Study On Genetic Improvement Of Seed Oil Content In Peanut (Arachis Hypogaea L.) By Overexpression Of AtLEC1 And AtMYB118 Gene

Using Fenghua No.1 (FH1), one of a cultivar of peanut (Arachis hypogaea L.), as a receptor, overexpression of AtLECl and AtMYB118 gene driven by different length of seed-specific NapinA promoter in peanut.Through PCR and the second-generation of high-throughput sequencing technologies, two of transgenic peanut lines including p230:AtLECl constructs, named 2011030801 and 2011031001, and one event transformed the frame of pB2F: AtLEC1 (with full-length-NapinA promoter), were obtained; as well as two of p270: AtMYB118 transgenic lines, denominated 2011040603 and 2011040201, were also achieved.The expression level of the foreign gene AtLECl or AtMYB118 in the transgenic peanut seeds was detected in transcription level and in protein level by QRT-PCR and Western Blot analysis respectively. Chosing three periods (15d,30d, and 60d after pegging) of seeds during seed development, the expression pattern of several genes that related to fatty acid biosynthesis, lipid accumulation and sugar metabolism, were analyzed by QRT-PCR. The results showed that the expression level of the genes detected are relatively low in the seeds of the transgenic AtLECl peanuts at 15d after soil penetration, have small difference with the FH1; at th development stage of 60d seeds, the expression level of most genes in transgenic peanut compared with those in control, increased significantly, including AhaccA Ⅱ, AhBccpl,AhBc4, AhKASⅡ, AhSAD, AhDGAT1-1, AhDGAT2a, AhOle2. The AhSuSy mRNA that related sugar metabolism was decreased significantly in 30d of transgenic seeds, and further reduced at 60 d after soil penetration. The expression levels of AhSAD and AhSuSy in the transgenic peanut line haboring AtMYB118, compared with those in FH1, were up-regulated in seeds at 15d and 30d after soil penetration. The transcipts of some genes that related to fatty acid biosynthesis, such as AhBccpl, AhBc4 and AhKASⅡ, were significantly up-regulated respectively at 30d And 60d, and while at 60d after soil penetration, exception for the down-regulation of three genes AhOle3, AhSAD and AhSuSy, the expression levels of the other 8 genes were enhanced significantly, compared with those in FH1 seeds.Using the methods of near infrared reflectance spectra (NIR), soxhlet extraction and GC-MS, the oil content and the content and composition of fatty acids in the transgenic peanut seeds and control seeds, were investigated. It was found that the seed oil content of all transgenic peanut lines detected were significantly improved, the highest content is over 20.2% than that of FH1.In peanut seeds, there are four kinds of major fatty acids like as oleic acid, linoleic acid,stearic acid,palmitic acid, and among them, the contents of oleic acid, stearic acid and palmitic acid in almost all transgenic lines are increased, and the content of linoleic acid was slightly increased in sedds of AtMYB118 transgenetic peanut.In short, overexpression of AtLECl and AtMYB118 in peanut seeds could increase the expression level of a lot genes that related to fatty acid synthesis and oil accumulation, and significantly improve the seed oil content of transgenetic line.