Manipulation Of Bn Vet4Encodingγ-Tocopherol Methytransferase In Oilseed Rape (Brassica Napusl)

Rapeseed that includes the species of Brassica campestris, Brassica juncea and Brassica napus, belongs to genus Brassica in the family of Cruciferous. It is one of the most important oil crops all over the world, it is also the one of most mainly economic crops in our country. In recent years, with the expansion of applications and increase of market demands, the acreage of rapeseed is still increasing. Brassica napus has the largest acreage due to the advantages as high oil content, and high tolerance to cold and disease and the high yield.The transgenic technologies and their applications have being become hot issues with the rapid development of molecular biotechnology, especially in the transgenic breeding of rapeseed. Some regeneration systems had been established using explants such as cotyledons, hypocotyls, stems, protoplasts and microspore. And the transgenic methods including Agrobacterium tumefaciens, gene gun, PEG, electroporation, laser microbeam, microinjection, pollen and floral-dip. Thus, the rapeseed breeding by transformation was improved quickly, for example, seed quality, resistance to disease, pest and abiotic stress, sterile breeding, etc.Rapeseed is abundant in Vitamin E, which is an essential lipid soluble antioxidant synthesized by plants and some cyanobacteria including tocopherols and tocotrienols. Tocopherols play a crucial role in the process of human growth and development, which include decreasing the risk of cardiovascular disease and cancer, improving the function of the immune system and slowing of the progression of human degenerative conditions. tocopherols also modulate the signal transduction pathways and transcription. a-tocopherol also helps in keeping on the redox capacity in chloroplasts, maintaining thylakoid structure and function during plant development, and enhancing tolenance to abitic stress in plant.α-tocopherol has the highest Vitamin E activity and the lowest a-tocopherol content in plant seeds. Natural a-tocopherol has twice the bioavailability of the synthetic isomer and lower cost. BnVET4was cloned from Brassica napus Zheshuang72, the expressed product of which is y-tocopherol methyl-transferase that catalyzes the synthesis of a-tocopherol. Then, BnVET4was constructed to the overexpression vector pCAMBIA1300and transferred into B. napus for increasing the content of a-tocopherol.Based on the understanding of a-tocopherol synthetical pathway, we manipulated the BnVET4gene in rapeseed (B. napus), and achieved the following results.1. The cloning of BnVET4gene. The primers were synthesized through retrieval of database NCBI. BnVET4gene was cloned by RT-PCR from B. napus leaves, and the the length of coding sequence is1044bp. The similarity is99%by the sequence alignment in genebank.2. The construction of binary overexpression vector. pCAMBIA1300and BnVET4was emzymed with Xba I and Sac I, and the constructed vector transferred into Agrobacterium of LBA4404.3. Establishment of transformation system in B. napus transformation system of B. napus was established through reseaching the seedling age of explants, the selectable concentration of hygromycin, the infected concentration of Agrobacterium, the induction of calli, the regeneration of shoots and initiation of roots, cotyledons of4-5day and and hypocotyls of6-7day has the highest effecency of regeneration. Cotyledons and hypocotyls were cultured in calli induced medium containing1.5mg/L2,4-D and1.5mg/L2,4-D+0.2mg/L6-BA respectively. Then, the explants were moved into shoot initiation medium containing4mg/L6-BA,0.02mg/L NAA,2.5mg/L AgNO3and10mg/L hyg for shoot regeneration and selection. After shoot outgrowth, the shoot was transferred to root initiation medium containing0.1mg/L NAA or IBA.4. Analysis of transgenic plants. The selectable plants were analyzed by PCR, and12plants were transgenic plants. BnVET4gene was intergrated into genomic DNA of B. napus by Southern blot. The expression level of BnVET4gene was increased significantly in some plants through the analysis of RT-PCR and Real time-PCR.5. Measure of α-tocopherol. The contents of Vitamin E were measured in the transgenic plant leaves with the highest expression level of BnVET4by HPLC. the content of α-tocopherol decreased as the total contents of VE reduced. The transgenic seeds are unmatured because of the effect of climate. The contents of VE will be measured when the matured seeds are harvested.