The Study Of Cryptochrome Gene Family From Isochrysis Galbana

Cryptochrome(cryptochrome)is an important type of blue light receptor,widely existing in various animals,plants,bacteria and other organisms.Cryptochromes play an important role in plant pigment accumulation,chloroplast formation and other related physiological and biochemical activities.Studies have shown that blue light can participate in regulating physiological processes such as the synthesis of Chlamydomonas reinhardtii chlorophyll and fucoxanthin.Isochrysis galbana is rich in metabolites such as fucoxanthin and polyunsaturated fatty acids.Blue light and cryptochrome genes should play an active role in regulating and accumulating the above metabolites of Isochrysis galbana.In this experiment,by searching for and identifying the cryptochrome genes of Isochrysis galbana and conducting gene cloning and electric shock transformation,it provides an important prerequisite for the subsequent research on the structure and function of cryptochromes;by ARTP mutagenesis and light quality cultivation of Isochrysis galbana,From the two aspects of genetic engineering and cultivation conditions,aimed at improving the ability of algae strains to produce fucoxanthin and fatty acid.Active and beneficial exploration in obtaining high-yield fucoxanthin,DHA fine algae strains and suitable culture conditions The main research contents and results of this article are as follows:1.Using the Isochrysis galbana genome and transcriptome database constructed in this laboratory,and then through a variety of bioinformatics analysis websites and softwares,find 5 genes of the cryptochrome gene family of Isochrysis galbana.Analyzing the physicochemical properties including the primary,secondary and tertiary structures,phosphorylation sites,transmembrane domains and subcellular localization of the five proteins encoded by Isochrysis galbana cryptochrome genes.Correlation analysis from the tree perspective.The results show that the 5 genes are particularly closely related to the cryptochrome genes of Emiliania huxleyi.Among them,IZCRY1 is closely related to Emiliania huxleyi.CRY1;IZCRY3 is the closest to Emiliania huxleyi.CRY3;IZCRY4and Emiliania huxleyi.CRY4 belong to the same branch and they have closely relationship.2.Using molecular biology methods,clone the full-length DNA sequence of the 3 cryptochrome genes of Isochrysis galbana.The full-length c DNA sequence of IZCRY1 gene was obtained by overlapping extension PCR,and then the gene overexpression vector p Pha-IZCRY1 was constructed by seam Less cloning Electric shock transformation of IZCRY1 gene and screening of positive clones were carried out on Isochrysis galbana,and an electric shock transformation system of Isochrysis galbana was established.The results showed that the growth of single algae colonies grown on the bleomycin-resistant plate with a concentration of 20 ng/m L after electroporation transformed algae strains was significantly better than that of wild algae strains.3.Through ARTP mutagenesis breeding experiment on Isochrysis galbana,several strains of mutagenic algae with rapid growth rate were screened out.Rescreening was based on biomass,fucoxanthin production,total fatty acid content and DHA content.In these four aspects,several strains of mutagenic algae strains that performed better than wild algae strains were selected.Among them,MT2,MT4,MT5,and MT7 were 34.07%,20.51%,66.85%,and 28.57% higher in biomass than wild algae strains.Three high-yielding fucoxanthin-induced mutant algae strains,MT4,MT5,and MT6,were selected.In terms of total fatty acid content,the mutant algae strains MT4 and MT5 were significantly higher than the wild algae strains.Among them,the MT3 and MT4 monounsaturated fatty acids and monounsaturated fatty acid contents were significantly better than the wild algae strains.In terms of DHA production,MT4 and MT5 are excellent.Compared with wild algae strains,the DHA content in each liter of algae fluid system is 37% and 41% higher,and the single cell DHA content is 17.19% and 39.18%higher,respectively.4.In the light qualitative incubator,two different light qualities,such as white light and blue light,are used to culture the flagellate algae,cultured for a week,Sampling every day during the experiment period for cell count and the difference detection of fucoxanthin,fatty acid and DHA content under different light qualities.The experimental results show that the growth state of cells under blue light culture conditions is slightly worse than that of white light,and blue light has no obvious promotion effect on increasing the total fatty acid content of Isochrysis galbana,but it can promote the content of fucoxanthin and DHA to a certain extent.The content of fucoxanthin and DHA in Isochrysis galbana showed a trend of increasing first and then decreasing under blue and white light culture conditions.