Cloning And Functional Characterization Of CsDGAT3 Genes In Camelina Sativa

Camelina sativa(L.)Crantz,as oil crop with low input and high output in the cruciferous plant,has agronomic advantages such as low fertilizer requirement,high resistance to pests and diseases,and strong environmental adaptability.The seed oil content of camelina accounts for47% of dry weight,and its fatty acid component(containing 90% unsaturated fatty acid)is suitable for aviation fuel,biodiesel and high-value industrial lubricating oil.In addition,the camelina seed oil is also rich in α-linolenic acid,an essential fatty acids for human body,accounting for 32-40% of the total oil content.As the market for camelina oil is substantially increasing,considerable efforts are focused to enhance camelina agronomic traits such as seed oil yield and quality by genetic engineering and genome editing technology.Nevertheless,intensive understanding the intrinsic molecular mechanisms underlying oil biosynthesis and other biological processes would lay the foundation for camelina genetic improvement and sustainable commercialization of high-value products.The accumulation and storage of seed oil of oil crops are mainly in the form of triacylglycerols(TAG).Diacylglycerol acyltransferases(DGAT)are the key rate-limiting enzymes in the final step of TAG biosynthesis(Kennedy pathway).Atleast three distinct classes of DGATs were identified in eukaryotes,designated as DGAT1,DGAT2,and DGAT3.DGAT1 and DGAT2 are integral membrane proteins of ER,while DGAT3 is a soluble enzyme protein without a transmembrane structure.Therefore,compared with DGAT1 and DGAT2,DGAT3 gene is more likely to be a molecular target for improving crop oil in genetic engineering.Atpresent,the homologous sequence of DGAT3 has been found in most oil crops,but there is no functional verification.In order to investigate the molecular mechanism of camelina oil synthesis,this study identified and cloned the CsDGAT3 gene of camelina and verified its function.The main research results are as follows:1.Using amino acid(AA)sequences of peanut(Arachis hypogeal)AhDGAT3 as the query sequence,three candidate genes were screened out from the camelina genomic database.They were named CsDGAT3-1,CsDGAT3-2 and CsDGAT3-3,encodeing 361,364,and 365 amino acids,respectively.Transcriptome data analysis showed that the transcripts of the CsDGAT3 gene contained complete open reading frame(ORF),indicating that they have expression activity.Bioinformatics tools were used to systematically analyze the structure and physical and chemical characteristics of the proteins encoded by these three genes.The results showed that all three contained the typical domains of the TRX-like Fd family,and they were all water-soluble enzyme proteins located in the cytoplasm and without transmembrane structure.Tertiary structure analysis showed that three proteins presented as a homodimer containing the [2Fe-2S] domain.Multiple sequence alignment analysis revealed that all three camelina CsDGAT3 proteins had a typical conserved region of DGAT and an acyltransferase binding site.Phylogenetic tree results showed that the three CsDGAT3 proteins had the highest homology with the Arabidopsis DGAT3 protein,suggesting functional similarity.2.RT-PCR and qRT-PCR were employed to investigate expression profiles of three CsDGAT3 gens in various tissues of camelina.The results found different patterns of transcript levels for the three genes.CsDGAT3-1 mainly expressed in roots while CsDGAT3-2 transcripts predominantly accumulated in flowers and germinating seeds.However,for CsDGAT3-3,its expression was highly increased in developing seeds.These results suggest that these three genes may play a role in different tissues.Using high-fidelity RT-PCR,three CsDGAT3 gene coding sequences were cloned from the developmental seeds of camelina,and their sizes were 1092 bp,1095 bp,and 1098 bp,respectively.3.In order to verify the function of the CsDGAT3 gene of camelina,we heterologously expressed CsDGAT3-1,CsDGAT3-2 and CsDGAT3-3 in TAG-deficient yeast H1246.Nile red staining of transgenic yeast cells and TLC analysis of cell lipids showed that the three camelina CsDGAT3 genes could restore the ability to TAG synthesize in H1246 yeast cells.And the accumulation of TAG in CsDGAT3-3 overexpressing yeast cells was significantly higher than the TAG content in the other two genes overexpressing cells,which further indicated that the CsDGAT3 protein had DGAT enzyme activity and the CsDGAT3-3 enzyme activity was stronger.Gas chromatography(GC)analysis of lipids in transgenic yeast cells showed that palmitoic acid(16: 1)and oleic acid(18: 1)contents of overexpressing CsDGAT3-3 yeast cells were higher than those of transgenic CsDGAT3-1 and CsDGAT3-2 genes.Further,by adding exogenous unsaturated fatty acids(18: 2,18: 3,and 20: 1 rich in camelina seeds)to the yeast medium of the CsDGAT3-3gene-transformed yeast,and the fatty acid composition of the yeast cells was detected.Compared with yeast cells that were not added with exogenous unsaturated fatty acids,yeast cells that were separately added with exogenous unsaturated fatty acids were significantly enriched in these unsaturated fatty acids,especially 20: 1 accumulated the most.It can be seen that CsDGAT3-3 has a higher substrate specificity for unsaturated fatty acids,especially C20: 1.4.To further identify whether the CsDGAT3 gene can function in somatic tissues of other higher plants,agrobacterium-mediated leaf disc method was used to transfer the constructed plant tissue-specific expression vector into Nicotiana tabacum.And transgenic tobacco plants were identified by DNA and RNA detection.Analysis of the total lipid and fatty acid components of transgenic tobacco leaves showed that overexpression of CsDGAT3 gene in tobacco could increase the total lipid content in leaves.And the content of C18: 1 and C20: 0 in CsDGAT3-1transgenic tobacco leaves increased,which were 0.75 and 1.53 times higher than those in wild tobacco,respectively.Transgenic tobacco leaves C18: 2 and C18: 3 with CsDGAT3-2 increased by50.74% and 31.24%,respectively,compared with wild tobacco leaves.The expression of CsDGAT3-3 can significantly increase the fatty acid accumulation of C20: 1,and it was 225.4%higher than that of wild tobacco,which was consistent with the results of accumulation of high amounts of C20: 1 in CsDGAT3-3 yeast cells cultured with exogenous C20: 1.It further indicates that the CsDGAT3-3 gene is more likely to have a preference for TAG synthesis using unsaturated fatty acids,especially C20: 1 as a substrate.The substrate specificity of the other two genes needs further study.To sum up,we have isolated,identified and cloned three CsDGAT3 gene encoding DGAT3 enzyme protein from camelina.The biological function of these three enzyme proteins was demonstrated for the first time through the TAG-deficient yeast H1246 functional complementation test and tobacco genetic transformation.In particular,the high enzyme activity of CsDGAT3-3 and its substrate selectivity for C20: 1 were clarified.These findings provide new insights for further understanding the mechanism of CsDGAT3 gene involved in seed oil biosynthesis and regulation,and also lay an excellent target gene element for other oil crops to improve seed oil production and enrich unsaturated fatty acids such as C20:1.