| In nature, microoganism play a critical role in the circulation of material and energy. Since Pringsheim et al. found bacteria isolated from soil can degrade cellulose, the studies about cellulolytic microoganism and the cellulose-decomposing enzymes producted by them have made a great progress in recent decades. It has been confirmed recently that there are abundant microbes in rumen of ruminants such as sheep or cattle. Therefore, exploration of rumen microbes for degradation of cellulose is a new path for biofule. Besides, the diversity and structure of microbes in rumen closely relate to animal’s diet, increase the percentage of cellulose in animal’s diet would benifit the finding of new cellulolytic microbe or enzyme.In the present study,16S rRNA and18S rRNA gene libraries were constructed, and the changes of diversity of bacteria, archaea and fungi induced by the diet of Miscanthus sinensis were analyzed. In addition, the metagenome library of ruminal microbes was constructed, one new p-glucosidase gene was screened and subjected to prokaryotic expression. The detailed results of our study were as follows.Firstly, as the comparision with commen mixed forage feeding library shown, the proportion of Bacteroidetes had increased by about12%, in contrast, the ratios of Firmicutes and Proteobacteria had declined by8%and7%, respectively, after feeding on Miscanthus sinensis for18months. Additionally, the proportion of uncultured bacteria was shifted obviously. At the same time, archaea community had been changed considerabely caused by the sole feedstuff of Miscanthus sinensis. The proportion of the Methanomicrobia class decreased from57.14%to12.22%, and the dominant group was replaced by Thermoplasmata. While, for fungi, there were Orpinomyces sp., Sarocladium zeae, Aspergillus niger, Piromyces sp., Alternaria alternata, Candida castellii, Ascomycota sp., Gibellulopsis nigrescens, Neosartorya udagawae in rumen of mixed forage feeding cattle, in contrast, the species in rumen of Miscanthus sinensis feeding cattle was concentrated relatively:Orpinomyces sp., Piromyces sp., Neocallimastix frontalis, Microdochium sp..Secondly, the metagenome library of ruminal microbes was constructed based on the pCC2Fosmid vector. This library contained20160clones with mean length of30.23kb, which covered595.2Mb totally. Besides, it was of strong stability through the testing by4times of continued subculture. Consequently, this library was of high quality for functional gene screening.Thirdly, one β-glucosidase functional clone was obtained from the metagenome library, named135B12. Furthermore, through subclone, one DNA fragment including3470bp was gained, which contain a predicted ORF with2340bp named unglu135B12. Unglu135B12encoded a protein (unglu135B12) with779amino acids, and its molecular weight was predicted as84162.2Daltons, and pI was6.0. According to the phylogenetic analysis of unglu135B12, the result showed it probablly derived from Bacteroides. The similarity of unglul35B12with its closest relatived sequence was only62%, indicated that this gene was probably a new β-glucosidase encoding gene. Moreover, after overexpressed by pET28a, the study of enzymatic properties of unglu135B12indicated its optimal reaction temperature was38℃, and optimal pH was5.0(specific activity at optimum condition:2.5×103U/mg), and the enzymatic activity of unglu135B12to pNPG was enhanced by Ca2+, K+, Na+, while was inhibited by Fe3+, Cu2+, Zn2+, Mn2+. Km was0.314mmol/L, Vmax was7.188μmol/min according to the Lineweaver-Burk method.The findings suggested that the cellulolytic microbiome in the local cattle’s rumen could be induced by sole diet M. sinensis, and one novel β-glucosidase encoding gene unglu135B12was screened from the metagenome library of ruminal microbes of M. sinensis feding cattle. Unglu135B12will be benefit to futher utilization of cellulose in M. sinensis. |
PDF Full Text Request