Genetic Analysis Of Archaeal Plasmid PRN2and Establishment Of A Vector-host System In Sulfolobus

Two cryptic plasmids pRNl and pRN2were isolated from Sulfolobus islandicus REN1H1, which may originated from commom ancestor, but replication independent. There are many similarities between pRN1and pRN2, which have three conserved genes: repA, copG and plrA. In the plasmid pRN1, repA is a large replication protein containing904amino acids, which of N terminal sequence coding Primase/Pol activity and C terminal sequence having putatived helicase activity. Lipps et al had cloned repA into Escherichia coli, and found the primase activity that can synthesis primer containing dNTP and NTP. Analysis of copG showed that the expression copG protein can conbine with its promoter region. Due to an overlap of copG and repA gene in pRN1, Lipps et al suggests that copG is a protein that regulates the plasmid copy number. plrA is highly conserved in the pRN family plasmid, heterologous expression experiments showed the plrA protein form dipolymer and can bind its promoter region. In view of the above results, Lipps et al constructed a expression vector based on pRNl, but because of plasmid instability and difficult transformation, the vector system had not been widely used in the field of related research. The plasmid copy number of pRN2is higher than pRNl, the study of pRN2plasmid can develop a more efficient archaea genetic operating system.In this study, using uracil mutant Sulfolobus islandicus E233as host and the complete pyrEF gene as a selection marker, we constructed a vector named pGEF,then the different fragment of plasmid pRN2were cloned into pGEF, and the derived plasmids were electrotransfomated into host E233. The minimum stable unit of replication in pRN2contains the sequences of orf89, copG, repA and ori, namely derived plasmid pZC1. The tests of recombinant plasmid stability after200consecutive genetations, the loss rate of pZCl was6%. The recombinant plasmid pSD which copG deletion was1.9%, however the recombinant plasmid pSX which deletion of orf89was47%. The results of qPCR showed that the pSD stability is the highest, about13-15copy, however the pSX was just the contrary. All the result indicated that copG may not be the key gene of regulating the copy number and stability of pRN2, it seems orf89was really the role. The tests of transcription of recombinant plasmid pZC1, the orf89and copG were co-transcription and there was a reverse transcription of orf89named ct89, moreover we determined the transcription terminator of orf980.In order to analysis the function of orf89protein, the gene of orf89was cloned into expression vector pET32a and successfully expression in E. coli rosetta. The result of EMSA indicated orf89protein can bind the upstream of orf89gene. Due to the co-transcription of orf89and copG, the transcription of ct89surely interfere the protein expression of orf89and copG, orf89protein and the transcription of ct89realize double control of pRN2from protein level and transcriptional level. Compared with CRISPR93sequence of Sulfolobus islandicus REY15A, the conservative helicase region of rep A gene in pRN1and pRN2were found in spacer56. The mutations were constructed based on the sequence similarity, and electrotransformated into host. The result showed that the negative correlationship between sequence similarity and the transformation efficiency of plasmid.