Studies On Incorporating Pta And Sb401 Genes Into Rapeseed (Brassica Napus L.) By Agrobacterium Tumefaciens

Rape is one of the most important oil crops with strong adaptability, extensive application, high economy worthiness and great developmental potential. There are more than 60% of people having rapeseed oil in our country, the cultivated area and gross production of rapeseed oil both occupy the first class in the world, but the yield and quality are far lower than some European countries such as Canada. The prevalence of disease and insect pest is one of the factors, which restrict the yield in rape production. Furthermore, the content of Erucic acid and Glucosinolates is too high in general rapeseed cake, which affects the quality of rape oil and the exploitation of rapeseed cakes extensively. So it is important to extend and popularize double low rape varieties, to improve their disease and insect pest resistance and promote the protein nutrition quality of rapeseeds.Gene technology, which has broken the limitation of traditional breeding, can transfer one or more genes into receptor crop genome simultaneously to improve one or more properties of crops. This has provided a new method for crop breeding and improving.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 rape transgenic system yet, many domestic and overseas reports on gene transformation in rape take a special variety as receptor, the result does not have universality. We transferred pinellia ternata lectin (pta) gene which resists Homopteran pests (such as rape aphides ) and a lysine-rich protein (sb401) gene into double-low rapeseed cultivars by Agrobacterium-mediated genetic transformation. The two genes were carried in the same T-DNA region of plasmid pDB13PS. The major results are as follows:1. Eight double-low general rapeseed cultivars (Brassica napus 1.) such as zhong Shuang 9 etc were selected to be receptors, an efficiency regeneration system of different rapeseed hypocotyls were preliminary established by exploring eight- hormone combinations; basic medium; asepsis seedling; hypocotyls cutting length. Selecting MS+6-BA1mg/L+2.4-D0.5 mg/L as pre-culture medium and MS+6-BA 1 ~4mg/L + NAA 0.05~0.4mg/L as seedling medium at last. Four varieties selected as transformation materials, which behaved higher regeneration frequency (40.67~69.67%).2. On the basis of explants regeneration system, we established their transformation system .The result are as following: Hypocotyls from 5~6 days old seeding were cut into0.5cm in length and pre-cultured on MS containing 1.0mg/L6-BA and 0.5mg/L2.4-D for two days, After quickly dipped into the Agrobacterium suspension of OD600 0.3(the same volume re-suspension after centrifugal machine collecting bacterium) for 30~60sec, the hypocotyls were co-cultured on the same medium (added 100n m/LAS) for 30~42h after absorbed superfluous bacterium. When the co-culture was over, the hypocotyls were washed in liquid MS containing 200mg/Lcb and 400mg/Lcef to kill overgrown Agrobacterium. Then the explants were dried properly and transferred to the delay selection medium to doff bacterium for 6~7d,At last riddling culture till the hyg-resistant bud appeared, the sprouts were cut and transferred inhomogeneous rootage mediums by different genotype. Experiment proved that the transgenic system selected materials expediently, transformed gene simple relatively, the effect distinctness.3. Pta gene and sb401 gene which were designed in the same T-DNA region of plasmid were introduced into zhongshuang9.7.6 and chuanyoull by the agrobacterium-mediated transformation system and 79 To generation anti-hyg plants were obtained.4. 26 To generation anti-hyg plants were amplified 832bp size bands by htp primer .5 htp positive plants selected randomly were amplified 417bp> 81 Obp size bands by sb401, pta primers. Total DNA of four transgenic plants identified by PCR and one non-transgenic plant was extracted to perform southern blotting assay. DNA hybridization results showed that the four transgenic plants have 1 to 2 hybridization bands, but the non-transgenic plant has no hybridization signals. So results of PCR and Southern-blotting confirmed that foreign genes had been integrated into the genome of receptors.5. Observing and recording the figure of young plants, florescence and resistance when the transgenic plants were challenged with rape aphids. All this work will establish the foundation for the next genetic analysis.