| As a microorganism that accounts for 50% of the total mass of rumen,rumen ciliates regulate the rumen micro-ecosystem through predation,competition and symbiosis with other rumen microorganisms,and play an important role in the degradation of carbohydrate.Rumen ciliates belong to unicellular eukaryote with binuclear characteristics.Because they are difficult to pure culture and have a large number of morphological species,their phylogenetic relationship and ecological functions have not been fully determined and analyzed.In this study,we analyzed the classification of rumen ciliates and their adaptive evolution in the rumen from the aspects of morphological observation,single cell sequencing,carbohydrate enzyme identification and functional identification.The main research results in this study are as follows:1.Classification and phylogenetic evolution of rumen ciliates.In this study,we collected 69 rumen ciliates from the rumen contents of 5 healthy Guanzhong dairy goats,6Holstein cows,and 3 Qinchuan beefs,representing 22 species of rumen ciliates with 97%genus abundance.Through observation with common light microscope,field-emission scanning electron microscope and confocal laser scanning microscope,two hidden species belonging to Isotrichidae were found,and then we made a detailed morphological description of 22 morphospecies of rumen ciliates representing 97% morphogenera abundance.Subsequently,we conducted a detailed morphological description of these 22 morphological species.The genomes of 69 rumen ciliates were obtained by single-cell sequencing technology,after assessing their integrity,52 of them belonged to high-quality genomes(BUSCO value>80%).Subsequently,combined with genome average nucleotide identity(ANI)and relative evolutionary difference(RED)analysis,we redefined 1 new family,2 new genera,and 2 new species of 52 rumen ciliates,totaling 19 species of rumen ciliates;Finally,through single copy gene tree construction,the overall phylogenetic relationship of rumen ciliates was clarified,and 19 species of rumen ciliates were classified into 2 orders,3 families,and 13 genera.2.Identification of new species of rumen ciliates and reconstruction of fermentation metabolic pathways.By using the micro-image recognition and identification website we developed earlier,we found a new species of rumen ciliates with triangular shape(named Isotricha＿A mobula);Through the construction of phylogenetic tree,the results show that Isotricha＿A mobula and Isotricha＿A.protoma is a sister branch of each other.The discovery of Isotricha＿A mobula has once again supplemented the species of rumen ciliates,providing the possibility for a deeper understanding of the evolution of Isotrichidae.Reconstruction of metabolic pathway of rumen ciliates found that about 80% of rumen ciliates can encode acetate production pathway,but lack of complete coding of butyric acid and propionic acid metabolic pathway.3.Investment tradeoff and source identification of rumen ciliates CAZymes.A total of43,901 CAZymes proteins were predicted in 53 rumen ciliates,and only 1% of CAZyme clusters were 95% or more similarity with the public database sequence through cd-hit de-redundant.These results indicate that rumen ciliates encode a large number of novel CAZymes.Especially for the Diplodininae and Ophrystocolecinae,which encode CAZymes equivalent to intestinal fungi,the CAZymes encoded by the species belonging to these two families are two and five times higher than those of Entodiininae and Isotropidae/Daystricidae,respectively.It is suggested that Diplodininae and Ophrystocolecinae play an important role in the degradation of carbohydrates.In addition,each family/subfamily of rumen ciliates can not only encode multiple enzymes to synergistically degrade a single type of polysaccharide,but also degrade different substrates between subfamilies to avoid niche overlap.This study found that Diplodininae and Ophrystocolecinae preferred the degradation of structural carbohydrates,such as cellulose,hemicellulose,and pectin,while Entodininae,Isotricidae and Dasytricidae preferred the degradation of non-structural carbohydrates,such as starch and fructan.Finally,combined with BLAST alignment and phylogenetic analysis,it was found that 60%of CAZymes coding genes of rumen ciliates were obtained by horizontal gene transfer(HGT).Among them,52% of HGT genes were obtained from bacteria,a total of 16,379,mainly from Firmicutes and Bacteroides.In addition,8% were from fungi(2,245).Secondly,we also found that the Isotricidae/Dasytricidae ciliates have more obtained through by HGT from fungi.4.Functional identification of rumen ciliate CAZymes obtained by HGT.From the rumen ciliates CAZymes identified as HGT source,we selected 35 genes for gene synthesis.In this study,9 genes were successfully expressed and tested for enzymatic properties,including 2 cellulases,4 xylanases,2 mannanases,1 pectinase,and 1 lysozyme.In addition,in order to compare the activity between HGT putative donor species and rumen ciliates CAZymes,we also successfully expressed CAZymes of two putative donor species.The results showed that the activities of cellulase OCCel1 A and xylanase OGXyn1 A obtained by HGT were 9 times and 2 times higher than those of the putative donor species,respectively;The sequence structure showed that the sequence of the ciliate CAZymes was one less motif than the hypothetical donor species.The results of enzyme kinetics showed that the substrate binding ability of the rumen ciliate OGXyn1 A was higher than that of the hypothetical donor species.The results showed that the CAZymes of the rumen ciliate obtained from HGT enhanced its carbohydrate degradation,and making it better adapted to the high carbohydrate environment in the rumen.Secondly,the enzymatic properties of the CAZymes obtained from HGT of the rumen ciliates were determined.The results showed that the optimum temperature of most of the CAZymes encoded by the rumen ciliates was maintained at 45～60°C,and the optimum p H was 5.0～6.5,which was close to the p H of the rumen environment of ruminants.In addition,after xylanase was treated in the optimal p H buffer at 40° C for 24 h,their enzyme activity remained above 65%.Finally,9.In addition to the predicted GH structure with EC enzyme,we have also demonstrated that a GH19 family protein has a high lysozyme activity,with a specific activity of 36,007U/mg(shake flask),which has a significant inhibitory effect on gram-positive bacteria.The results of fermentation showed that the enzyme activity gradually increased with the passage of time in the upper tank.To sum up,the high-quality genome catalog of rumen ciliates obtained in this study provides the possibility for the determination of its phylogenetic relationship and the analysis of its ecological functions.A large number of CAZymes enzymes with high activity encoded by rumen ciliates are of great significance for their adaptive evolution.In addition,as a new source material of CAZymes,rumen ciliates provide great potential for biomass conversion and development of antibacterial drugs in the future. |
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