Functional Analysis Of LuAccD From Linum Usitatissimum On Seed Oil Accumulation

The lipid is one of the three major nutritional elements required by the human body and plays a variety of important functions in human health.Flax(Linum usitatissimum)is an important oil crop in our country.Studying the function of its lipid metabolism genes is of great value for clarifying the regulatory network of oil formation and breeding flax varieties with high oil content.TheAccD gene encodes the β-CT subunit of acetyl-Co A carboxylase,which has the function of catalyzing the formation of malonyl-Co A from acetyl-Co A,and catalyzes the first key step in the process of fatty acid synthesis.At present,there is no related research on the role ofAccD gene in the metabolism of linseed oil in flax.In this study,the LuAccD gene was cloned from the flax variety "Longya 10",and it’s Arabidopsis thaliana transgenic line was obtained by Agrobacterium-mediated method.The function of LuAccD in A.thaliana seed oil accumulation was preliminarily studied,and its effect on seed germination under abiotic stress conditions was discussed.The main results are listed as follows:(1)Amino acid sequence analysis showed that the proteins encoded by AtAccD and LuAccD both had typical carboxyltransferase domains with conserved sequences.Phylogenetic analysis showed that there is a certain distance in the genetic relationship between theAccD gene of flax and homologous genes in other species.These results indicated that there are both similarities and possible differences in biological functions between LuAccD and AtAccD.(2)Bioinformatics analysis showed that both AtAccD and LuAccD were hydrophilic proteins without transmembrane domain and signal peptide.In the secondary structure,the α-helix,β-turn,extended chain and random coil structure of AtAccD protein accounted for33.61%,7.58%,18.24% and 40.57%,respectively.LuAccD protein α-helix,β-turn,extended chain and random coil structure accounted for 34.85%,8.18%,18.79% and 38.18%,respectively.The phosphorylation modification sites of AtAccD proteins are 68 and the Nglycosylation modification sites of AtAccD proteins are 2.The phosphorylation modification sites of LuAccD proteins are 47 and the N-glycosylation modification site of LuAccD proteins is 0.AtAccD and LuAccD may interact with proteins containing biotin carboxylase domain and carboxyltransferase domain.(3)We overexpressed LuAccD in the A.thaliana wild-type(Col-0)background due to the loss of AtAccD to death in Arabidopsis plants.Overexpression of LuAccD did not change the characteristics of seed morphology(length,width,weight and seed coat color).The total fatty acid content and various fatty acid components in the seeds of A.thaliana transgenic plants were significantly higher than wild-type.The heterologous expression of LuAccD is positively regulating the content of total fatty acids and fatty acid components in A.thaliana seeds.Meanwhile,overexpression of LuAccD significantly increased the expression levels of genes such as At BCCP1,At BCCP2,At MCAT,At KAS1,At KAS2,At SSI2,At FAD2,At FAD3,and At PDAT2 in developmental seeds.These results indicate that the LuAccD gene promotes the expression of genes related to oil accumulation in A.thaliana seeds and then promotes the accumulation of fatty acids in seeds.(4)The seed germination experiment showed that under 150 m M Na Cl and 300 m M mannitol stress conditions,the seed germination rate of the Col-0 35S:LuAccD-6HA transgenic strain was significantly higher than that of the wild-type.The real-time quantitative PCR detection showed that under salt stress conditions,the expression levels of ABA synthesis genes,such as At NCED3,At AAO3,and ABA signal response genes,such as At ABI3,At EM1,and At EM6 in the germinated seeds of the Col-0 35S:LuAccD-6HA #4 transgenic strain were significantly higher than those of the wild-type.These results indicate that overexpression of LuAccD can enhance the tolerance of seeds to salt and osmotic stress by regulating ABA synthesis and signal transduction pathways.In summary,this research analyzed the physicochemical properties and protein structure of LuAccD protein,and preliminarily explored the function of LuAccD in the oil accumulation and abiotic stress response of seeds.The results of this study further lay a molecular foundation for exploring the regulatory network of flax seed oil accumulation and stress response,and for breeding high-quality varieties with high oil content,high resistance,and high content of unsaturated fatty acids such as α-linolenic acid.