Transformation Research On RNAi Suppressed Expression Of AhFAD2 Gene Of Arachis Hypogaea L.

Peanut (Arachis hypogaea L.) is the fourth largest oilseed crop in the world, which originated from South America. Peanut oilseeds consists mainly of palmitic, oleic and linoleic acids. The content, composition and condition of the polyunsaturated fatty acids in oilseeds are the most important determinant factors of oil quality. Plant oils rich in oleate are considered superior products compared to oils rich in polyunsaturated fatty acids. Oils with higher oleic acid and lower polyunsaturated fatty acids are good for health , and satisfy desirability commercially and nutritionally.Δ¹²- oleic acid desaturase which places the second double bond in the fatty acid chain, is the key enzyme which desaturates oleic acid to linoleic acid. The ?12- oleic acid desaturase gene contains a putative intron, the coding region at the 3′end, and an open reading frame (ORF) of 1140 bp encoding 379 amino acids. There exist two homeologous genes(AhFAD2A, AhFAD2B) forΔ¹²-oleic acid desaturase in peanut, which derived from the original diploid progenitor species. A mutation in ahFAD2A and significant reduction in levels of the ahFAD2B transcript together cause the high oleate phenotype in peanut varieties, and that one of them expressed a functional enzyme appears to be sufficient for the normal oleate phenotype. The expression of ?12- oleic acid desaturase gene also are affected by temperature. All in all, the two homeologous genes, FAD2A and FAD2B, are the key genes which detemine the concentration of oleic acid in peanut, although other genes or control mechanisms may also play an important role.Δ¹²- oleic acid desaturase has become one of the major researching enzyme in the area of microbial engineering , oilseeds crop gene engineering and plant lipid modification.We linked the inverted repeat sequence of partialΔ¹² fatty acid desaturase gene AhFAD2 with CaMV35S promoter and inserted them into binary plant vector pCAMBIA1300, and linked the inverted repeat sequence of partial AhFAD2 gene with strong seed specific soybean Lectin promoter and inserted them into binary plant vector pCAMBIA1302. The resulting plasmid, named pCSF and pCLF, respectively, were mobilized to Agrobacterium tumefaciens strain LBA4404 and EHA105, used for plant transformation. We used hypocotyls and cotyledons of matured peanut seeds of Luhua No.14 and Fenghua No.1 as recipients, the inverted repeat sequence of partialΔ¹² fatty acid desaturase gene AhFAD2 was introduced into Arachis hypogaea L via Agrobacterium tumefaciens infection. Because there are only a little reportes about the screening concentration of hygromicin B in dicotyledonous plants, especially in Arachis hypogaea L, furthermore, there are big sensitivity differences between different species and different part of Arachis hypogaea L, we designed a gradient screening tests before transformation. Finally, we determined the screening concentration of hygromicin B for hypocotyls is 25mg/L and for cotyledons is 10mg/L. In addition, to differnent species of plants, there are big transformation efficiency differences between different strains of Agrobacterium tumefaciens. It was reported that Agrobacterium tumefaciens strain LBA4404 and EHA105 both had high transformation efficiency to Arachis hypogaea L. In order to determine which strain had higher transformation efficiency to Arachis hypogaea L, we made a contrast experiment between the two strains. The result showed that compared with LBA4404, EHA105 had higher transformation efficiency to Arachis hypogaea L. Transformants were selected for their ability to grow on medium containing hygromicin B, more than 400 lines that were all tolerant to hygromicin B were selected, 180 of them were driven by CaMV35S promoter, and 230 of them were driven by strong seed specific soybean Lectin promoter. The transformants used for further molecular determination. PCR showed that most of the transformants had strong positive signals, and no signal was shown in wild type plants. This indicated that the inverted repeat structure had introduced into Arachis hypogaea L successfully. PCR Southern blotting analysis also indicated that the structure had been integrated into all of the transgenic plants'genomes.As control, we cultured the regeneration plants at the same time which haven't been transformated. Compared with regeneration plants, some of the transformants showed abnormal form, such as leaves dark green and wrinkle, the base of plant swollen, the space of branch shortened, root abnormal, and so on. Although the form of others were normal, most of them showed slow-growing and the branches of them were weak. Tried many methods to improve, but the effcets were not noticeable. Fatty acids matebolism is one of the basic vital processes, and fatty acids are imprtant component part of membranous lipids, so the phenomenons above may result from the supressed expression ofΔ12 fatty acid desaturase gene AhFAD2. And also there are reportes showed that hygromicin B has many disadvantages for plant develepoment. Thus, the reasons which result in the phenomenons above, still need further research.