Construction And Transformation Of DGLA Expression Vector In Cotton

Polyunsaturated fatty acids(PUFAs) refers to straight chain fatty acids containing 18-22 carbon atomsand two or more double bonds. Polyunsaturated fatty acids play important role in human nutrition and health. Recently, polyunsaturated fatty acids come mainly from deep sea fish. But due to overfishing and pollution of ocean, fish oil output and quality can’t meet the needs of human beings(Domergue et al.,2005; Drexler et al,2003). So it is necessary to find asafe and economic way to solve this problem. In recent years, along with research advances in polyunsaturated fatty acid metabolism and gene cloning of related key enzymes,it is considered a possible way to obtain PUFAs through genetic modified higher plants,especially oil crops. Cotton is not only an important economic crop but also an important oil crop, with relatively high seed linoleic acid content( about 49.5%-49.5%)(Dong et al.,2010),which can be used in genetic engineering to product PUFAs.In this work, expressing vector for DGLA synthesis was constructed and transformed into Hanwu 198 and shannongmian 9. EDA and DGLA were detected in T3 transgenic seeds. The main results were as follows:1. An auxiliary vector PAUX(α) by replace the 35 S promoter with a cotton seed specific promoter α.2. On the basis of the auxiliary vector PAUX(α), using the theory of constructing multiple gene expression box, a multigene expression vector p Cam BAR-(α-△9Elo)-(α-△8Des) was built, which could be used to synthesize DGLA from LA through EDA.3.The expression vector p Cam BAR-(α-△9Elo)-(α-△8Des) was transformed into shannongmian9 and Hanwu 198 through agrobacterium mediated method and ovary injection transformation method. And 18 positive plants were identified through PCR of the herbicide resistance gene BAR, the seed specific promoter α and the two key genes △8Des and △9Elo in the vector respectively.4.Fatty acid components analysis of four samples indicated that transgenic cotton seeds contain OA, LA, EDA and DGLA while DGLA and EDA were not detected in the control seeds. In the transgenic cotton seeds, the EDA and DGLA content is 0.28% and 0.22%respectively, with decreased LA and OA contents. From LA to EDA, the conversion efficiency is 51.02%, from EDA to DGLA is 44%.This work provides useful base for further study in synthesis of PUFAs in cotton seeds.