| Non-coding RNA(lncRNA)is a kind of long chain length is greater than or equal to 200 bp and coding of RNA and protein in the previous studies are considered to be "noise" in the gene expression,but with the completion of human genome project,the researchers gradually deepened the understanding of and lncRNA,more and more has the important function in the life activities of lncRNA was found and research,study of lncRNA has also gradually become more.LncRNA is of low evolutionary conservatism in different species and has great species diversity.It is expressed in most tissues and organs of living organisms,but the transcription levels of different tissues and organs are greatly different.In current studies on lncRNA,it has been found that lncRNA plays an important role in many important life activities of living organisms and the occurrence and development of some diseases,which are specifically reflected in the development of organisms,cell differentiation,innate immunity,and the occurrence and development of tumors.LncRNA is mainly involved in gene imprinting,cell cycle regulation,transcriptional regulation and other functions in cell regulation.It is mainly manifested in three aspects: epigenetic regulation,transcriptional regulation and post-transcriptional regulation.In the present of lncRNA research techniques,in addition to the early prediction of lncRNA gene Chip and transcriptome analysis of bioinformatics methods,from the analysis results of gene Chip and RNA-seq still need from molecular biology experimental verification,validation methods including Q-PCR,gene knock down,and FISH,hybridization,RIP,RIP-Chip and RIP-seq,CLIP,etc.Grass carp is one of the four great fishes in China.Grass carp farming accounts for about 20% of the freshwater fish farming in China.It is an important part of China's agricultural economy.At present,the cultivation of grass carp is carried out in different scales all over the country.With the expansion of the cultivation scale and the increase of the cultivation density,the influence o f fish diseases on the cultivation is growing,among which the most influential fish disease is grass carp hemorrhagic disease.Therefore,studies on the immune mechanism of grass carp hemorrhage are particularly important.However,studies on the immune-related lncRNA of grass carp are still rare in China.The recently published complete genome atlas of grass carp provides a cornerstone for the study on lncRNA of grass carp.In order to study the lncRNA related to innate immunity of grass carp,we used TopHat2 software to compare transcriptome data to grass carp genome,and then used Cufflinks to reconstruct the TopHat2 results to obtain new transcriptome data.Non-coding RNA that was not marked as a gene by grass carp genome was selected as candidate from grass carp genome new transcripts.Transcripts larger than 200 bp were selected from non-coding RNA as candidate lncRNAs.These lncrnas were predicted through the CPC software based on the support vector machine algorithm to obtain the final lncRNA set related to antiviral.The screened lncRNA CiISG152-L2 was co-expressed through WGCNA software,and the network of related genes was obtained.The functional enrichment analysis of these genes showed that the main functions were concentrated in the immune functio n.In order to verify the result of bioinformatics analysis,the first cloned grass carp lncRNA CiISG152-L2 and the length of the gene CiISG152 sequence features,the length is 1802 bp and 1161 bp respectively,which contains CiISG152 genes within a 104-bp 5 'UTR,a 586 bp 3' UTR sequence and a length of 471 bp ORF region,ORF encoding a polypeptide chain length of 156 aa,its relative molecular mass of the polypeptide chain is 17.58 kD,and cloning of the gene promoter.Through RT-PCR,the expression of lncRNA CiISG152-L2 in different tissues of grass carps was analyzed,reflecting the tissue differences of lncRNA,and the expression characteristics of lncRNA CiISG152-L2 and CiISG152 in CIK cells were analyzed,and the expression levels of both showed similar changes after virus induction.In order to analyze the functions of the lncRNA,RNAfold WebServe was used to analyze the secondary structure of the RNA sequences obtained.The predicted results based on the minimum free energy and the thermodynamic set were obtained,and the possible functional regions were truncated.To analyze the correlation between lncRNA CiISG152-L2 and CiISG152 at the transcriptional level,the expression of CiISG152 was up-regulated and down-regulated synchronously by RT-PCR after overexpression and interference of the lncRNA.In order to further analyze the transcriptional regulation of CiISG152 by the lncRNA,the dual luciferase reporter gene experiment showed that the lncRNA could significantly enhance the promoter activity of CiISG152,and through the predicted functional region truncated by the RNA secondary structure,it was found that the region that promoted the promoter activity was located at 620-1190 bp of the lncRNA.In order to further explore the function of this lncRNA in innate immunity,we proved that CiISG152 can be combined with CiIRF3 through immunoprecipition experiments.In order to further explore the function of this lncRNA in innate immunity,we proved that Ci ISG152 can be combined with CiIRF3 through immunoprecipation experiments,and Ci IRF3 can effectively enhance the expression intensity of CiIRF3 in cells after ISG modification after Ci ISG152 binding,thus playing a more important role in antiviral response.In order to verify the function of this lncRNA in fish,CiIRF3 in viral response was quantitatively analyzed by overexpression and interference of lncRNA CiISG152-L2.It was proved that after overexpression of this lncRNA,the protein level of CiIRF3 in cells was significantly enhanced,while interference of this lncRNA was the opposite.In summary,the transcriptional regulation of lncRNA CiISG152-L2 in innate immunity was studied,reflecting the important role of lncRNA in the antiviral response of grass carps. |
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