M.barneyi were summarized,and the?-1,3(4)-glucanase and chitinase genes were cloned and expressed.Besides,we analyzed its enzymatic property and the protein structure of Mbbgl with homologous modeling. |
PDF Full Text Request