Screening And Analysis Of Related Genes During MBL Anti-infection For Mycoplasma Pneumonia In Sheep Based On Transcriptome Sequencing

Mycoplasma pneumonia(MP)threatens the development of sheep industry in Xinjiang and even in the world.It is a chronic respiratory disease caused by infection of Mycoplasma ovipneumoniae(MO).The pathogenesis of the disease is complex and not yet clear,so it is difficult in diagnosis,treatment and prevention.Mannan-binding lectin(MBL)is an important component of the body natural immune system and plays a role in the MO anti-infection of sheep.Object: In order to explore the MO pathogenic mechanism of and the MBL anti-infection mechanism for MO,the differentially expressed genes were screened from the sequencing results of the transcriptome and the partial differential genes were verified at the cellular level.Methods:(1)The recombinant MBL protein was prepared by eukaryotic expression,and MBL nature protein was extracted from sheep serum.PCR-SSCP was used to classify sheep MBL exon 1 gene for 2-month-old Chinese Merino sheep.Five resistant-type sheep and five susceptible-type sheep were randomly selected according to the analysis results.We infected the sheep with MO.We injected MBL to sheep intravenous after 14 day and 21 day.We slaughtered the sheep and send their liver to biological company for mRNA sequencing after 42 days.The data of sequencing feedback were analyzed by bioinformatics analysis software,and the differentially expressed genes were screened out.(2)Sheep respiratory epithelial cells were obtained from tissue blocks.We isolated and purified sheep epithelial cells and finished morphological identification and immunohistochemical identification.(3)Cell viability was measured at 8h,16 h and 24 h after MO infection.Fluorescence quantitative PCR was used to detect the transcription of FGF1,C-Myc and c-jun gene 24 hours after MO infection.Results:(1)SDS-PAGE gels can detect MBL monomers and polymers at 28 kD and higher molecular weight.The recombinant protein showed a single clear band at about 32 kD.Monosaccharide ligand binding assay showed that both of the two protein had good biological activity.PCR-SSCP was used to isolate four MBL exon 1 genotypes of Chinese Merino sheep.Construction of a library of MO infection and MBL treatment model by liver sampling sequencing.The data of the transcriptome library were analyzed,and 325 genes differentially expressed genes were screened out successfully.We completed the analysis of bioinformatics classification and transcript expression levels.(2)We isolated and purified sheep epithelial cells and finished morphological identification and immunohistochemical identification.(3)The survival rate of sheep respiratory epithelial cells was decreased in susceptible and resistant groups at 8h,16 h and 24 h after MO infection.But there was no significant difference between susceptible group and disease resistance group(P>0.05).The addition of MBL to cell culture medium could significantly decrease the apoptosis rate 24 hours after MO infection(P<0.05).The transcription level of FGF1 gene was significantly higher than that of untreated cells after MO infection(P<0.01).However C-Myc and c-jun gene were notsignificantly higher than that of untreated cells after MO infection(P>0.05).Conclusion:(1)The transcriptome library about MO artificial infection and MBL treatment for Chinese Merino sheep was successfully constructed by high-throughput sequencing.325 differentially expressed genes were obtained from the transcriptome analysis.(2)Sheep respiratory epithelial cells and fibroblasts were successfully isolated.(3)There was no significant difference between the cells of different genotypes faced with MO infection.The transcription of FGF1 gene was significantly increased,C-Myc and c-jun gene were not significantly higher than that of untreated cells after MO infection,after sheep respiratory epithelial cells infected by MO.FGF1 may play an important role in the process.