Characteristic Analysis Of GⅡ Intron Gene In Arthrospira

Tranposons which are able to move and insert into new positon of DNA,is an important part of the genome.It can be divided into DNA transposon and Tetrotransposon,GⅡintron is a member of retrotransposon,and has peculiar structue and function.The GⅡintron was first found in the mitochondrion and chloroplast DNA of the plant.As the development of prokaryotes molecular biology,GⅡintron was found in Escherichia coli and Lactobacillus delbrueckii.lt is identified to be the ancestor of the intron,because they have the same mechanism of splicing.Arthrospira is a kind of noticeable cyanobacteria not only in their nutritional but in their special biological characteristics,yet it was unkown about GⅡintron gene in Arthrospira.Three fosmid clones which contained a GⅡintronl gene was isolated from a fosmid DNA library of Arthrospira FACHB 438 by PCR-pooling based on partial known sequence.The full lenth of GⅡintronl gene was cloned by sequence walking and consists of 2287bp nucleotide acids.Based on BLAST,ORF Finder at NCBI website and the alignment among homologous genes,the gene we got has a construct of an integrated GⅡintron gene:Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ,Ⅵ,six domains.After transcripting domainⅣcan translated into reverse trancriptase domain,GⅡintron-type maturase domain and HNH endonuclease domain,the other five domains can be folded into a conserved RNA secondary structure,domainⅠ,Ⅱ,Ⅲlocate in the upstream of protein-coding region with a length of 583bp,domainⅤandⅥlocate in the downstream of the protein-encoding region with a length of 91bp.The average GC contents of ORF region and intron encoded region is close,respectively tobe 43.03% and 43.01%.The alignment of protein-encoding sequence in GⅡintron gene among Arthrospira FACHB 438 and other cyanobacterial species shows that there are two bases deletion at 648bp positon of Arthrospira FACHB 438 GⅡintron gene.Amino acid analysis demonstrates that it has every constructional site of reverse transcriptase,GⅡintron-type maturase and HNH endonuclease.Reverse transcriptase contains eight subdomains of 0,l,2,3,4,5,6,7,and subdomain0 is a domain of the non-LTR reverse tranposase differing from other types.YADD which located in subdomain5 is the sign of reverse transcriptase.GⅡintron-tyipe maturase which have not a conserved sequence can help RNA splicing.HNH endonuclease which has conserved amino acid H568,N582,H591,assist intron inserting into target DNA.Secondary structure analysis shows that the protein has 23 a helixes and 13βstrands.GⅡintron gene is usually multicopy in prokaryotes especially cyanobacteria,in this study we used real-time PCR to calculate the copy number of GⅡintron in Arthrospira maxima FACHB 438,as the kaiB,kaiC which is single copy in Arthrospira maxima FACHB 438 to be the referrence genes.Result revealed that the copy number of GⅡintron gene in Arthrospira Maxima FACHB 438 is 28-29.Besides,we also designed primers at the upstream and downstream of GⅡintron ORF region to PCR and sequence of Arthrospira FACHB 438,Arthrospira platensis FACHB 341,350,790 and 793.Alignment throws out that the GⅡintron ORF regions are conserved,identities are all above 90%,and they all have two bases deletion at the homologous region.Moreover,438GⅡintron2,793,341,790 GⅡintron have about 54-60bp deletion at 222bp positon,compared with Arthrospira FACHB 43SGⅡintronl.It illuminates that the deformity of the gene may be caused by the retrotransposition during the evolution.