Optimization Of Cotyledon Node Gene Transformation System In No.5 Jidadou And Functional Analysis Of GmPEPC4 Gene

Soybean is one of the main oil crops in the world,and the breeding of high oil varieties has always been an important work of soybean breeders.However,oil content is a quantitative trait controlled by multiple genes,which requires multiple generations for genetic stability.Traditional breeding methods have long time to breeding and heavy work.Gene engineering can be used to obtain genetically stable transgenic lines and make breeding efficiency.PEPC gene is a key enzyme in plant photosynthesis.Previous studies have proved that PEPC gene,as a key node in the central carbon metabolism pathway,is related to lipid content.It can lead the metabolic flow to the tricarboxylic acid cycle,and make the carbon flux flow to the non-lipid accumulation direction,which is not easy to accumulate lipid.Interference with the expression of the gene can increase oil content,which has been confirmed in rapeseed and peanut crops.In this study,Gm PEPC4 gene was cloned and RNAi vector was constructed to silence Gm PEPC4 gene in high-oil soybean variety No.5 Jidadou in order to further improve the oil content of soybean.At the same time,the genetic transformation system of No.5 Jidadou was optimized to provide technical support for soybean variety breeding.The following results are obtained:1.In this study,soybean Gm PEPC4 gene was cloned by PCR from Williams 82.Overexpression vectors 3300-PEPC4 and 1301-PEPC4 and RNA interference vectors PFGC-PEPC4 i were successfully constructed.2.Transgenic soybean hairy roots were transformed into positive materials by molecular identification,and the effects of the exogenous gene on the content of oil and fatty acid in roots were detected.Soxhlet extraction results showed that silencing Gm PEPC4 gene could increase crude fat content of hairy roots by 0.97%,and overexpression of Gm PEPC4 gene could increase crude fat content of hairy roots by 0.19%,but did not reach significant level.The results of GC-MS analysis showed that five main fatty acids were isolated from fresh hairy roots of soybean,and the crude fat content of hairy roots of soybean increased slightly but did not reach a significant level due to the gene Gm PEPC4.Fatty acid content identification showed that Gm PEPC4 gene was silenced,oleic acid and linoleic acid content increased significantly,and linoleic acid content increased to a significant level.Overexpression of Gm PEPC4 gene resulted in a significant decrease in linoleic acid.3.Jida 5 is selected as a transformation receptor.Thirty seconds with 75% alcohol,4-5 times with sterile water,20 minutes with 10% sodium hypochlorite,4-5 times with sterile water,1 day with germination,0.6 with OD600,200 mg/L with dithiothreitol and 250 mg/L with sodium thiosulfate at co-culture stage to reduce browning,150 mg/L with Cef and 200 mg/L as bacteriostat induction stage,10 mg/L with ZT and IAA as the optimum concentration for long-term bud elongation.The optimum concentrations were 1.5 mg/L and 0.2 mg/L,and GA31.0 mg/L.