Regulation Of Fatty Acid Metabolism In Seeds Of Soybean With Gene Silencing To Get High Oil And High Oleic Acid Materials

It is an effective and efficient way for the study of soybean fatty acid synthesisand quality breeding using soybean genetic engineering regulation of lipid metabolicpathways and to explore mechanisms of gene regulation. In this paper, Jilin soybeanswere chosen as materials, base on protein and fatty acid metabolism of the "substratecompetition" hypothesis and using genetic engineering techniques to explore roles ofsoybean fad2 (fatty acid desaturase) gene and pepc (phosphoenolpyruvate carboxylase)gene in the regulations of soybean seeds fatty acid synthesis metabolism, and thenselect high-oleic and high oil breedings of genetically modified soybean. In this study,gene silencing vectors pBIGFP-FAD2、pBI-FAD2 and pBI-PEP were constructedaccording to gene silencing principle in plant, which contained fatty acid metabolismgenes fad2 (oleic acid dehydrogenase) and pepc (Phosphoenolpyruvate carboxylase),They were transformed into soybeans to regulate fatty acid matabolizm withenhancing fatty acids synthesis and oleic acid accumulation, and then, to screen outhigh oleic and high oil soybean materials.The pBIGFP-FAD2 vector was transformed into Jilin 35, Jilin 47 and Jike 1 usingAgrobacterium tumefaciens mediated method espectively, which were three high-oilcultivars in Jilin Province. The expression of GFP showed that, Jilin 35 is the mostsensitive cultivar in embryonic tip regeneration system. Transformation efficiency wasup to 42.9%. In this study, we optimized kanamycin concentration in embryonic tipregeneration system and attempted to applicate graft technology instead of thetraditional rooting method in soybean regeneration system for the first time. Graftingmethod survival rate can be up to 80% compare to 10% in root acclimatization method,the cycle was deduced from 45 days to 10 days. Total 180 regenerated plants wereobtained in the pBI-FAD2 transformation Jilin 35 soybean study. Molecular detectionshowed that the exogenous DNAs intergrated in 34 transgenic positive plantsgenomes. Southern hybridization, Northern hybridization, fatty acid contents and oleicacid proportion (ODP) assays indicated that fad2 gene expression was inhibited by theihpRNA construct, which consistant with high oleic acid and low linoleic acid in theseeds and a single-gene segregation pattern of Mendel. The fatty acid analysis of thetransgenic positive seeds showed that oleic acid content increased from 17.5% to 41-52%. Expression vector pBI-PEP was transformed into Jilin 35 soybeans by A.tumefaciens method. Total 265 regenerated plants and 64 PCR-positive transgenicplants were obtained. Southern hybridization showed that targeted genes wereintegrated into the plants genomes. 48 regenerated plants with different degrees of oilcontent, number of them accounted for 75% in the positive transformation materials.Nine materials of these soybeans had 22.55% oil content in T1-T4 on average, 1.8%higher than the control (20.75%). The average protein content of these 9 positivematerials was 34.07%, down 2.7% compared with the control (36.77%). Four seedpositive materials were chosen to analyze fatty acid composition in T3 showed littledifference between the controls. At five different developing stages, four positivematerials were detected PEPC activity. The result showed PEPC activitys of positivematerials at different stages were lower than that of control respectively. Expressionsof pepc and acc in developing seeds assay using Real-time PCR showed that, pepcexpressions at all detected stages were lower than the expressions of the control at thesame stage respectively, the expressions of them were 0.9 times, 0.78 times and 0.56times, 0.15 times and 0.03 times of the controls. Acc expressions of transgenicmaterials at all stage are larger than the control respectively, the expressions of themwere 1.23 times, 1.56 times and 3.01 times, 1.72 times and 18.78 times of the controls.The positive materials were assayed by Southern blot, fluorescence quantitative geneexpressions of pepc and acc, fat composition, protein content and enzyme activityanalysis, the results confirmed ihpRNA silence pepc genetically improved oil contentand decreased protein content with fatty acid metabolism in plants, it provided newevidence to verify the "substrate competition" hypothesis.Overall, we constructed two silence vectors contained fad2 and pepc andtransformed them into soybeans. We obtained better silencing effect on fad2 and pepcexpression then had screened high oleic acid and high oil soybean materials. Itprovided a foundation and basis materials to study lipid metabolism and breeding highoil, high oleic acid soybeans. For the first time we showed the graft could be apply inembryonic tip regeneration system in soybean seedlings. Graft improves the frequencyof embryonic tips regeneration system. It is provides a new approach for optimizatingof soybean regeneration system.