Identification Of Group Ⅱ Introns In Trichodesmium Erythraeum And Study Of Its Self-Splicing Mechanism

GroupⅡintrons are large catalytic RNAs.They are found in mitochondria and chloroplasts genomes of plants,fungi,protests and algae,as well as bacterial and archaebacterial genomes.GroupⅡintrons are not conserved in primary structure.The signature secondary structure of groupⅡintrons consists of six stem-loop domains denoted DI～DVI.DI is the largest domain and DV is the most conserved domain.DI and DV both involve in the catalysis of groupⅡintrons. Many groupⅡintrons can encode maturases in DIV which help self-splicing and mobility of groupⅡintrons.In vivo,the splicing reactions of many,not all groupⅡintrons are assisted by proteins encoded by either the introns themselves,or other genes of the host organisms.GroupⅡintrons can integrate efficiently into intronless alleles of the same gene or at much lower frequencies into ectopic sites. It is thought that both nuclear spliceosomal introns and non-long terminal repeat retrotransposons evolved from mobile groupⅡintrons.This research identified groupⅡintrons in the genome of Trichodesmium erythraeum and analyzed the distribution and structure of these groupⅡintrons by bioinformatics tools,such as BLAST and CLUSTAL.Then the effect of maturases encoded by groupⅡintrons on the self-splicing of groupⅡintrons were analyzed. The coding sequences of different groupⅡintrons were PCR-amplified and inserted in pDrive plasmid vector.The maturase coding sequences of different groupⅡintrons were PCR-amplified and inserted in a modified pAR3 plasmid vector.Individual recombinant plasmid was introduced into E.coll cells through electroporation.For co-expression experiments,two plasmids were introduced sequentially into the same E.coli cells,and transformed cells were selected and maintained in the presence of both ampicillin and chloramphenical.For RNA splicing assays,E.coli cells containing the specified plasmid were grown in liquid LB medium at 37℃to mid-log phase.L-arabinose and IPTG were then added to induce transcription of the maturase-containing gene and intron-containing gene. Total RNAs of the induced cells were extracted and treated with RNase-free DNase using the RNeasy kit(Qiagen).Reverse transcription and subsequent PCR were carried out using specified primers and the reverse transcriptase M-MLV(Shanghai Tiangen).The resulting DNA products were identified by their predicted sizes and confirmed through cloning and DNA sequencing.In T.erythraeum,about 50%groupⅡintrons insert in highly conserved protein-coding genes.Several conserved genes contain multiple groupⅡintrons. GroupⅡintrons and inteins coexsist in one conserved gene.Sequence alignments indicate that there is high homology between 20 different groupⅡintrons.But the insertion sites of these groupⅡintrons are variable very much.Maybe retrotransposition of groupⅡintrons happened multiple times in T.erythraeum. Our experimental results indicate that maturase proteins encoded by TerⅡ6,TerⅡ7,TerⅡ18 and TerⅡ24 do not help self-spicing of TerⅡ1,TerⅡ2,TerⅡ3,TerⅡ4,TerⅡ6,TerⅡ7 and TerⅡ16.And maturase proteins encoded by TerⅡ8 and TerⅡ10 do not help self-spicing of TerⅡ6,TerⅡ7 and TerⅡ16.And maturase protein encoded by TerⅡ16 can help self-spicing of TerⅡ6 and TerⅡ16,but can not help self-splicing of TerⅡ1,TerⅡ2,TerⅡ3,TerⅡ4 and TerⅡ7.