| As a marine microalgae,Phaeodactylum tricornutum has a high-content of EPA,It's content of more than 30% of total fatty acids.EPA plays an important role in improving human immunity?lowering blood pressure etc.It is difficult from deep-sea fish oil to extracting EPA.Besides,the application of fish oil has been limited.However,the brown-leaved algae easy to culture and odorless and has a fast breeding,simple extraction and purification process.Therefore,it is selected as an advantageous strain to increasing EPA production.In this study,we studied algae strain(FACHB-863)by identification.It is proved P.tricornutum at finally.The bacteria were sterilized by coating,scribing,capillary microscopy and antibiotics.There are three ways of alage preservation,For example:liquid,solid-liquid two-phase and low temperature glycerol/dimethyl sulfoxide preservation were compared.The solid-liquid two-phase preservation has an best effect,and the cryopreservation was the worst.And the cells preserved of protective agent failed to survive by the low concentration.The FAD gene family of Arabidopsis thaliana was downloaded from TAIR,and the FAD gene of P.tricornutum was screened by blastp.A total of 15 family genes were identified.?5,?6,?4,and ?12-desaturase involved in fatty acid synthesis process.Among them,the ?5-desaturase is located on chromosome 11.Due to the ?5-desaturase gene is a key gene in the EPA synthesis pathway,so we studied the encoded protein(ptd5?).The protein belonged to a hydrophilic protein and has four transmembrane domains.There are many potential phosphorylated sites to change their phosphorylation status during interaction with other enzyme proteins,thereby regulating their biological activities and functions.This paper has carried out researching on high-yield EPA of P.tricornutum.Firstly,the ?5-desaturase gene was cloned located upstream and the structure and function of the expressed protein were predicted and analyzed.Secondly,the recombinant plasmid p Pha-T1-?5 was constructed and insert to alaga's cell for overexpresion.Finally,three mutant strains named 5D1,5D2,5D3 were screened by resistance screening and PCR sequencing.Through a series of physiological and biochemical indicators,the growth cycle of mutant algae strains and wild algae strains was not significant.The fatty acid content of the three mutant algae strains showed that the fatty acid content.Especially the EPA content,was significantly higher than that of wild algae strains.The mutant strain(5D3)increased to31.1% compared with the wild algae strain(WT).Real-time PCR showed that the relative expression of mutant strains(5D2,5D3)increase about85.3% compared to wild algae(WT).The re-sequencing of the mutated algae strain and the wild algae strain genome revealed that there were 1-4 differential genes in the In Del and SNP variations of each group.The atmospheric pressure plasma mutation(ARTP)was carried out on the wild algae strain of P.tricornutum.The mortality test showed that the cell death rate was 80%.After initial screening and rescreening,four mutant algae strains(SJA1,SJA3,SJA4,SJA6)were screened.The difference between the wild algae strain and the mutant algae strain was not significant by growing delection,but the content of polyunsaturated fatty acid EPA compared with wild algae strain increase about 25.6%?17.4%?15.5%?21.6%?... |
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