Cloning And Expression Analysis Of Enoyl-acyl Carrier Protein Reductase Gene And Fatty Acids Assay Under Environment Stress In Isochrysis Galbana

Enoyl-ACP reductase (ENR) plays a crucial role in metabolism of fatty acid synthesis. Itmakes the substrate enoyl-ACP reductive into acyl-ACP, and catalyzes unsaturated C=C bondback into saturated C-C bond. This is the final step in fatty acid. ENR is an enzyme thatcatalyzes enoyl-ACP back into acyl-ACP; but also can adjust to the content of saturated andunsaturated fatty acids, participate in the catalytic synthesis of long chain fatty acids.The full-length cDNA and DNA sequences of IgENR were obtained in RT-PCR withprimers designed according to a partial IgENR cDNA generated in the transcriptome analysisof I. galbana CCMM5001and deposited in GenBank (GenBank accession number:JQ309976and JQ903585). RNA was extracted and separated from I. galbana at first. Andthen, primers for PCR were designed. IgENR was successfully cloned by RapidAmplification of cDNA Ends (RACE) technique, and characterized by bioinformaticsanalysis. IgENR was1053bp in total length, containing an open reading frame (ORF) of1044bp, a5′-UTR of14bp and3′-UTR of445bp. IgENR was deduced from the ORF, whichcontained347amino acids residues with a predicted molecular weight of36.3kD and a pointof electricity (pI) of5.24. The DNA sequence is2253bp in length (JQ903585) and isinterrupted by four introns, with canonical GT/AG splicing signals found at the ends of theintrons. IgENR is very similar to the known ENRs of Nicotiana tabacum, Arabidopsisthaliana, Thalassiosira pseudonana and Phaeodactylum tricornutum.The expression patterns of IgENR under the challenge of low temperature, hightemperature and different concentrations of sodium nitrate (0mg/L、75mg/L、150mg/L) wereexamined in I. galbana CCMM5001by Real-time PCR analysis. Under temperature stress,the expression level of the IgENR increased with increasing temperature, when thetemperature is35℃, IgENR gene transcription level higher than15℃and25℃, themaximum value of66.6times that of the control group was much higher than15℃and the 25℃IgENR gene transcription level. Under different nitrogen concentration of stress, with amedium level of expression of the elevated concentration of nitrogen IgENR elevated nitrogenconcentration in excess (150mg/L), IgENR gene expression levels in48h to achieve themaximum, and then gradually decreased expression amount. Thus, the heating temperature orincrease the concentration of nitrogen can to increase IgENR gene expression.In this study, we analyzed the influence of different culture conditions on its growth andlipids accumulation, which of lipids-riched marine microalgal I. galbana. The resultsindicated that low nitrogen concentration and the temperature of25℃were suitable for lipidsaccumulation，but the lipids content reduced if nitrogen concentration and temperature wastoo high. In addition, in the sight of fatty acid composition of microalgae, the conditionconducive to the accumulation of PUFAs is low temperature (15℃), relative content of57.56%. The optimum conditions for growth were25℃and nitrogen concentration75mg/L,the optimum conditions for lipids accumulation nitrogen concentration0mg/L.Results obtained from this study provide valuable information on further investigatingthe expression of thiolase related genes and lipids accumulation on different conditions of I.galbana CCMM5001. There is not only being physiologically and ecologically significant forthose interested in algal adaptation to their environment, but also an important theoreticalsignificance to comprehensively understand the structure, function and features of enoyl-ACPreductase related enzyme genes.