Full-length Transcriptome Sequencing Of Yak Abomasum And Mutation Expression And Antibacterial Research Of Stomach Lysozyme

Yak（Bos grunniens）is a unique breed of cattle in alpine areas.For a long time,it has provided effective living guarantee and economic source for herdsmen in plateau areas,such as food,housing and transportation.As the only true stomach of newborn Yak with digestive function,yak abomasum can not only secrete gastric acid to digest protein and fat in food,but also secrete lysozyme with anti acid and anti pepsin ability.Lysozyme（LYZ）is a key innate immune factor in animals.It has the functions of sterilization,antiviral,anti-inflammatory and immune regulation.In this study,the full-length transcriptome of yak abomasum was sequenced by PacBio SMRT sequencing technology,and the full-length transcriptome data of yak abomasum were obtained.Yak stomach lysozyme 1a（ysLYZ1a）and amino acid mutant yak stomach lysozyme 1a（mysLYZ1a）were eukaryotic expressed and purified.The inhibitory effects of ysLYZ1a and mysLYZ1a on Staphylococcus aureus,E.coli O₁₁₁ and Salmonella enterica were studied in vitro.The therapeutic effects of ysLYZ1a and mysLYZ1a on E.coli O₁₁₁ diarrhea in mice were explored by in vivo test.The main results are as follows:1.The full-length transcriptome data of yak abomasum were obtained by sequencing on PacBio Sequel platform,and a total of 14 467 420 subreads were obtained.After filtering and de redundancy,8 556 non repetitive sequences（unigenes）were finally obtained by comparing the reference genome.The transcriptome data were annotated with seven databases:NR,Swiss-Prot,KEGG,KOG,egg NOG,GO and Pfam,and 8 544,8 475,1 721,6 572,8 491,7 725 and 8 162 unigenes were annotated respectively.943 TFs were obtained by transcription factor（TF）annotation analysis,which were distributed in 51 TFs families.CDS predicted that 8 544 gene fragments could be regarded as CDS coding regions,and a total of 82 were annotated to lysozyme sequences.3 596 SSR loci were retrieved by SSR analysis,and 1 825alternative splicing events were obtained by alternative splicing analysis.2.By consulting the literature,the amino acid mutation sites of mysLYZ1a were determined:A18D、K43T、Q102S.The eukaryotic expression vectors p PICZαA-ysLYZ1a and p PICZαA-mysLYZ1a of ysLYZ1a and mysLYZ1a genes were constructed using a synthetic method.Then it was transformed into Pichia pastoris strain X33 by chemical method.After induced expression and purification,the specific enzyme activity of ysLYZ1a was 1 424.17 U/mg and that of mysLYZ1a was 2 649.28 U/mg.The results showed that the specific enzyme activity of mysLYZ1a was 1 225.11 U/mg higher than that of ysLYZ1a.3.The bacteriostatic test in vitro showed that the bacteriostatic effects of ysLYZ1a and mysLYZ1a on the three tested bacteria were:Staphylococcus aureus>Salmonella enterica>E.coli O₁₁₁.In the in vivo model test,the diarrhea model of mice was established by intraperitoneal injection of E.coli O₁₁₁.Diarrhea mice were treated with antibiotics,yslyz1a and mysLYZ1a respectively,and compared with normal group and infection group.Compared with the normal group,the weight of mice in the infection group decreased significantly,the number of leukocytes increased significantly,the indexes of lung and spleen increased significantly（P<0.05）,and the duodenal injury was serious.After treatment with antibiotics,ysLYZ1a and mysLYZ1a,the indexes of each group were improved compared with the infection group.The intestinal flora of mice in each groupαDiversity andβDiversity analysis showed that E.coli O₁₁₁ infection could significantly reduce the diversity and richness of intestinal flora.In the infection group,the relative abundance of Proteobacteria and Firmicutes increased significantly,while Bacteroidota decreased significantly.After treatment with mysLYZ1a and antibiotics,the proportion of Bacteroidota,Firmicutes and Proteobacteria basically returned to normal,and Campilobacterota increased slightly.After ysLYZ1a treatment,proteus bacteria still increased.MysLYZ1a and antibiotics can restore the flora structure by increasing the abundance of beneficial bacteria and reducing the abundance of harmful bacteria.Ys LYZ1a group also had a small amount of Enterobacter,increased harmful bacteria and a small amount of beneficial bacteria.The therapeutic effect of ysLYZ1a group was worse than mysLYZ1a group and antibiotic group.