Studies On Germplasm Diversity And Biological Functions Of Extrachromosomal DNA In Arthrospira

1. Studies on germplasm diversity of Arthrospira exDNAThe exDNA of seven Arthrospira strains were detected by the improved method, and the results showed that all the strains contained exDNA, moreover, exDNA from different strains differed in their types. Combined the analyses of molecular phylogenetics based on the sequences of 16S rRNA gene and cpcHID operon of seven strains, we found that the exDNA type was correlative with Arthrospira germplasm diversity, strains with close genetic relationships contained similar exDNA type and vice versa.2. Cloning and sequence analysis of Arthrospira exDNAThree different methods were applied to clone the exDNA (exDNA-L and exDNA-S) of Arthrospira platensis Sp-3. As a result, five exDNA segments (exDNA-L1, exDNA-L2, exDNA-L3, exDNA-S1, exDNA-S2) were obtained. Further bioinformatics analyses revealed that: (1) exDNA-L1, whose size was 2589 bp, had two putative open reading frames (ORPs). The deduced amimo-acid sequences of these two ORPs contained conservative domains of histidine protein kinase and response regulator, respectively. (2) exDNA-L2, whose size was 1216 bp, had one putative ORF. The deduced amimo-acid sequence contained CheW domain and CheY domain. These two domains were necessary for bacterial chemotaxis two-component signal transduction. (3) exDNA-Sl, whose size was 936 bp, had one putative ORP. The deduced amino-acid sequence contained the PAS/PAC domain of histidine protein kinase. (4) exDNA-L3, whose size was 900 bp, contained many direct repeats and inverted repeats. Moreover, at 5' end of exDNA-L3, there was an incomplete ORF, whose deduced amino-acid sequence had high similarity with some transposase. (5) Although exDNA-Sl had one putative ORF, there was no known domain existed in this ORF.The full-length sequences of exDNA-L and exDNA-S were not obtained in thisstudy. However, the bioinformatics analysis of five exDNA fragments indicated that exDNA-L and exDNA-S possessed some genes related with two-component signal transduction, and these genes may be involved in adaptation regulation of Arthrospira, such as light regulation, chemotaxis regulation.3. Expression of exDNA-Sl-ORF and purification of the productIn order to explore the biological function of Arthrospira exDNA, the exDNA-Sl-ORF was cloned into pET-30a(+) expression vector. Then the recombinant vector was transformed into E. coli BL21 and the target ORF was induced to express by IPTG. The SDS-PAGE analysis showed that the target protein was successfully expressed. But further analysis indicated that the target protein formed inclusion body. After dissolved in 6 mol/ L Guanidine hydrochloride solution, the target protein was purified by Ni-NTA affinity chromatography and gradient dialysis. Finally, the target protein with electrophoresis degree was obtained.4. Preparation of antibody against exDNA-Sl-ORF and immunological analysisThe purified exDNA-Sl-ORF protein was used as antigen to immune New Zealand rabbit to prepare specific antiserum. ELISA assay showed that the titer of the antiserum was up to 1: 204,800. Further western blot analysis revealed that the antiserum had specific reaction with whole-cell soluble protein of Arthrospira platensis Sp-3. So it was suggested that the Arthrospira exDNA was not redundant DNA, but possessed specific genetic function.