| Circadian rhythms help organisms keep the track of daily cycle.Circadian clock genes generate and control the running of the circadian rhythms. Cyanobacteria which are the simplest organism known to exhibit circadian rhythms have provided expriment model systems. More and more research find the cyanobacteria clock is known to control many physiological processes an other gene expression. Arthrospira is a kind of noticeable cyanobacteria not only in their nutritional potential but in their special biological characteristics. Although the molecular genetic studies of Arthrospira initiated earlier, it lagged behind in circadian clock gene research and it was unknow about circadian rhythms in Arthrospira.Four fosmid clones which contained a circadian clock gene kaiC was isolated from a fosmid DNA library of Arthrospira maxima by PCR-pooling based on using partial known sequence. The full length kai gene cluster was cloned by sequence walking and the coding region of kai gene cluster consists of 3820bp nucleotide acids. The clock gene cluster consists of 3 genes that they are kaiA, kaiB, and kaiC. The length of encode region of kaiA, kaiB, and kaiC genes are 858bp,315bp,1554bp respectively. The average GC content of the coding region of kaiA, kaiB, and kaiC gene are close, respectively, 39.86%,39.37%,41.76%. The condon usage analysis found the third position of codon of kai genes had a strong codon usage bias towards U, especially for kaiC gene. The promoters of kaiA ,kaiB and a possible kaiC promter have been found and some regulation elements have been also found in their respective promoter region. The result of KaiA amino acid sequence analysis demonstrated KaiA protein contain some functional sites and 14αhelixes and 6βstrands. And three dimension of KaiA protein is similar to KaiA protein of Thermosynechococcus elongatus bp-1, identity 64.5%. The result of KaiB amino acid sequence analysis demonstrated KaiB protein contain some functional sites and 5αhelixes and 5βstrands. And three dimension of KaiB protein is similar to KaiB protein of Anabaena PCC7120, identity 96.7%. The result of KaiC amino acid sequence analysis demonstrated KaiC protein contained not only some functional sites but some important motif such as walker A motif, imperfect walker motif B, DXXG motif, two deduced catalytic Glu residues and so on. KaiC protein contained 18αhelixes and 21βstrands. And three dimension of KaiC protein was similar to KaiC protein of Synechococcus PCC 7942, identity 81.6%. The analysis of Kai protein phylogenetic trees showed that Kai protein were conserved, especially for KaiC protein.The southern blot result showed that kaiA, kaiB, kaiC genes were all present as a single copy in the genome of Arthrospira maxima. This demonstrated there was only one kai gene cluster in the genome without separate kai genes. And it indirectly proved that kai genes was functional genes in Arthrospira maxima.The kai genes expression of Arthrospira maxima for 92 hours in different environment was monitored by real-time quantitative PCR. The results showed that kai gene cluster expression in constant light had obvious circadian rhythm about 12 hours. The control group (12L/12D) had similar genes expression trendline in first 36 hours, after that the phases of kai genes expression changed. Although there had differences between two test groups, three genes expression were always synchronous and their relative genes expression amount were approximative. The difference of the rhythms between two test groups suggested that the light had played an important role in circadian rhythm of Arthrospira maxima.
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