Construction Of The Small RNAs Library From Rhizobium Leguminosarum Biovar Viciae Strain 248

Small RNAs(sRNAs) in prokaryotes seem to be far more abundant than previously believed.By a variety of mechanisms,including changes in RNA conformation, protein binding,base pairing with other RNAs,and interactions with DNA,sRNAs can be involved in multiple biological processes,such as transcription,translation, mRNA stability,and DNA maintenance or silencing.The number of sRNAs confirmed in E.coli through various approaches is about 80,with several hundred more sRNAs candidate genes under biological validation.With the concept of RNomics,to search new sRNAs candidates in model organisms,both bioinformatical and experimental methods are involved.The corresponding projects are labor-cosuming,yet of great importance in future application.Symbiotic association between leguminous plants and rhizobia bacteria is of great importance in agriculture.To further investigate their molecular mechanism, genomic screens in search of microRNAs have been performed in different leguminous plants.In rhizobia bacteria,search of novel sRNAs,mainly in intergenic regions on the basis of informatical screen,has also begun.By using the Experimental RNomics method,we have built a sRNAs library of Rhizobium leguminosarum biovar viciae strain 248.We had extracted the total RNA from 248 cells in three different phases,and harvested about 18,000 clones within the cDNA library.After the following screen work and data analysis,we had obtained a final set of 363 contigs.According to their location within genes or in the intergenic regions in Rhizobium leguminosarum biovar viciae strain 3841,the contigs had been separated into seven classes:The most strange class is the "No significant similarity was found" one,which has 83 members that represent the contigs that have no match in all rhizobia bacteria genome,although some of them were included mainly because of their short length that resulted in no credible matches.The three most possible classes of contigs in which there may be novel small RNAs candidates,according to previous research results,were 69 "Intergenic regions" contigs,25 "Antisense within gene" contigs and 34 "overlapping reading frames,or overlap of mRNA with intergenic region" contigs.Finally,we got three classes of contigs representing the open reading frame(ORF) of known genes:"Entire mRNA or mRNA middle part" contigs(95),"N-terminal part of mRNA" contigs(30) and "C-terminal part of mRNA" contigs(27).These three classes are quite possibly fragements processed from the mRNAs of genes. And the numbers of contigs exhibited an obvious bias towards the "Entire mRNA or mRNA middle part" class.