| The horse industry is an industry that China has vigorously supported in recent years.It is of great significance to study the biological characteristics of crude feed tolerance in domestic local horses for improving the utilization of forage and promoting the transformation,upgrading and sustainable development of China's horse industry.Mongolian horse is native to the Mongolian Plateau and is an ancient grassland horse breed in the world.It is one of the important and excellent local horse breed resources in China and has strong adaptability,disease resistance and crude feed tolerance.The feature of crude feed resistance in Mongolian horse is inevitably closely related to its unique gastrointestinal microbes.In order to further unravel the underlining mechanism of crude feed resistance in Mongolian horse,a systematically designed study was conducted from the following aspects:firstly,the gastrointestinal tract bacterial population composition diversity in Mongolian horse was characterized;secondly,the cellulolytic bacterial strains in gastrointestinal contents of the experimental horses were isolated and screened;thirdly,the enzymatic properties of the cellulase produced by the isolates were evaluated;in addition,the codon optimization and cloning of the cellulase gene were performed,and the food-grade engineering lactic acid bacteria expressing cellulase was constructed.The detailed results were described as followed:(1)A total of five free-grazing Mongolian horses in grassland were selected to compare the differences in bacterial community composition and diversity between six different segments of the gastrointestinal tract(stomach,jejunum,ileum,cecum,ventral colon and dorsal colon)by using high-throughput sequencing technology with Thermo Ion S5 XL platform.It was found that the total number of reads of the 16S rDNA V3-V4 hypervariable region obtained from the contents of the gastrointestinal tract of Mongolian horses was 1 355 813,with an average of 45 194,and the number of OTUs clusters(similarity was 97%)was 24 602,with an average of 820.There were significant differences in microbial communities in different segments of the gastrointestinal tract of Mongolian horses.The differences in microbial composition in stomach was the largest among horse individuals.The microbial composition between the small intestine and the large intestine was clearly distinguishing,and the microbial structure in the cecum and colon was similar.In the foregut contents,the highest proportion was observed in Firmicutes(65%)and Proteobacteria(23%),and Firmicutes(45%)and Bacteroidetes(42%)were dominated in the hindgut contents.At the level of the family,the mainly found microorganisms in hindgut included Ruminococcaceae(P=0.203),Laschnospiraceae(P=0.157),Rikenellaceae(P=0.122),and Prevotellaceae(P=0.068).At the genus level,the relative abundance of cellulose-degrading bacteria such as Fibrobacter in the cecum and Ruminococcus in the ventral colon was higher than that in the other parts of the gastrointestinal tract,indicating that the plant fiber fermentation occurred manly in hindgut.Akkermansia spp.which was associated with health status in the abdominal colon,had a relatively higher abundance of 5.7%.In terms of functional prediction,the microbial functional abundance in different parts of gastrointestinal tract of Mongolian horses was observed similar,which might be able to characterize the microbial community structure in the gastrointestinal tract of Mongolian horse.It could be seen that the herbivore and adaptability of Mongolian horses were not only associated with their unique gastrointestinal microbial community structure,but also might be attributed to the specific physiological features induced by long-term feeding and digestion of roughage.(2)Separation and screening of cellulose-degrading bacteria from cecum content samples of Mongolian horses were performed by using CMC-Na plate method and Congo red staining method.Two isolates possessing quite potent cellulose-degrading activities were obtained,which were identified as Microbacterium arborescens(denominated as C6)and Bacillus amyloliquefaciens(denominated as C14)by Gram staining method,physiological and biochemical identification method and 16S rDNA PCR and sequence analysis,respectively.The C6 strain had a slow growth rate and a long fermentation cycle.After start of incubation,its growth entered a logarithmic phase ranging from 8 to 120h,a stationary phase ranging from 120 to 124h,and a gradually decreasing phase after 124 h,respectively.The C14 strain showed a rapid growth rate whose growth entered a logarithmic phase ranging from 8 to 36 h after start of incubation and a stationary phase after 36 h,respectively.No recession phase was observed in growth of the C14 strain.The cellulase produced by the C14 strain exhibited the highest enzymatic activity under the incubation conditions of pH 4.8,55? and reaction for 5 min.Furthermore,it had strong acid-and alkali-resistance and thermal stability,indicating a promising prospect in deep development and industrial production.(3)A cellulase gene of egl-BA with length of 1 500 bp was cloned from the genomic DNA of the Bacillus amyloliquefaciens C14 strain of Mongolian horse which encoded 499 amino acids.Sequence analysis predicted that the cellulase encoded by egl-BA gene was composed of a transmembrane domain(7-29 aa),a structural functional domain of cellulase(50-297 aa),and a cellulose-binding domain region at the N-terminus(356-437 aa).It was a hydrophilic and secreted protein with complicated spatial structure and contained a signal peptide.Finally,codon optimization of the egl-BA gene using lactic acid bacteria as host was conducted,laying a foundation for heterologous expression in further study.(4)A stain of food-grade engineering lactic acid bacteria with strong cellulose-degrading ability,denominated as NN1-EGL(pMG36e-Emr+p32+usp45+melA+NisI+egl),was successfully constructed.The enzymatic activity of the cellulose produced by NN1-EGL was 0.92 U/m L,which was higher than that of the original strain(0.60 U/mL).The obtained results laid the foundation for the further development of food-grade engineering bacteria,and also provided the necessary experimental basis for its industrial development. |
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