Transformation Of LRP Gene In Brassica Napus Mediated By Agrobacterium

Rapeseed is one of the most important oil crops with strong adaptability, extensive application, highly economical value and greatly developmental potentiality. There are more than 60% of people taking rapeseed oil in our country, Both the cultivated area and gross production of rapeseed oil rank one in the world, but the yield is lower and oil quality is not good compared with that of some European and American countries. So it is important to improve their resistance to diseases, pest and to promote the protein nutrition quality of rapeseed.In the aspect of quality improvement of rapeseed, one of the effective ways is the application of genetic engineering in the change of chemical composition of the seed. And one of crucial technologies is to regulate the expression of key genes.The gene regulation of the high plant mainly happens in the transcription level and is affected by the interaction of various cis-acting and trans-acting factors. The gene promoter is an important cis-acting factor in plant, a DNA sequence which is identified and combined by RNA polymerase and thus initiating the gene transcription.Agrobacterium-mediated transformation is widely adopted by plant breeders because of its many advantages, such as simple operation, high transformation efficiency, low copy number of introduced genes and simple integration pattern. But the transformation via A. tumefaciences is a complex course with interaction between bacteria and callus, every factor that is relevant with the activation bacteria and the state of callus tissue may affect transformation. So far there are many questions in rapeseed transgenic system yet, many reports on gene transformation in rapeseed take a special variety as experimental material, and the result does not have universality. Here we transferred LRP ( lysine rich protein) gene into double-low rapeseed cultivars by Agrobacterium-mediated genetic transformation.This study begins with establishing highly effective regeneration systems, constructing a vector including seed-specific promoter of napin and a LRP gene,then introducing this vector into Brassica napus via Agrobacterium. The results are as follows:1. Cloning of the promoter of napin gene in B. napus. A pair of primers was designed according to 5'non-encoding region of the napin sequence reported in GenBank. Using genomic DNA of B. napus as the template, the amplified products by PCR were cloned into pMD18-T Vector.2. Constructing an expressing vector by enzyme digestion, linking, transformation and a series of molecular cloning methods.3. Optimizing the regeneration system by adjusting the days of explants, the ratio of hormones, and the cultivating condition.4. Transforming exogenous gene by Agrobacterium, a lot of regeneration plants were obtained, PCR and PCR-Southern identification of LRP gene proved to be transgenic plants and showed the foreign genes have been integrated into B. napus. We've got 34 positive transgenic plants, 19 of them were grown in greenhouse to maturity, and 15 of them were cultured in vitro for rooting.