Overexpression Of GmDof4 And GmDof11 Influences The Seed Fatty Acids Composition Of Brassica Napus

Rapeseed(Brassica napus L.)is one of the most widely cultivated oil crops.Breeding of varieties with high quality and yield of rapeseed oil is one of important direction.The DOF(DNA binding with One Finger)transcription factor family involves in vital processes in higher plants.The GmDof4 and GmDofll transcription factors were isolated from soybeans(Glycine max),and involved in the biosynthesis and accumulation of seed oil in soybeans,Arabidopsis thaliana,and might participate in the responses to biotic and abiotic stresses in plants.In this study,using B.napus cultivar "Yangyou 6" as transgenic receptor,GmDof4 and GmDofll overexpression lines were generated by using the Agrobacterium tumefaciens-mediated transgenic technique.The influence of the GmDof4 and GmDofll genes on the seed quality characters was investigated.The major results are summarized as follows:(1)We explored the genetic transformation of B.napus by using cotyledon explants.Firstly,we demonstrated that the callus differentiation rate and seedling regeneration rate of "Yangyou 6"cotyledon explants was as high as 94.9%and 71.4%respectively.Secondly,we demonstrated that the optimum concentration of selective agent of Kan(kanamycin sulfate)in seedling medium was 40 mg/L.Finally,genetic transformation of cotyledon was performed with binary expression vector pCambia-1303,which contains a GUS-GFP fusion gene as report gene,and the accumulation of GUS in cotyledons 1 to 4 weeks after infection was tested,we found that,the expression of GUS was detected in cotyledons and the cells near the incision 1 week after infection;and 4 weeks after infection,most of the cells were positive transformants in healthy callus.In addition,6 of 1135S:GUS transgenic plants were detected positive by GUS staining,with a positive rate of 54.5%.(2)Genetic transformation of pBi438/GmDof4 and pBin438/GmDof11 vectors was carried out.PCR analysis showed that 9 of the regenerated seedling were pBin438/GmDof4 transformants,and 8 of them were pBin438/GmDof11 transformants.Analysis of the 17 PCR positive lines by semi-quantitative PCR revealed the GmDof4 gene expressed in 6 of the transformants,and GmDofll gene expressed in 3 of them.(3)There were no significant changes in the content of lipid and glucosides in T2 transgenic seeds of GmDof4 and GmDofll.One of GmDof4 line showed lower seed protein content than wild type.Fatty acids composition of seeds was analyzed by near infra-red reflectance spectroscopy(NIRS)and Gas Chromatography-Mass Spectrometer(GC)simultaneously,and we found the oleic acid content was increased,while the linoleic acid and linolenic acid content were decreased in both GmDof4 and GmDofll T2 transgenic seeds than those in wild type seeds.(4)The expression of genes associated with the synthesis of protein and FA was analyzed by qPCR.Results showed that the expression of accD gene could be activated by GmDOf4 and GmDof11,but LACS was not regulated;the expression of FAB2 gene and FAD8 gene was up-regulated by GmDof4;GmDofll could down-regulate the expression of FAD2 and FAD6 genes;while the expression of the CRA1 gene might be activated by GmDof4.(5)By yeast-one hybrid,we verified whether the different expressed genes we detected were regulated by GmDof directly.It was found that GmDof4 protein interacts with the cis-elements on the promoters of CRA1,FAB2 genes;GmDofll protein interacts with the promoters of CRA1,FAD2,FAD6 genes.