Study On Lipid Metabolism And Secretion Of Chlamydomonas Reinhardtii By Multi-gene Regulation

With the depletion of fossil fuels and environmental pollution intensified,a new generation of microalga biofuel technology has been widespread concerned.In order to reduce the cost and increase the lipid content of biofuel producing microalga,it is important to develop new strategies to regulate the key genes of microalga fatty acids(FAs)synthesis and secreation pathway.Our previous studies have successfully increased FAs content in Chlamydomonas reinhardtii through two ways,either by the overexpression DOF transcription factor,or by the gene silencing of ACS1 and ACS2,two long-chain acyl-CoA synthase genes.In this study,we further inverstigated the possibility of regulating the two pathways simultaneously and successfully constructed a series of transgenic alga.In transgenic alga C.reinhardtii Mq12,both ACS1 and ACS2 genes were co-silenced by continuous transformation.Compared with single gene silencing alga,the intracellular lipid content of the co-silencing alga Mq12 was significantly improved.Based on this,we also constructed the transgenic alga C.reinhardtii Mc7 with silencing ACS2 and overexpressing DOF at the same time.Compared with single gene regulated alga,the intracellular lipid content of Mc7 also increased significantly,and the secretion of extracellular fatty acids improved as well.The significance of multi-gene co-control strategy in the regulation of microalga production was confirmed.The results of this paper are as follows:1.The RNA interference vector pMaa7 IR / ACS2 IR of ACS2 gene was constructed.The vector showed a Paromomycin resistance in C.reinhardtii and contained a 421 bp fragment from ACS2,which was regulated by RBCS2 promoter of C.reinhardtii and could effectively inhibit the expression of endogenous ACS2 gene in C.reinhardtii expression level;2.The pMaa7IR/ACS2 IR was transformed into the alga which the ACS1 gene knocked down by continuous transformation,and the transgenic alga Mq12 was secreened by BODIPY505/515 staining.Fluorescence quantitative PCR showed that the expression level of ACS1 and ACS2 genes in Mq12 were reduced by 63% and 82%.Compared wih wild type strain CC849,ACS1 knockdown strain Q15 and ACS2 knockdown strain P13,the intracellularTAG content of strain Mq12 was increased by 200%,73.5% and 46.6% respectively.The total lipid content of Mq12 was 167.1 ?g/mg,which was 65%,23% and 38% higher than CC849,P13 and Q15,respectively.The C18: 3n3 and C18: 1c contents in Mq12 were 6 times and 2.5times higher than that in CC849.These results suggested that co-silencing of ACS1 and ACS2 could significantly increase the intracellular TAG and total lipid content of C.reinhardtii.The extracellular fatty acid data showed that the secretion of fatty acids in Mq12 was 19% higher than that in CC849,but not significantly different with P13 and Q15;3.The pMaa7IR/ACS2 IR was transfected into DOF overexpressed transgenic alga C12 by continuous transformation.The transgenic alga Mc7 was obtained by BODIPY staining.The results showed that the intracellular glyceride content of Mc7 was significantly higher than CC849,C12 and P13 by 91%,40% and 48% respectively.The intracellular total fatty acid content of Mc7 increased by 90%,29% and 42%,respectively,compared with CC849,C12 and P13,and there was a significant increase in unsaturated fatty acids compared with CC849.Moreover,the total extracellular fatty acid amount in Mc7 was significantly higher than that of CC849,C12 and P13(67.4%,35.88% and 21.5%,respectively).Compared with CC849,the extracellular unsaturated fatty acids content in Mc7 was also greatly improved.The results showed that co-regulation of ACS2 gene and DOF transcription factor could significantly increase the intracellular lipid content,especially TAG content,and the extracellular secretion of fatty acids was also significantly increased.In this study,we developed a multi-gene co-regulation method in C.reinhardtii,and contructed a transgenic C.reinhardtii strain that simultaneously regulating ACS gene and DOF transcription factor.The results showed that ACS and DOF had a synergistic effect in improving the intracellular lipid production and fatty acid secretion in C.reinhardtii,which provided a new idea and research basis for microalga biofuel production.