Study On Improving RNA Splicing Of Npu RIR Intron By Directed Evolution And Homing Mechanisms Of Ter DnaN-1 Intron

Ribozymes(Catalytic RNAs) has been found that they self-splice from their pre-mRNAs with the flanking exons spliced.Groupâ… introns and groupâ…¡introns are two abundant kinds of self-splicing introns.The typical secondary structure of a groupâ… intron has approximately ten paired elements.The typical groupâ…¡introns fold into a conserved secondary structure which has six double-helical domains (Dâ… -Dâ…¥),of which domainâ…£encoded multiple functional protein with reverse transcriptase,maturase and endonuclease activities.These activities have been found to play important roles in splicing and mobility of groupâ…¡introns.Groupâ… introns catalyze a two-step transesterification reaction using a guanosine molecule as a cofactor.Groupâ… introns are distinguished from the groupâ…¡introns,which generally use an internal bulged adenosine to initiate self-splicing.On one hand,the splicing mechanism of a groupâ… intron of Nostoc punctiforme has been studied in Escherichia coli.A rare groupâ… intron,which is 383 nucleotides long,has been found in the cyanobacterial ribonucleotide reductase(RIR) gene of N. punctiforme.A novel insertion sequence element inside the groupâ… intron has been identified.Then,its RNA splicing activity was demonstrated in E.coli cells.The intron-coding DNA was inserted into the Kanamycin-resistance gene of the plasmid pDrive self-ligation.The resulting plasmid pKR-12 was transformed E.coli,however, this intron couldn't splice in this strange system,likely because of the intron not adapting to the kanR gene.Therefore the first thing to do is to introduce random mutations into the intron using error-prone PCR to get an adapted intron that can splice and allow the E.coli cell to grow on a LB plate containing kanamycin.Through error-prone PCR,we had constructed a recombinant plasmids library containing a lot of mutational introns.After transformation into E.coli,42 colonies grown on a plate containing kanamycin was obtained.Finally,a mutant intron that could allow the E. coli cell to grow on a plate containing 20μg/ml kanamycin was screened.This work have laid solid foundation for further research on revealing the splicing mechanism and searching for critical nucleotides affect the splicing of Npu RIR intron.On the other hand,the retrohoming mechanisms of a groupâ…¡intron in Trichodesmium erythraeum are also studied in E.coli.This groupâ…¡intron have been found in theβ-subunit of DNA polymeraseâ…¢gene of T.erythraeum.The donor plasmid vector pDN1-C with Intron-containing DNA sequence and the recipient plasmid vector pAM1-H2 had been constructed in our lab.Notably,the intron was inserted with a KanR gene inside on the donor expression plasmid which also has an ampicillin-resistance(AmpR) gene on it,while the recipient expression plasmid has a chloramphenical-resistance(CamR) gene on it.The individual recombinant plasmid was introduced into E.coli cells by electroporation.For co-expression experiments,two plasmids had been introduced sequentially into the same E.coli cells,and E.coli cells containing the specified plasmid was selected.After improving the grown conditions(e.g.dissolved O₂),we got two"well-grown" E.coli strains containing those two plasmids.E.coli cells containing the specified plasmid were induced firstly by L-arabinose and then by IPTG.Total plasmids of the induced cells were extracted and then digested the donor plasmids mixed in these plasmids.Then transform E.coli,screen the cells on plates containing both kanamycin and chloramphenical.Extract the plasmids of the resulting colonies,respectively. Subsequent PCR were carried out using specified primers outside the intron insertion site.The resulting PCR products were then confirmed through the agarose gel electrophoresis.There are no bands on the agarose gel stained by EB under UV light after electrophoresis.Maybe homing of this groupâ…¡intron had occurred in a ratively low frequency.Further research was required in this project,the strain containing the donor and the recipient plasmids would greatly help the subsequently research on the mobility pathways of the Ter dnaN-1 intron.