Study On The Co-regulation Of Lipid Accumulation By Three Genes DOF-LACS2-CIS1 In Chlamydomonas Reinhardtii

Chlamydomonas reinhardtii is a model organism for studying lipid biosynthesis and engineering strains with high lipid accumulation.The results showed that the regulation of long-chain acyl-CoA synthetase(LACS2),citrate synthase(CIS 1)and DOF recording factor(DOF),respectively,could increase the content of fatty acids LACS2 activates fatty acids into ? oxidation.CIS1 catalyzes the first step of the TCA cycle.DOF transcription factors regulate carbon and nitrogen metabolism.We synthesize three different ways to increase oil accumulation.In this study,LACS2-CIS1,a double silencing gene,was transferred into the DOF transcription factor engineering algae to obtain three genes.And the growth status,starch,protein,fatty acid,and the changes of several key genes in the transcription level of the engineering algae were analyzed.The specific research results are as follows1.Firstly,the RNAi interference vector pMaa7IR/LACS2-CIS1IR of the LACS2-CIS1 gene was constructed.The vector can be selected by hygromycin resistance,which could effectively inhibit the gene expression of endogenous LACS2 and CIS1 of Chlamydomonas reinhardtii2.The pMaa7IR/LACS2-CIS1IR was transferred into DOF engineering algae to obtain 150 strains of transformants,and 33 strains of algae with high oil content were screened by Nile red staining.Analysis of DOF,LACS2,and CIS1 gene expression of engineered algae DLC-4 showed that after heat induction,LACS2 and CIS1 genes were down-regulated by 58%and 26%,respectively,compared with DOF engineered algae.Compared with WT,DOF,LACS2,and CIS1 genes were up-regulated by 121%and down-regulated by 53.3%and 46.9%,respectively.These data indicate that the engineered alga DLC-4 achieves three genes co-regulation3.The intracellular fatty acid content of DLC-4 increased by 31%and 52%before and after heat induction,respectively.Among them,C16:0,C18:0,C18:1 and C18:3 have higher fatty acid components.After heat induction,the intracellular fatty acid content of engineered algae DLC-4 increased by 25%compared with DOF engineered algae.Nile Red further analyzed that the DLC-4 TAG content increased by 142%under nitrogen depletion.Starch and protein content showed that the starch and protein content of DLC-4 decreased slightly compared with WT before heat induction;after heat induction,the starch content of DLC-4 decreased by 45%and protein content by 24%compared with WT.These results indicate that after heat induction,there is a decrease in starch and protein that provides energy and precursors for fatty acid synthesis4.To further understand the mechanism of oil accumulation of engineered algae DLC-4,transcriptome analysis of gene expression of DLC-4 and WT after heat induction.The results showed that 5 of the 7 proteins involved in the catabolism of branched-chain amino acids were up-regulated and 4 starch synthases involved in starch synthesis were down-regulated and ?-amylase was up-regulated,which indicated that the protein and starch were degrading.Subsequently,the increase in the content of acetyl-CoA and the up-regulation of fatty acid synthase activity and the degradation of some phospholipids increased the activity of acyl CoA:diacylglycerol acyltransferase(DGAT)and phospholipid diacylglycerol acyltransferase(PDAT),thereby increasing the accumulation of TAG5.The results of gene expression related to lipid synthesis indicate that,under the co-regulation of the three genes,the up-regulation of acetyl-CoA carboxylase(ACCD)and acyl carrier protein thioesterase(FAT1)genes increased fatty acid synthesis.Four DGAT2 genes except for the DGTT1 gene have no expression,and the other three genes are up-regulated,of which DGTT4 is up-regulated by 84%,and C18:1 is the preferred substrate for DGTT4.The expression levels of phospha-tidic acid phophyhydrolase(PAP2),PDAT1,and DGAT1 were all up-regulated.The up-regulation of these genes increases the synthesis of TAG.It was confirmed that the three-gene regulation strategy can slow down the speed of the TCA cycle,reduce the degradation of fatty acids,and use carbon flux for fatty acid synthesis to accumulate TAG.Therefore,this study achieved the co-regulation of three genes LACS2-CIS1-DOF by the way of polygenic regulation of microalgae,and finally obtained the engineering algae to improve the oil content.The method of polygenic regulation has a higher potential for improving microalgae oil production and provides a new idea and basis for microalgae oil production.