Characterization Of CRY-DASH Gene And Expression Analysis In Saccharina Japonica

Blue light is predominant wavelength below seawater surface, and it plays a critical role in regulating growth and development of photosynthetic stramenopile such as Saccharina japonica. The cryptochrome/photolyase family (CPF) is a diversified protein family which can be induced and activated by blue light. It has been confirmed that CRY-DASH, a new member of the cryptochrome/photolyase family,can repair the cyclobutane pyrimidine dimer (CPD) in ssDNA and loop structures of dsDNA, though its other functions are less investigated.Based on the transcriptome data, we characterized the full-length cDNA of CRY-DASH from S. japonica (designated as SjCRY-DASH) by rapid amplification of cDNA ends (RACE). Sequence analysis showed that the full-length ORF was 1.779 bp, encoding a protein of 592 amino acids. By multiple alignments of protein sequences, conserved residues that interact with two crucial cofactors, MTHF and FAD, were detected between SjCRY-DASH and several DASH-type proteins of other algae and higher plant. Prediction of protein structure showed SjCRY-DASH shared high similarity with other CPF members dissolved already. Two kinds of prokaryotic vectors were applied for SjCRY-DASH expression in vitro, and objective protein of high purity was obtained through affinity chromatography.Juvenile sporophytes were induced by light of different wavelengths. Both blue light and white light upregulated the transcriptional level of SjCRY-DASH in 1 h,though stronger response to blue light was found for SjCRY-DASH than white light.Nevertheless, red light had no notable influence on the transcriptional level. The SjCRY-DASH transcription variation persisted with 24-hour periodicity when juvenile sporophytes were grown under various daylength conditions. Besides, the time of onset of the transcription was reset by different daylengths, indicating SjCRY-DASH is closely related to circadian control. Furthermore, the rhythms of SjCRY-DASH transcription under different temperature treatments were temperature compensated,verifying that it was a crucial component of the circadian clocks. This study is expected to enrich our understanding of CRY-DASH function, and lays foundation for exploring the molecular mechanism of responses to light and temperature in S.japonica.