Functional Study Of BnVIK And BnNAC13 In Regulation Of Oil Content In Seed Of Brassica Napus L.

Rapeseed oil is the main source of China’s self-produced edible vegetable oil,and the stable development of the rape industry is vital to satisfy the growing demand for edible vegetable oil in China.Increasing the seed oil content of rapeseed is one of the important goals of rapeseed breeding.The main metabolic pathways of plant seed lipid synthesis are relatively clear.There are many genes involved in lipid synthesis.Simply increasing the expression of a single lipid synthesis gene has a limited effect on the seed oil content.However,there are genes that regulate the accumulation of oil in the process of seed oil synthesis,such as WRI1,LEC2 and so on.Studies have shown that these genes are vital in seed oil synthesis.At present,there are limited reports on genes regulating the oil synthesis of rapeseed.Therefore,it is important to discover new genes that regulate the oil content of rapeseed to increase the oil content of rapeseeds.Using GWAS results for 5-year oil content of 505 oilseed rape materials and TWAS results for seed oil content of 309 oilseed rape materials,our group previously identified27 significant QTL loci and 692 candidate genes that were significantly associated with oil content.In this study,transcriptomic data of high and low oil content varieties at three different developmental periods were referenced based on the existing data in our group,and bioinformatics analysis was used to predict two candidate genes that might be involved in regulating oil content of rapeseed:Bna A09.VIK and Bna A07.NAC13.Among them,Bna A09.VIK was located in the QTL q OC.A09.5 interval.The Bn VIK（Bna A09.VIK,Bna C08.VIK,Bna A06.VIK,Bna C05.VIK）and Bn NAC13（Bna A07.NAC13,Bna C07.NAC13）genes were overexpressed in the rapeseed materials Westar and Columbia wild-type Arabidopsis,and the CRISPR/Cas9 technology to mutate Bn VIK and Bn NAC13 in rapeseed Westar,and finally transgenic lines of rapeseed and Arabidopsis were obtained.By analyzing the seed oil content and fatty acid content of the transgenic materials,we initially verified that Bna A09.VIK not only affected the oil content of rape seeds,but also affected the fatty acid composition of oil.Bna A07.NAC13 and Bna C07.NAC13 could also affect the oil content and change the fatty acid composition of oil in rapeseed and Arabidopsis seeds.The main findings were as follows:The oil content of T₁generation rapeseeds overexpressing Bna A09.VIK was 7.19%-7.57%higher than that of WT,while by mutating Bna A09.VIK and Bna C08.VIK simultaneously,the oil content of rapeseeds was 4.37%-8.69%lower than that of WT.The C18:1 content and C18:0 content in T₁generation rapeseeds by overexpressing Bna A09.VIK were higher than WT,while the C18:2 content was lower than WT.By overexpressing Bna A07.NAC13 and its homologous gene Bna C07.NAC13 in rapeseed respectively,the oil content of T₁generation rapeseeds was 2.52%-6.51%higher than that of WT;after Bna A07.NAC13 and Bna C07.NAC13 genes were simultaneously mutated,the oil content of T₁generation transgenic.rapeseeds is 2.93%-6.37%lower than that of WT.In addition,by overexpressing Bna A07.NAC13 and Bna C07.NAC13 in Arabidopsis respectively,the oil content of transgenic Arabidopsis seeds was 1.74%-3.90%higher than that of WT.After mutating Bna A07.NAC13 and Bna C07.NAC13 simultaneously,the C16:0,C18:2 and C18:3 contents of T₁generation transgenic rapeseeds were higher than WT,while the C18:1 content was lower than WT.Compared with WT,the C18:1 and C18:2 contents of T₂generation Arabidopsis which overexpressing Bna A07.NAC13 increased,while the C18:3 content decreased.This study showed that VIK and transcription factor NAC13 could affect the oil content and fatty acid content of rapeseed The results showed that VIK and NAC13 may be genes regulating seed oil synthesis,and the resolution of VIK and NAC13 gene functions is beneficial to our further understanding of the regulatory mechanisms in seed oil synthesis,laying a theoretical foundation for further refining the molecular mechanisms of oil synthesis,as well as providing new genetic resources for breeding high-oil-content varieties.