Screening Of Non-coding RNAs Of Spiroplasma Sinensis And Study Of The Interaction Between SR05 And Targets

Aquaculture industry is an important part of China's national economy,of which Crustacean is an important part of aquaculture and have huge economic value.However,as the degree of intensive aquaculture continues to increase,various diseases continue to emerge,among which tremor disease(TD)is a serious aquatic disease that has a serious impact and causes huge economic losses.Spiroplasma eriocheiris is the pathogen of “tremor disease”.It often exists in aquatic environment and aquatic animals.It infects a wide range of hosts.It can not only infect Chinese mitten crabs,but also to Procambarus clarkii,Litopenaeus vannamei,Macrobrachium rosenbergii and Macrobrachium nipponensis.In order to adapt to the complex environment in various hosts and changes in the external environment,spiroplasma needs to quickly and efficiently adjust its related gene expression.Therefore,there may be regulatory factors related to growth and disease-causing gene expression in its genome.Non-coding RNA,also known as small RNA(sRNA),has been proven to be an important regulatory factor in many bacteria.It is widely involved in bacterial growth stress,quorum sensing,virulence regulation,and metabolic processes.However,the related reports are mostly concentrated in model bacteria,while the study on spiroplasma sRNA is rarely reported.In view of the important role of sRNA in bacteria,based on the previous genome sequencing of spiroplasma and the differential expression of sRNA in vivo infection and in vitro culture of spiroplasma,the existence of sRNA related to the survival and virulence of spiroplasma has been confirmed by our research group.In this study,through the use of bacterial sRNA strand-specific transcriptome sequencing,RNA pull down combined with RNA-seq and LC-MS/MS and other technologies,combined with bioinformatics methods,the screening and identification analysis of sRNA and its potential interacting RNA and protein were carried out.Exploring the potential regulatory function of sRNA in the growth and metabolism of spiroplasma provides a basis for studying the growth regulation and pathogenic mechanism of spiroplasma.This research is mainly carried out from the following three aspects:1.sRNA strand specific transcriptome sequencing and analysisFirstly,on in vitro culture in different periods of the original body of the screw for chain specific transcriptome sequencing,combined with bioinformatics methods,to analyze the transcript,found 81 new sRNA with promoter,which,according to the results of screw of chlamydial sRNA regulation at key nodes in the network sRNA appeared difference,confirmed the previous research results.At the same time,27 new sRNAs were identified,including 37 forward transcripts and 43 reverse transcripts.Differential transcriptome analysis of spiroplasma cultured at different times in vitro showed that a total of 70 sRNAs were differentially expressed in the stationary phase relative to the logarithmic growth stage,of which 25 were up-regulated and 45 were down-regulated.A total of 70 sRNAs were differentially expressed in the decay stage compared with the logarithmic growth stage,of which 20 were up-regulated and 50 were down-regulated.The total number of differentially expressed sRNAs in the decay stage was 64 compared with that in the platform-stage,of which 11 were up-regulated and 53 were down-regulated.The results showed that sRNA was differentially expressed in different growth phases of spiroplasma in vitro.2.RNA-RNA pull down combined with RNA-Seq was used to identify the sRNA interacting RNAIn vitro transcription of Spiroplasma sRNA SR05,using RNA-RNA pull down method to screen the SR05 interacting RNA,and classify the potential interacting RNA and predict the target.The results showed that not only sRNA interacting mRNA was identified,but also non-coding RNA was identified.The total number of differentially expressed genes was 53,and 30 genes were significantly up-regulated and 23 genes were significantly down-regulated in Sense group compared with Antisense group.The total number of differentially expressed lncRNAs was 6,and all of them were significantly down-regulated.The total number of differentially expressed sRNAs was17,12 of which were significantly up-regulated and 5 of which were significantly down-regulated in the Sense group compared with the Antisense group.The GO functional annotation and KEGG pathway enrichment of the interaction genes and noncoding RNA targets showed that the potential SR05 targets involved multiple signaling pathways and biological functions.3.RNA-Protein pull down combined with LC-MS/MS was used to screen and identify sRNA interacting proteinsSR05 was transcribed in vitro,and the interacting proteins of spiroplasma sRNA were pulled down by RNA-Protein pull down method.After SDS-PAGE,silver staining was performed,and LC-MS/MS was performed to identify the interacting proteins.The results of mass spectrometry showed that a total of 120 potential interacting proteins were obtained,covering proteins related to nucleic acid metabolism,energy conversion,material transport and other life activities.ABC transporter permease,a potential interacting protein,negatively regulates biofilm formation.Redox-regulated ATPase YCHF is preferred for the hydrolysis of ATP and is involved in a variety of cellular processes and pathology,including DNA repair,tumorigenesis and apoptosis.And the 50 S ribosomal protein subunit L11 and L28,which are related to the synthesis of ribosomes and proteins.To a certain extent,it has been confirmed that SR05 plays an important role in the regulation network of sRNAprotein.The selected 120 potential interacting proteins laid an important foundation for the further study of spiroplasma sRNA-protein interaction.In summary,through the use of bacterial sRNA strand-specific transcriptome sequencing,RNA pull down combined with RNA-seq and LC-MS/MS and other technologies,combined with bioinformatics methods,to screen out SR05 that is differentially expressed at different stages of spiroplasma growth.Multiple sRNAs,using SR05 as the target non-coding RNA to screen 53 genes,6 lncRNAs,17 sRNAs,and 120 proteins that can interact with them.These substances are regulating nucleic acid metabolism,energy conversion,and material transportation,plays an important role in this life activity.