Development Of An Genetic Transfomation And Genetic Transformation Of PWY86 And TLW10 Expression Vector In Brassica NaPus L.

Rape is one of the world's four oil-bearing crops, which occupies an important position in the agricultural production and industrial purposes. Currently, Brassica napus 1 is the rape of three types of the highest grain yield and the main production cultivation rapeseed varieties. Some traditional hybrid breeding methods cannot have satisfied foster resistant to diseases and insect pests, resistance to fight/weeds and improve quality. The development of transgenic technology has offered us a new way. Currently import of heterologous gene method is mainly agrobacterium of variations. In the transformation of brassica napus 1. experiments, agrobacterium explant strains, the concentration of the choice, infection, medium hormone screening of formula and antibiotics will influence the transgenic plants of pressure obtained. The experiment based on these factors do comparative experiments, choose the suitable condition and concentration, optimize the regeneration of agrobacterium transformation system. In addition to the TLW10 carrier and the construction of the PWY86 respectively by agrobacterium catalysis brassica napus l.'zhongyou 821 transformation in the transgenic plants', and the molecular appraisal, so as to prove the feasibility of this regeneration conversion system.1.TLW10 transformation vector construction:The Frt-GFP-Tnos fragment inserted into the Hindâ…¢site with the binary vector pBI121. It is successful to constructed TLW 10 transformation vector By PCR and restriction enzyme digestion test.2. Regeneration system: EHA105 by agrobacterium strains infect has higher efficiency; Zhongyou 821 brassica napus 1.'with'the child with cotyledons for receptors, and MiaoLing petiole when for higher conversion 4D; Infection of agrobacterium diluted into OD value equal to 0.1, AS the concentration of 15mg/L, for 4-dimethylaniline Dl mg/L AgNO3 concentration for 5 mg/L,6-A and B, the concentration of NAA would 3mg/L and combination of 0.1 mg/L; The grass with screening ammonium phosphate pressure for 7 mg/L herbicide, kanamycin as 13mg/L.3. PWY86 and TLW10 carrier of genetic transformation and molecular detection:"Zhongyou 821 in brassica napus 1. for receptor materials, import" carrier, will be transformed TLW10 PWY86 and get positive plants molecule detection, experimental results are as follows:(1) Test the transgenic plants which import PWY86 vector by PCR and PCR-southern blot hybridization assay,456bp specific bands were got in PCR experiments by primers designed to be specific bands Bar gene. This is consistent with the idea of the expected initial successful transfer of materials that PWY86 carrier in vivo. The specific bands FLP sequence were obtained of PCR-southern blot hybridization experiments.All of the results show that resistant plants were obtained by import PWY86 vector to Brassica napus'zhongyou 821'. (2) Test the transgenic plants which import TLW10 vector by PCR experiments and GUS staining.672bp specific bands were got in PCR experiments by primers designed to be specific bands NPTII gene, This is consistent with the idea of the expected initial successful transfer of materials that TLW10 carrier invivo The result of GUS staining is that GUS gene expression in the roots, stems, leaves and flower buds of transgenic plants. The experiment Show that resistant plants were obtained by import PWY86 vector to Brassica napus'zhongyou 821'.