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Study On The Transcription And Expression Of Critical Lipase Genes In Schizochytrium Sp.S31

Posted on:2018-06-14Degree:MasterType:Thesis
Country:ChinaCandidate:F LouFull Text:PDF
GTID:2310330518986401Subject:Food Science and Engineering
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Schizochytrium is the most high-yield microorganism for DHA industrial production.Although its high-density fermentation has already been realized,little is known about the lipid metabolism mechanism.In order to gain a more particular knowledge about the regulatory mechanism of Schizochytrium lipid metabolism,Schizochytrium sp.S31 was used as research subject.Firstly,the whole genome sequence of Schizochytrium sp.S31 was acquired and then some critical lipase genes were identified.The relative expression levels of these genes in different fermentation conditions were measured.The inner link between fatty acid changes and the relative expression levels of these genes was discussed,which,could thereafter provide theoretical foundation for future improvement of Schizochytrium sp.S31 by genetic engineering for higher DHA algal oil output and better quality.Firstly,the whole genome sequencing work of Schizochytrium sp.S31 was finished.After wipe out of low quality data,18,216,884 high quality reads and overall 454.5 Mb data were acquired.Its genome size was about 42.99 Mb and GC content was 56.58%.The number of genes annotated was 11,404.All the protein-encoding genes were blasted to different databases and further annotated.Genes responsible for lipid metabolism were found out with Kyoto encyclopedia of genes and genomes.Then these genes were further blasted in national center of biotechnology information and three lipase genes and three phospholipase genes were identified.Secondly,the transcriptional levels of two fatty acid biosynthesis genes in different fermentation temperature were measured.The result showed that cells entered lipid accumulation stage after 72 h fermentation in shake flask cultures with glycerol as carbon source.For the up-shift to 30oC group,the ratio of PKS product to FAS product decreased from 1.129 to 0.758.And three hours after temperature shift,the transcriptional level of FAS gene increased two folds,while transcriptional level of PFA1 decreased 20%.But for the down-shift to 20 oC group,the ratio of PKS product to FAS product increased from 1.129 to 1.834.Three hours after temperature shift,the transcriptional level of PFA1 and PFA2 increased eight and nine fold,respectively;while FAS only increased two fold.So,it was speculated that the fatty acid biosynthesis was transcriptionally regulated by FAS and PKS genes in Schizochytrium S31.Then,the changes of fatty acids of triacylglycerol and phospholipilds during late fermentation process were measured.The result showed that lipolysis of phospholipids mainly concentrated on saturated fatty acids,and the lipolysis of triacylglycerol mainly happened to C14:0 and C16:0.RT-qPCR result showed that the changes of phospholipid were regulated by SPLA,SPLB and SPLD genes.And the lipolysis of triacylglycerol was mianly mediated by STGL1 and STGL2 genes,while STGL3 might also take little part in this process.Finally,one of three critical phospholipase genes SPLB was successfully cloned and heterologous expressed in Pichia pastoris.
Keywords/Search Tags:Schizochytrium, lipase, phospholipase, transcriptional regulation, heterologous expression
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