The Analysis Of Cryptochrome/photolyase Family In Eukaryotic Algae And Functional Study On Cryptochrome From Haematococcus Pluvialis

Light serves as one of the most engrossing influential factors for plants,which provides not only an energy source for photosynthesis but also a crucial signal to control plant growth and development.Plants respond for light that is regulated by a precise program.Aiming at perceiving and transducing the light signals,photoreceptors have an ability to control signaling processes and are direct charge of the traditional reprogramming,it has been a gripping and hot research field in plant biology.The Cryptochrome/Photolyase Family(CPF)is a kind of widely distributed UV-A/blue-light perceiving photoreceptors protein.Eukaryotic microalgae are a kind of photoautotrophic single-cell eukaryote and origin from cyanobacteria by primary and secondary endosymbiont events.It is an ideal experimental material to study the origin,evolution,structure and functional diversity of photorecept families.Haematococcus pluvialis is a freshwater single-cell green alga and rich in astaxanthin content,which is considered as the best ideal sources of natural astaxanthin.It has been proven that blue light can effectively induce the accumulation of astaxanthin and the genes involved in astaxanthin biosynthesis are up-regulated,however,the regulation mechanism of this induction process remains unknown.In this study,comparative and functional genomics analyses were applied to study CPF gene family and to explore their distribution,classification,phylogeny,evolution,and structure in eukaryotic microalgae.Plant-type cryptochrome(CRY),an important gene,from Haematococcus pluvialis,was examined.Totally,195 members of the CPF family were identified from the genomes of 27 eukaryotic algae.According to phylogenetic evolution analysis,these CPF proteins could be divided into 4 branches,named CRY,CPD,6-4 PHO,and CRY-DASH.CPD was further divided into CPD-like and CPDII subgroups.Guillardia theta has the fewest CPF numbers and types,only 2 CPDII type,3 Plant-CRY-like type and 1 CRY-DASH type.There are many types and numbers of CPF in Cyanidioschyzon merolae,such as 1 6-4 PHO,1 CPDII,3 CPDII-like,2 Plant-CRY,1 Plant-CRY-like and 3 CRY-DASH.Large numbers and types of CPF were rich in eukaryotic green algae,such as 8 Chlamydomonas reinhardtii,9 Coccomyxa C-169,and 10 Volvox carteri.Plant-type CRY was lineage specific distributed and was only present in green and red algae such as V.carteri,Chlorella variabilis,Chlorella NC64A,C.reinhardtii,C.subellipsoidea C-169,and C.merolae.CRY-DASH was existed in all algae examined in this study.Based on the transcriptome data,7 CPFs were identified from H.pluvialis,which included 2 plant-type CRYs,1 PHO type,and 4 CRY-DASH types.Compared with white light control,the results from express heat map indicated that transcriptional expression of plant-type CRY(Hae-P-CRY)was up-regulated under high blue light treatment.Interestingly,high blue light stress also could effectively induce the astaxanthin synthesis(included free astaxanthin,astaxanthin monoester and astaxanthin diester)in H.pluvialis,which was consistent with the up-regulation expression of Hae-P-CRY gene.These results implied that this gene(Hae-P-CRY)might be involved in high blue light induction process.A full-length complementary DNA(cDNA)sequence of plant type CRY(designated Hae-P-CRY)was cloned from the green alga H.pluvialis by combined homology cloning and rapid-amplification of cDNA ends(RACEs)method.The Hae-P-CRY cDNA sequence was 3608 base pairs(bp)in length,which contained a 2988-bp open reading frame,a 294-bp 5'-untranslated region(UTR),and a 198-bp 3'-UTR with the characteristic of the poly(A)tail.The deduced protein(995 amino acids)had a calculated molecular mass of 107.7 kDa with an estimated isoelectric point of 6.19.Multiple alignment analysis revealed that the deduced amino acid sequence of Hae-P-CRY shared high identity of 46-66%with corresponding plant type CRYs from other eukaryotes.The catalytic motifs of plant type CRYs were detected in the amino acid sequence of Hae-P-CRY including the typical PHR and CCT domains.Phylogenetic analysis showed that the Hae-P-CRY was grouped together with other plant type CRYs from green microalgae and higher plants,which distinguished from other distinct groups.Under four light conditions of red light,white light,blue light and ultraviolet light,Hae-P-CRY was more sensitive to blue light and ultraviolet light,strongly decreased after 0-4 h under HL stress.In addition,the Hae-P-CRY gene was heterologously expressed in Escherichia coli BL21(DE3)and successfully purified.The typical spectroscopic characteristics of plant type CRYs were present in Hae-P-CRY indicated that it may be an active enzyme,which provided valuable clue for further functional investigation in green alga H.pluvialis.These results lay the foundation for further function and interaction protein identification involved in CRY mediated signal pathway under HBL stress in H.pluvialis.