Seed-specific Expression IhpRNA Vector Construction And Genetic Transformation Of Rapeseed Pepc Gene

Rapeseed (Brassica napus L.) is one of the most important oilseed crop plantedwidely in the world and as a major source of edible vegetable oil and protein forhuman. Vegetable oils are main production of rapeseed. They are not only the mainsources of necessary fatty acid for human being and animals, but also the industrialmaterials of plastics, textile dyes, printing ink, leather, detergents and leather andlubricants. Rape cultivated area of our country exceeds 100 million must (mainly inYangtse Valley), accounts for 1/3 of cultivated area the world,and has been providedexceeding 60% edible oil giving our country people. However, compared with othercountries, most of rapeseed cultivars in China contain lower amounts of oil content:international rapeseed average oil content approximately 42%, our country rapeseedaverage oil content is 35% only. Therefore,it has significant meaning to improve oilcontent of rapeseed.Phosphoenolpyruvatecarboxylase(PEPC) showclosely correlation with vegetableoil content. It controls indirectly the direction of the flow of phosphoe-nopyruvatewhich is the common substrate of protein and lipid biosynthesis and determines theratio of the protein/lipid content of plant seed. We make use of the RNAi technologyto restrain PEPC, make solubility sugar be so as to flow into Metabolic Pathway onFatty Acid, and gain Genetically-modified rape with high oil content. The mainresults were as followings:1.The napin promoter (seed-specific promoter) and pepc gene was isolated fromB.napus "Xiangyou15"by PCR methods.The sequenced results indicated the clondfragment shared a homology of 100% and 93%.According to the design principle ofihpRNA expression vector, the products was cloned into pBI121 expression vector.Then, the seed-specific ihpRNA expression vector pBI121NP~+IP~- was constructedfinnally by Digestion of pBI121NP~+IP~- with restriction Endonuclease.Transfor-mation of Agrobacterium strains LBA4404 with pBI121NP~+IP~- was carried out andidentified by PCR.The pepc sense and antisense gene sequence was registered in GenBank, with theAccession NO.being EF539209 and EF565860.2.The cotyledons node was dipped into the Agrobacterium strainsLBA4404 withpBI121NP~+IP~-. By optimizing the parameters affecting the regeneration of rapeseedcotyledons node and transformation mediated by Agrobactenium tumefaciens,we haveestablished a high-efficiency Agrobacterium-mediated co-transformation system, andThe regenerated seedlings with Kanamycin resistant was obtained.PCR analysis oftransgenic plants showed that the napin and pepc gene integrated into genome ofB.napus "Xiangyou 15".The successful experiment has paved a way for breedinghigh oil content rapeseed in the near future.