| Phaeodactylum tricornutum is a kind of marine diatom which is easy to be cultured and propagated fastly.It is rich in fucoxanthin with antioxidant activity.And it has a large number of fatty acids,especially eicosapentaenoic acid(EPA).It is a model organism of diatoms.Cryptochrome / photolyase family(CPF)is an ancient protein family,which has the functions of photoreceptor,clock protein and DNA repair enzyme.By studying the function of cryptochrome / photolyase gene family from Phaeodactylum tricornutum the basis is provided for the study of the function of CPF and the downstream signal response.By studying the production of fucoxanthin and fatty acids from Phaeodactylum tricornutum,the content of fucoxanthin and EPA can be improved which contributed to its industrialization.The main research contents and results are as follows:1.Firstly,the whole genome of Phaeodactylum tricornutum from NCBI database is downloaded.Secondly,six members of cryptochrome / photolyase family are found in the genome of Phaeodactylum tricornutum using DNA photolyase pfam(pfam00875)by BLASTP searching.It is found that the amino acid number of six CPF genes is not significantly different,the number and type of motif contained were similar,and there is no tandem clustering phenomenon on the chromosome.According to the homology of protein amino acid sequence,phylogenetic tree is constructed to classify six members of CPF gene family from Phaeodactylum tricornutum.The cis acting elements of CPF genes promoter from Phaeodactylum tricornutum contain many light response elements,plant growth hormone response elements and abiotic stress response elements.2.The CDS sequence of CPF6 gene of Phaeodactylum tricornutum is cloned by means of molecular biology,and the recombinant plasmid p Pha-T1-CPF6 is constructed.The recombinant overexpression plasmid is transferred into algal cells by the method of electric transformation.It is confirmed by ble resistance screening,PCR sequencing and q PCR.Three transformed algae strains(CPF6#1,CPF6#2,CPF6#3)are screened out.There is no significant difference in the cell growth trend,the dry weight and the fucoxanthin accumulation between the three transformed strains and the wild strains.3.In this paper,the effects of different light quality on the cell growth,the accumulation of fucoxanthin and fatty acids from Phaeodactylum tricornutum are studied.The cells of algal can continue to grow under the condition of white light,while the cell density of algal is decreased under the condition of blue light.So blue light inhibits the growth of algal cells.When the cells are treated with different light quality for 1-3 days,the content of fucoxanthin in blue light culture system is higher than that in white light culture system.Therefore,it is speculated that short-term blue light stimulation could increase the accumulation of fucoxanthin.It also is found that blue light could stimulate the accumulation of EPA and the total fatty acids in a single cell.4.The effects of glucose and sugarcane molasses on the cell growth the accumulation of fucoxanthin and fatty acids from Phaeodactylum tricornutum are studied.When the concentration of sugarcane molasses is 0.5g/L and 1g/L,the growth,the content of fucoxanthin,the content of total fatty acids and the content of EPA are significantly higher than that of glucose.5.In this paper,the atmospheric room temperature plasma(ARTP)mutation breeding of Phaeodactylum tricornutum was carried out.320 W,60s and 400 W,45s were selected as the best mutagenic conditions of ARTP.Through ARTP mutagenesis,the mutants A6,A7,B3,B27,C2 and E23 were screened out and the contents of fucoxanthin are significantly increased(p<0.001).The contents of fucoxanthin are 1.53 times,1.45 times,1.40 times,1.33 times,1.41 times and 1.39 times of wild strain respectively.Among them,the content of fucoxanthin of mutant A6 is the highest,which is 3.59mg/L,1.53 times of that of wild strain,1.24mg/L higher than that of wild strain. |
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