Sequence Analysis And Expression Of Lignocellulose Degradation Related Enzymes From The Midgut Of Macrotermes Barneyi

Termite is a social inesect which can also be called as ecosystem engineers for its important role in ecosystem of soil. According to whether prosists exist in their guts or not, termites can be divided into lower termite and higher termite. In taxonomy, only Termitidae belongs to higher temite, while the amounts of higher termites occupy75%of all the termite species. Relative to the study of lower termites, at present, the study of higher termites lignocellulose degradation mechanism is still less.Macrotermes barneyi is a kind of higher fungus-growing termite widely distributed in southern China, which is symbiotic with fungi. Study has shown that the symbiotic fungi own the ability of lignocellulose degradation. In addition, Study has also shown that there are endogenous cellulase in fungus-termite gut which may play important role in cellulose degradation. In our previous study, the highest activities of lignocellulolytic enzyme were found in the midgut. The RNA-seq analysis discovered relative unigenes predicted for lignocellulose degradation, including laccase, β-glucosidase, peroxidase, feruloy esterase ect. To better understand the function of these unigenes and their role in lignocellulose degradation in fungus-growing termite, the full length of cDNA of these unigenes were cloned and expressed using the prokaryotic and e eukaryotic expression system. The detailed contents are presented as follows:The genome walking technique was used to amplify the laccase DNA and about10kb length of DNA sequence was obtained, which composed of8introns and9exons. Two regions are still unknown for the predicted complex secondary structure. Both the5’and3’end flanking sequence of laccase were amplified to prove the laccase’s origin. Only the3’flanking sequence was obtained, which contains transposon of insect origin, therefore the laccase is proved to be an insect origin enzyme. Since cDNA of laccase could not expressed in E.coli and Pichia pastor is, here we tried to express it using an insect baculovirus expression system. However the cDNA of laccase was not expressed.In previous transcriptome sequencing of M. barneyi, three P-glucosidase unigenes (10737,10810and59997) were predicted. The unigene10737belongs to GH30family, while the other two (10810and59997) belong to GH1family. The full-length cDNA of three unigenes containing the complete open reading frame were successfully amplified by RACE. Sequence alignment analysis found that10810and59997has a conservative structural loci of family of GH1, while the structure loci of the10737has no tbeen found. The phylogenetic tree indicated that10810and59997sequences are all insect origin. Heterologous expression studies had found that10810was expressed in the prokaryotic expression system but detected no activities. While in eukaryotic expression system, the expressed protein was not purified. Both59997 and10737can not be expressed in E.coli and P. pastoris. After sequence optimization of BG-59997and BG-10737according to the codon preference, the10737was expressed and purified, but activity was not detected and59997was still not expressed. Further MbBG, BG-10737,59997and10810were tried to express using insect baculovirus expression system, but no signal was detected in the western blot test. The reason needs to be explored.The full length cDNAs of75774and82227, which were predicted as peroxidase and ferulic acid esterase, respectively, were also successfully amplified by RACE. The sequence results showed that the75774coded for peroxicentin protein from invertebrate animals peroxidase family, which participates in cell adhesion, while the82227is juvenile hormone esterase of Esterase_lipase superfamily, which is an important enzymes involved in metamorphosis and the development of insects.In conclusion, the DNA sequence of laccase was amplified and proved to be insect origin laccase, the expression of laccase in insect baculovirus expression system was failed. Simutanously three full-length cDNA of predicted P-glucosidase unigenes were obtained, but the functions was not certified. Further two cDNA sequence that predicted for invertebrate peroxinectin from animal peroxidase family and juvenile hormone esterase of esterase_lipase superfamily were obtained.