| With the rapid increase of the world population and the continuous improvement of people's requirements for food,the demand for oil in domestic and foreign markets is becoming higher and higher.The topic about how to improve the oil content of crops has become an important goal of plant breeding.Soybean(Glycine Max L.)is one of the most important oil crops and food crops in the world,which contains various unsaturated fatty acids,vitamins,inorganic salts and other rich nutrients needed by human beings.It is a kind of very important high-quality vegetable edible oil.However,compared with other oil crop plants,its oil content still has much room for improvement.Peanut(Arachis hypogaea L.)is recognized as a crop with high oil content in the world,and its seeds contain about 50% oil content,which is of high research value.The purpose of this study was to develop a new high-oil content soybean line by genetic engineering,and to reveal the regulation mechanism of lipid metabolism pathway using quantitative proteomic and lipidomics analyses,so as to provide an important theoretical and practical basis for soybean quality improvement.Diacylglycerol acyltransferase(DGAT)is the only rate-limiting enzyme required for the formation of triacylglycerol TAG(Triacyglycerol)in Kennedy fatty acid metabolic pathway.In the early stage of this study,10 Gm DGAT genes of soybean and Ah DGAT3 gene of peanut were over-expressed in Arabidopsis thaliana,and it was found that the functions of Gm DGAT1-2 and Ah DGAT3 were stronger.Therefore,Gm DGAT1-2 and Ah DGAT3 genes were transferred into soybean by agrobacterium tumefaciens mediated soybean cotyledon node.The further functions of two genes were verified,revealing the internal molecular mechanism of the changes of fatty acid components and the increase of total oil content in soybean,which provided a powerful condition for the cultivation of soybean lines with high oil content.The main experimental results are as follows:1.Ten genes in the DGAT family of soybean diacylglyceryl transferase were cloned from high oil soybean variety “JD5”.Three genes in the first subfamily were Gm DGAT1-1,Gm DGAT1-2 and Gm DGAT1-3.The second subfamily contains five genes Gm DGAT2-1,Gm DGAT2-2,Gm DGAT2-3,Gm DGAT2-4 and Gm DGAT2-5.And the third subfamily contains two genes: Gm DGAT3-1 and Gm DGAT3-2.It was found that each subfamily contained a special conserved domain and motif by protein sequence alignment.The amino acid sequence of Ah DGAT3 gene in peanut was aligned with other plants,and it also contained special conserved amino acid sequence and active site.2.The expression patterns of 10 Gm DGAT genes in different soybean tissues were analyzed by q RT-PCR,and it was found that the expression of three genes in Gm DGAT1 subfamily were the highest in soybean seeds.The five genes in Gm DGAT2 subfamily was expressed in different tissues of soybean.However,two genes in Gm DGAT3 subfamily were highly expressed in soybean leaves and mature seeds.3.Ten resultant p CHF3300-Gm DGATs and p CHF3300-Ah DGAT3 were heat shocked into A.tumefaciens EHA105 and then transferred into Arabidopsis Col-0 using the floral-dip method.The contents of five key fatty acid compositions(palmitic acid,stearic acid,oleic acid,linoleic acid and linolenic acid)and total fatty acids were measured.It was found that the content of 18:1 composition was significantly higher in the over-expressing transgenic Arabidopsis seeds than in the WT seeds,especially in Gm DGAT1-2 whose contents were 1.5 times higher,respectively,than in WT and total fatty acid content was significantly increased.However,the fatty acid content of atdgat1 and atdgat2 Arabidopsis mutants showed an opposite trend.Compared with the WT,the contents of oleic acid and total fatty acid contents in Ah DGAT3 transgenic Arabidopsis were also significantly increased,while linoleic acid content was significantly decreased.4.The resultant p TF101-Gm DGAT1-2 and p TF101-Ah DGAT3 was heat shocked into A.tumefaciens EHA105 and then transferred into cotyledon nodes of the soybean.The receptor variety used for soybean transformation is JACK,which is internationally recognized as a soybean receptor variety with high genetic transformation efficiency.Finally,we got 24 Gm DGAT1-2 transgenic soybean lines and 10 Ah DGAT3 transgenic soybean lines were confirmed using Bar strip,Southern blot,western blot and Elisa.In addition,the whole genome of Ah DGAT3 transgenic peanut soybean was resequenced,and Ah DGAT3 gene was confirmed to be integrated into soybean genome by T-DNA single copy insertion.Then the contents of five key fatty acid components(palmitic acid,stearic acid,oleic acid,linoleic acid,linolenic acid)and total fatty acid in T3 Gm DGAT1-2 and Ah DGAT3 transgenic soybean lines were measured.It was found that the content of oleic acid(18:1)and total fatty acid were significantly increased,while 18:2 was decreased in over-expressing transgenic soybean lines compared to wild type.5.The expression levels of key genes in the fatty acid metabolism pathway in T3 transgenic soybean lines and wild-type were detected,and it was found that these key genes had different degrees of change compared with the WT(control).In Gm DGAT1-2 transgenic soybean lines,FATA,SAD and other key genes which controlled the oleic acid rate significantly increased,while the expression level of FAD2 gene which controlled the linoleic acid rate decreased.This is consistent with the changing trend of fatty acids in Arabidopsis.6.The agronomic traits of Gm DGAT1-2 and Ah DGAT3 transgenic soybean plants harvested from T2 and T3 generations were investigated for two consecutive years.Compared with wild type soybean,plant height,effective branch number,pod number per plant,grain number per plant,grain weight per plant and other traits were improved to different degrees.And the growth was significantly better than the control(WT).It is speculated that the two DGAT genes may also have different effects on the growth and development of soybean7.50-day-old nearly mature pods of Gm DGAT1-2,Ah DGAT3 transgenic soybean and the WT(JACK)were sampled and analysed using quantitative proteomic and lipidomics.A total of 436 differential expressed proteins(DEPs)and 180 differential expressed metabolites(DEMs)were identified between T3 Gm DGAT1-2 transgenic soybean and the WT(JACK).Through integrated proteomic and lipidomics analyses,it was found that over-expression of Gm DGAT1-2 gene resulted in significantly downregulated expression of lipoxygenase gene,which was enriched in linoleic acid metabolic pathway,and then changed the content of fatty acid components.The content of linoleic acid(18:2)was significantly decreased,while the content of oleic acid(18:1)was significantly increased.In addition,four significantly up-regulated oleosin genes were screened.Over-expression of soybean Gm DGAT1-2 gene led to an increase in TAG content,while up-regulated expression of oleosin gene precisely catalyzed more triglyceride formation into final oil body,thus improving the total oil content of transgenic soybeans.161 differentially expressed proteins and 119 differentially expressed lipid metabolites were screened between Ah DGAT3 transgenic soybean lines and the WT(JACK).Through integrated proteomic and lipidomics analyses,it was found that over-expression of Ah DGAT3 gene resulted in significant up-regulation of the selected lipase gene,thus increasing the content of total oil content and oleic acid(18:1)composition. |
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