The Construction And Screening Of Natural Antisense Transcripts Library Of Pseudomonas Aeruginosa: The Identification And Functional Verification Of MucD-ATS

As the discovery of many epoch-making RNA, such as small interference RNA and micro RNA, we find that there are so many non-coding RNAs naturally existed in biology. There are a great deal of evidence to confirm that the regulation acted by ncRNA is prevalent and they constitute a very complicate RNA regulation network. NcRNA is very important in the regulation of gene expression and in the process such as development, evolution and many physiological activities. In fact, when compared with protein, RNA has advantages in many aspects, such as the recognition and interaction between RNA and RNA or DNA is more accurate and special, the synthesis and catabolism are more rapid. All of these advantages make RNA a more ideal and more simple regulator than protein for exact and rapid regulation during some processes such as cell differentiation and embryo development.Natural antisense transcripts(NATs) is one of ncRNA which has important regulation function of gene expression in prokaryon and eukaryon biology. NATs binds with its target by sequence complementation and forms natural sense-antisense transcripts(NSATs) double strand RNA(dsRNA), so the identification of NSATs is very important to the systematical comprehension of gene regulation mediated by RNA. Considered the widespread of the RNA mediated regulation in biological events, it seemed that high-throughput screening and identification of NATs are necessary and urgent. Until now, several groups have focused on this field and their results suggest that NATs are much more than we have expected before. However, bioinformatics methods or microarray analysis used in all these work have a serious limitation that they just mean there were two RNA molecules with the potential to form dsRNA, but could not confirm the reality, for two dissociative single stranded RNA might locate in different organelles in a cell, or different cells in a given tissue or cell pool. Furthermore, most of their work focused on eukaryon such as human, mouse, and few of them focused on prokaryon. Pseudomonas aeruginosa is a common conditional pathogenic bacteria which causes many kinds of infection, and our laboratory researched PA for long-term. At present, the sequencing of PA genome had been complemented and which afford the foundation for the systematic screening of NATs and illuminate the regulation mechanism of gene expression of PA. To overcome the shortcoming of above methods and screen NATs of PAO1, we established a novel strategy which is simple and reliable to high-throughput screen NATs by experimental approaches and make sure that all the NSATs we obtained existed in the form of dsRNA in cells naturally. This strategy resolved the limitations in previous works thoroughly and it based on the fact that RNase I digest only single strand RNA while double strand RNA can resist to the digestion. We extracted total RNA of Pseudomonas aeruginosa and it was treated with DNase I to remove the DNA contamination, then the remains were digested by RNase I to separate NSATs from total RNA, and we constructed a cDNA library of the separated NSATs to get its sequence information as follows: at first, NSATs were reverse transcriped into cDNA by M-MuLV, then polyA and a specific primer were added to 3'and 5'end repectively of all cDNA sequence by terminal transferase and T₄ RNA ligase. Following these work, PCR amplfication was conducted using the sequence of both ends of cDNAs as primers, the products were ligated with pGEM-T vector and transfected E. coli to get the NATs library. 90 randomly selected clones were sequenced after they were confirmed by digestion of restriction nuclease. Then the analysis of the sequence by means of bioinformatics strategies were carried out and the candidates were confirmed by cDNA and Northern hybrid-dization. Finally, we got 4 NATs which have not been reported yet, and the result confirmed the reliabity and effectiveness of this strategy. By analyzing the function of NATs related gene, we found that they were involved in the metabolism, the biosynthesis of antigen and virules effectors. These results indicated that NATs may have important and extensive regulatory functions in PAO1 and provided key clues for elucidating the gene regulatory mechanisms in PAO1.Among the identified 4 NATs molecules, 3 of them were not suitable for further research for the function of their corresponding genomic gene were uncertain, so we selected a molecule named mucD-ATS which hits in the open reading frame of mucD as the target for further research because the function of mucD had been clarified. MucD encodes a serine proteinase in PAO1, which inhibits the activity of transcription factor algU whose activity is essential for the transcription of a alginate biosynthesis operator. To identify the target and biological effects of mucD-ATS, we constructed a mucD gene partial knockout suicide plasmid and transfected PAO1, then screening the mutant strain by PCR and southern blot method. At the same time, a completely mucD gene knockout strain was presented from Prof. Lynn Wood, Boston general hospital, America.After being ligated with a shuttle plasmid and being transfected into PAO1, mucD-ATS was induced to express by IPTG and the changes of mucD gene expression amount were measured by semi-quantitation RT-PCR; The changes of algU expression amount were measured by the same method after the recombinant shuttle plasmid pUMA was transfected into PAO1; The biofilm appearance of above strains were surveyed by laser microscope following the PI staining, and the alginate concentration was surveyed by Carbazole method. The result suggested that: firstly, mucD-ATS functions with mucD mRNA and has no direct interaction with algU; secondly, mucD-ATS may causes the mucoid mutantion of PAO1 through the downregulation of the expression of mucD.In conclusion, 4 NATs molecules had not been reported were screened and identified by RNase I protection assay and molecule hybridization methods, and it bring light to the research of gene express regulation mechanisms mediated by NATs in PAO1; The function and molecular mechanism of mucD-ATS in the alginate biosynthesis of PAO1 was clarified in this research, and this afford new standpoint and evidence for the research of alginate biosynthesis of PAO1.