[Identification and expression analysis of gene family in eukaryotic algae].

WRKY is a superfamily of plant-specific transcription factors, playing a critical regulatory role in multiple biological processes such as plant growth and development, metabolism, and responses to biotic and abiotic stresses. Although genes have been characterized in a variety of higher plants, little is known about them in eukaryotic algae, which are close to higher plants in evolution. To fully characterize algal WRKY family members, we carried out multiple sequence alignment, phylogenetic analysis, and conserved domain prediction to identify the genes in the genomes of 30 algal species. A total of 24 WRKY members were identified in Chlorophyta, whereas no WRKY member was detected in Rhodophyta, Glaucophyta, or Bacillariophyta. The 24 WRKY members were classified into Ⅰ, Ⅱa, Ⅱb and R groups, with a conserved heptapeptide domain WRKYGQ(E/A/H/N)K and a zinc finger motif C-X4-5-C-X22-23-H-X-H. , a high producer of natural astaxanthin, contained two WRKY members (HaeWRKY-1 and HaeWRKY-2). Furthermore, the coding sequences of - and - genes were cloned and then inserted into prokaryotic expression vector. The recombinant vectors were induced to express in BL21(DE3) cells and the fusion proteins were purified by Ni-NTA affinity chromatography. - had significantly higher expression level than - in . cultured under normal conditions. High light stress significantly up-regulated the expression of - while down-regulated that of -. The promoters of genes contained multiple -elements responsive to light, ethylene, ABA, and stresses. Particularly, the promoter of - contained no W-box specific for WRKY binding. However, the W-box was detected in the promoters of - and the key enzyme genes (β-carotene ketolase) and (phytoene synthase) responsible for astaxanthin biosynthesis. Considering these findings and the research progress in the related fields, we hypothesized that the low expression of - under high light stress may lead to the up-regulation of - expression. HaeWRKY-1 may then up-regulate the expression of the key genes (, , etc.) for astaxanthin biosynthesis, consequently promoting astaxanthin enrichment in algal cells. The findings provide new insights into further analysis of the regulatory mechanism of astaxanthin biosynthesis and high light stress response of . .