Studies On Cloning, Structure And Function Of Extrachromosomal DNA In Spirulina

In this study, an efficient method was established for the extraction and purification of extrachromosomal DNA (exDNA) from Spirulina platensis strains, such as Sp-HO1, Sp-D, Sp-S, Sp-T etc. An investigation on the essential molecular characteristics of exDNA was carried out. The homology of several exDNAs, as well as between exDNA and chromosomal DNA is analyzed by Southern blotting;Partial sequences of exDNA from Sp-S were cloned. And the primary and secondary structures, open reading frames (ORFs) of the cloned fragments, and function of proteins encoded by ORFs were analyzed through bioinformatics method. Results got from the study are listed as following: 1. Establishment of methodology for exDNA extraction and purification in SpirulinaOn comparison basis of methods for extraction of exDNA from Spirulina, an improved and efficient method—CTAB-Proteinase K method was established. During the steps of cell lysis and dissolution of crude DNA extracts, Proteinase K was added into the solution and followed by phenol/chloroform/isoamyl alcohol extractions for final DNA purification. Through this operation, the contaminants of proteins can be removed efficiently, and the extraction efficiency and quality of exDNA was improved significantly. The method was used for exDNA extraction from more than ten strains of Spirulina, and good results were got. The results showed that: (1) most of S. platensis strains contain only one kind of exDNA, for example, Sp-HOl with a 0.75 kb exDNA, but exDNA size of Sp-D, Sp-J and Sp-Z ranges from 1.1 kb to 1.2 kb;(2) a few strains contain two kinds of exDNA, for example, Sp-S, one kind of the exDNA is 1.8 kb (named exDNA-S-S) and the other is 3.6 kb (named exDNA-S-L);similarly, Sp-T possesses two kinds of exDNAs with the size of 1.9 kb and 3.8 kb.All of exDNAs are with a very low abundance, and also very difficult to enrich and purify. For solving these difficulties, an effective method for enriching and purifying of exDNA from total DNA was established. The results revealed that three methods, freeze-thaw extraction, DNA gel extraction kit and alkaline method, can be used for exDNA purification, however, the freeze-thaw extraction method is the best one. It is with a recovery efficiency over 90%.2. Molecular characteristics of exDNA in SpirulinaThrough studies on the genetic stability, probable molecular conformation and restriction enzyme patterns of the exDNA, we found that: (1) exDNAs in Spirulina were stable after multi-generation culture;(2) exDNAs were easily renatured after denaturation treatment with alkali or heat, however, when exDNAs were exposed to UV, we did not find new exDNA bands in agarose gel electrophoresis. The enzyme digestion experiment of the exDNA indicated that exDNA can be digested by Dral incompletely, but no restriction patterns were found. Based on these results, we speculated that exDNA may be linear DNA with complicated structures;(3) by using two-dimensional agarose gel electrophoresis, the relationship between large exDNA and small exDNA of Sp-S or Sp-T was studied, and results suggested that small exDNA and large exDNA of Sp-S or Sp-T are two independent genetic elements, and that not two forms of one plasmid.3. Analysis of restriction endonuclease digestion of genomic DNA from SpirulinaThe modification of the CTAB extraction procedures includes adding proteinase K to lyse cells, high NaCI concentrations in the buffer to remove polysaccharides, and an additional proteinase K digestion of crude DNA extracts to remove any excess proteins. The so-called CTAB-Proteinase K method is efficient to prepare digestible genomic DNA from S.platensis. The genomic DNA can be digested by more than 12 restriction enzymes. The proper reaction condition is : (1) an enzyme concentration of 5 U///g DNA, and (2) a digestion time of 4 h.4. Homology analysis of exDNA of SpirulinaThe probes prepared from exDNAs were used to hybridize with various exDNAs and chromosomal DNA. The conclusions are as follows: (1) not only exDNAof Sp-HOl has no homology with exDNA of Sp-D, but also with exDNA of Sp-S or Sp-T;(2) there is partial homology between exDNAs of Sp-S and Sp-T. Furthermore, the homology also exists between large exDNA and small exDNA of Sp-S or Sp-T;(3) besides the homology with their own chromosomal DNA, exDNAs of Sp-HOl, Sp-D, Sp-S , Sp-T also have wide homology with to other chromosomal DNA It appeared that some strains may contain copies of several kinds of exDNA in their chromosomal genome.5. Cloning, structure and function analysis of Spirulina exDNAA method to clone fragments of exDNA was constructed. Two fragments, named SSI and SS2, whose sizes were 936 bp and 1091 bp, were cloned from exDNA-S-S of strain Sp-S. Moreover, a fragment, named SL1, whose size was 2589 bp, was cloned from exDNA-S-L of Sp-S. The resultsof Southem blotting showed that there is homology between SS2 and exDNA-S-L, but no homology between SSI and exDNA-S-L, videbcet, exDNA-S-L is likely to contain homologous sequences of SS2. And, we are sure that there are at least one copy of SSI and SLl and more than 4 copies of SS2 in chromosomal genome. Northern blotting of cloned fragments indicated that SSI and SLl is actively transcript, whereas SS2 is not.Analysis of DNA sequences and conserved domains of cloned fragments revealed that: (1) SSI has one potential open reading frame (ORF), and SLl has two (ORF1 and ORF2). These ORFs all encode histidine kinase, which contain some functional sites and several conserved domains, such as PAS, PAS/PAC and GAF domain. They are sensors and response regulators in two-component signaling systems, which play an important role in light-regulated signal transduction, photosensory perception and signal transduction of photochromes, and osmotic regulation;(2) There is a short and incomplete ORF at 3'-terminal of SS2, which encodes the polypeptide with functions in cell motility;(3) The sequence analysis indicated that many direct repeats and inverted repeats occur in SS2 induced complex construction of hairpin loops at 5'-terminal of exDNA. This structure is consistent with the molecular characteristics that exDNA can renature rapidly after denaturing with alkali or heat. According to the determined sequences of exDNA, we presume that exDNA might be an insertion-sequence (IS) - like element.