| Modifications of the fatty acid composition in rapeseed to meet the consumer demand have been very successful utilizing traditional breeding methods. By using sexual hybridization and other techniques such as X-irradiation or ethyl methanesufonate (EMS) treatment, it has been possible to modify the proportions of fatty acids that exist within the Brassica species. This has allowed the production of rapeseed lines with high palmitic acid content, high or low content of linoleic acid , high oleic acid and also rapeseed without erucic acid .Nevertheless, limitations in the rapeseed gene pool have caused plant breeders to search for alternative sources of other commercially interesting fatty acids.The development of the new gene cloning and genetic transformation technologies for genetic modification in the past two decades has opened the possibility of engineering a wide range of new oil qualites in rapaseed. The most important goal is to alter the content and composition of oil in the seeds. The paper reports the part of high oleic acid gene engineering breeding high oleic acid in rapaseed. The purpose of this research is to achieve a new variety of B. napus containing more oleate and fewer polyunsaturated lipid by co- suppression of the FAD2 gene of rapeseed.1. Cloning and sequence analysis of nap in gene promoter from Brassica napus.A pair of primers were designed according to the DNA sequence reported in GenBank. The upstream regulatory region of a seed-specific gene was isolated from the genomic DNA of B. napus "Xiangyou 15"by PCR amplification. The PCR products were cloned into pGEN-T Easy vector and sequenced. The results indicated the cloned fragment contained 1147 nucleotides, and shared a homology of 99.6% and 99.1% with the reported nap A and napB, respectively. The putative TATA box showed at the position-29. Further comparisons of the new promoter to the 5' -upstream region of the previously published napin genes reveal that the certain sequence motives are evolutionary conserved and may be implicated in seed-specific expression,, such as G-box, ABA-responsive element, RY repeat, AT sequence, (CA)n element.To investigate the function of the napin gene promoter, the promoter was fused tothe β -glucuronidase gene to form pBI121.N. Via a particle bombardment, pBI121.N was introduced into the seeds of Brassica napus which were treated with abscisic acid for 3d before bombardment, also into roots, stems, and leafs of oilseed. Transient expression was measured histochemically as spot number 24h after bombardment. The results indicated that the expression of napin gene promoter was embryo specific.The novel napin gene promoter sequence was registered in GenBank,with the Accession No. being AF420598.2. Cloning of FAD2 gene cDNA fragment from Brassica napus and construction of seed-specific antisense FAD2 gene expression vector.FAD2 is one of the most important enzymes for production of polyunsaturated fatty acids in plants. A pair of primers were designed according to the sequence described by Marillia(1999). The FAD2 gene cDNA fragment was isolated from B. napus "Xiangyou 15" by RT-PCR. The products were cloned into pGEM-T vector to form pTF. The Not I fragment from pTF was inserted into pBluescriptll SK+ cut with the same enzyme to form pSKF and sequenced. The amplified fragment showed to be 654bp in size, which was consistent with data reported. A 0.65kb fragment, which obtained from pSKF digested by BamH I /Sac I,was inserted into BamH I/Sac I site of pBI121.N. Then ,the antisense expression vector pBI121.NF was constructed finally. Transformation of Agrobacterium strains LBA4404 with pBI121.NF was done and vertified by plaque-PCR.3. The genetic transformation and obtaining the transgenic B. napusBy optimizing the parameters affecting Agrobacterium strain infection, we have established a high-efficiency Agrobacterium-mediated genetic transformation system. The transformation efficiency is higher than 10%.Meanwhile, we studieded the genetic transformation of particle bo...
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