Construction Of A CDNA Library Of E.Coli And Screening Of Proteins Interact With RNase Ⅲ

The basic RNAi process can be divided into three steps. First, a long double-stranded RNA (dsRNA) that is expressed in, or introduced into the cell is processed into small RNA duplexes by a ribonuclease III (RNaseⅢ) enzyme known as Dicer. Second, these duplexes are unwound, and one strand is preferentially loaded into a protein complex known as the RNA-induced silencing complex (RISC). Third, this complex effectively searches the transcriptome and finds potential target RNAs. The loaded single-stranded RNA (ssRNA), RISC to cleave mRNA that contain sequence homologous to the ssRNA.2002, Yang D discovered the use of Escherichia coli RNase III to cleave doublestranded RNA(dsRNA) into endoribonuclease-prepared siRNA(esiRNA) which mediates effective RNA interference in mammalian cells.In this study cDNA library of Escherichia coli was constructed, then we screened of proteins which interacted with RNase ⅠⅢ by Bacterio Match two hybrid system, to research the function of RNase III and whether prokaryote possesses RNA interference pathway, similaring to eukaryote by analysing them. A cDNA library of Escherichia coli was constructed using the cDNA library construction kit purchased from Stratagene. In this study, the cDNA library of E.coli was constructed using the following method:the total RNA of E. coli was extracted using Trizol kit, and the poly(A) tail was specifically added to mRNA with E.coli poly(A) Polymerase. mRNA was separated, purified, and reverse-transcripted into double-strand DNA. The ds-cDNA termini was blunted with pfu DNA polymerase. The blunted cDNAs were added EcoRI adaptors and then digested by Xho I. cDNA size was fractionated by CHROMA SPIN-400column. These purified fragments were ligased into the pTRG XR vector and transformed into XL1-BLUE MRF’ competent cells, which generates the oriental unamplified cDNA library. Unamplified library contained about3×106recombinants. PCR results showed that the percentage of positive recombinant clones was100%. The length of the inserted cDNA fragment was over300bp. The titer of the amplified library was3.15X1010pfu/mL. The quality of the constructed E.coli library was excellent and helpful to screen new genes.Screening the library by cotransforming pBT-RNaseⅢ and pTRG-cDNA plasmid into XL1-BLUE MRF screening competent cells, getting379clones at dual selective medium(5mM3-AT+strep) flat plates. These clones were performed by colony PCR, then digested by MSPI. Positive clones were classfied according to the clone’s characters,169positive clones were classfied. There are35clones repeated more than three times, sequenced and Blasted in NCBI nucleotide databases. This study has discovered that most clones are16S rRNA and23S rRNA and some glycolysis enzymes such as pyridoxal kinase, transketolase, isocitrate dehydrogenase and so on. Therefore we hypothesize that RNase III maybe involved in the glycolysis of E.coli.This study focus research area on16S rRNA. The interaction between RNaseⅢ and16S rRNA was confirmed in vivo by GST Pull-down assay. However, the interaction intensity between two proteins was weaker when RNase III separated to C-terminal domain and N-terminal domain than whole RNase III, demonstrating both of the two domians of RNase III was necessary to the interaction between RNaseⅢ and16S rRNA. This study also discovered that16S rRNA can enhance the ability of RNase III cleavage by semi-Quantitative RT-RCR...