The Regulation Research Between RNase H And Small RNA In Bacteria

RNase H is a kind of Ribonucleic acid hydrolase which extensively exists in prokaryotic and eukaryotic cells.It can effectively degrade RNA in the RNA and DNA hybridization chain.RNase H is ubiquitous in prokaryotic cells,and for its unique substrate specificity,RNase H plays a key role in the biochemical processes involved in DNA replication,gene expression and DNA repair.As a special class of oligonucleotides,sRNA regulates transcription,translation,mRNA stability,DNA stability,and gene silencing in cells by altering RNA conformation,binding to proteins,and interaction with nucleic acids and has great regulating effect on biochemistry reaction.Studies have shown that ribonucleases play a key role in the generation and processing of small RNAs and regulate their cellular levels by regulating the turnover of small RNAs.So this study speculates that ribonuclease H may be related to the regulation of small no-coding RNA in bacteria,and makes experiment to knock out the RNase HI and RNase HII genes of E.coli DY329 strain,and analyzes sRNA in the cell through RNase H expression defective strain.In this study,we first obtain the △rnhA strain,△rnhB strain and △rnhA & △rnhB strain of the three ribonuclease H-deficient strain with the wild type strain under the same culture condition,and find the difference of growth curve.The growth of the strain at 32℃ is not significantly affected compared with the wild type strain.After careful comparison and analysis to the data,it is found that the growth status of the strain after knocking out RNase HII is worse than that of RNase HI,and the growth of the strain is the worst after both RNases are knocked-out.However,knockout of ribonuclease H in the cells doesn’t significantly affect cell growth as a whole.Then cultivate the four strains,the expression levels of TFF,ryeA,GlmZ,SgrS and SraF were detected by RT-Q-PCR.The results show that there are changes in the small RNAs in the strains after RNase H knocked-out,among which the variation of SraF is the most obvious.The expression level of SraF of the strain which △rnhA and △rnhB are both knocked-out is 11.9 times of that of wild type.It shows that the expression of SraF has a significant upward trend in RNase H-deficient strain,which means RNase H do have regulation relationship with small RNA in bacteria.Because of the low copy number,the short length and zero translation,some small RNAs are extraordinary difficult to be detected.In order to solve this problem,this study designs a new kinds of stem-loop primers which contains dU,which can be used in the quantitative determination of small RNAs by RT-Q-PCR.The thymine(T)base is replaced by a uracil(U)base near the stem-loop portion of the stem-loop primers near the template RNA pair,and after the reverse transcription reaction is over,uracil-DNA glycosidase is used to remove the stem loop at the end of the primer which eliminates the effects of base stacking and steric hindrance on the polymerase chain reaction and improves the specificity and sensitivity of small RNA detection.We used this method to detect two small RNAs in Escherichia coli: TFF and ryeA.The results confirmed that UDG-treated chimeric dU stem-loop primers improved the specificity and sensitivity of small RNAs.