Analysis Of The Accumulation Of Fucoxanthin And Fatty Acids Mediated By Light Quality And The Structure Of Blue Light Receptor IgCRY1 With Isochrysis Galbana

Isochrysis galbana is found in the ocean widely,which is rich in fucoxanthin and polyunsaturated fatty acids.I.galbana is the ideal extraction source of algae and fucoxanthin and polyunsaturated fatty acids as the preferred economic bait for mariculture fish,shrimp and shellfish.As a primary producer in nature,I.galbana is characterized by rapid growth,easy cultivation and reproduction.It was found that different light quality culture of I.galbana could increase the fucoxanthin content and fatty acid accumulation.However,it is not clear whether different light quality affects the growth amount of I.galbana or the metabolic pathway of target products to increase fucoxanthin content and fatty acid accumulation.Cryptochrome,first discovered in Arabidopsis thaliana,is an important blue light receptor.With the deepening of research,cryptochrome has been found in animal and plant microorganisms.Cryptochrome belongs to the photochromic homologous gene family,but it does not have the DNA repair function of photochromic enzyme.Cryptochrome plays a key role in light regulation of plant growth and development,which is closely related to its structure.The Photolyase-Homologous Region(PHR)domain on the N-terminal and the Cryptochrome C-Terminal Extension(CCE)domain on the C-terminal are the two domains of cryptochrome.In the evolutionary process,cryptochrome with a highly conserved PHR domain will bind to downstream proteins after blue light activation and undergo highly conserved FAD photoreduction and oligomerization,and regulate plant growth and development.In order to investigate the effects of different light quality on the growth and development of Isochrysis galbana and its metabolites,and whether cryptochrome can regulate the growth amount of I.galbana in this process.In this experiment,I.galbana was cultured in white,red,green and blue light,and the structure of cryptochrome protein was analyzed.The main research contents and results of this paper are as follows:1.Isochrysis galbana was cultured with white,red,green and blue light,with white light as control and red,green and blue light as experimental groups.The effects of different light quality and different incubation days(1,3,6,9 and 12 days)on cell density,dry weight,fucoxanthin content and fatty acid accumulation of I.galbana were investigated.The results showed that blue light culture was beneficial to the increase of cell density compared with control group.There was no significant difference in cell density under white light,red light and green light.I.galbana cells had the maximum dry weight on day 12 under different light quality.The cell dry weight increased most obviously under blue light culture,which was positively correlated with cell density.The fucoxanthin content and fatty acid accumulation of I.galbana increased with the increase of time under different light quality culture.Blue,green and red light promoted fucoxanthin production by Dinoflagellates.The highest fucoxanthin content was 2.45mg/L,2.30 mg/L and 1.56 mg/L on day 12,respectively.The total fatty acid accumulation of white light,red light and green light was 4.374 mg/L,4.627 mg/L and 4.510 mg/L at the 12 th day respectively.Analysis of total fatty acid accumulation per unit cell and dry weight showed that green light promoted the accumulation of total fatty acid in I.galbana.On day 9,the total amount of DHA and the accumulation of DHA in unit cell and unit dry weight were higher,and blue light played an obvious promoting role.2.We compared the PHR domain and FAD domain obtained from the analysis of cryptochrome protein structure in Arabidopsis thaliana to find cryptochrome family genes in the Isochrysis galbana protein database,which was assembled by our research group earlier.Four cryptochromes were found to be Ig004702,Ig005899,Ig007083 and Ig011451,named IgCRY1,IgCRY2,IgCRY3 and IgCRY4 respectively.Phylogenetic analysis of cryptochrome family was carried out,including protein conserved domain analysis,gene structure analysis,protein first,second and third level structure analysis,predictive analysis of protein subcellular localization,protein conserved motif analysis,and searching multi-species homologous sequence to construct phylogenetic evolutionary tree.The results showed that IgCRY1,IgCRY2,IgCRY3 and IgCRY4 were all hydrophilic proteins.In protein tertiary structure analysis,the consistency rate of homology modeling was above 30%.Phylogenetic tree was established to analyze the relationship between I.galbana and cryptochrome family.In I.galbana,four cryptochrome genes were located in different clusters.IgCRY1 is closely related to animal cryptochrome.IgCRY2 and IgCRY4 are closely related to algae.Igcry CRY3 is in the same clade as multiple species.3.IgCRY1 was cloned and expressed in escherichia coli by molecular biological methods.After protein purification,IgCRY1 crystal was prepared,and the structure of IgCRY1 was analyzed by X-ray crystal diffraction.The IgCRY1 c DNA was 1560 bp in size.The recombinant plasmid was expressed in Escherichia Rosetta(DE3)to obtain a yellow-green protein solution with a molecular weight of about 60 Kda.After purification,the peak diagram is single and shows monomer structure.The expression level of the strain was higher,and the minute Absorbance Unit(m AU)was up to 2500.The crystal structure of IgCRY1 can be divided into two parts as a whole.The N-terminal is α/βdomain,which is composed of 9 α helices and 6 β folds,and the C-terminal is an αdomain composed of 15 α helices.FAD is shaped like "U" and located in the cavity of cterminal α domain,which is highly similar to the structure of rice cryptochrome protein.