Expression Analysis Of Gmubi Promoter From Soybean And Patatin2Promoter From Potato In Mustard

The plant genetic engineering technique is an important assistant approach to improve characters of crops, which can accelerate the process of crop breeding. In the area of the plant genetic engineering, except the study isolation and clo-ning of genes, the research of promoter for exogenous gene expression regulation is also an important subject. The promoter can control the regulation of downs-tream gene at the transcription level, as to generate new plant varieties with en-hance ability to external environment stress, improved stress resistance and higher nutritional value.In the research of plant genetic engineering, it is necessary to make target gene expression efficiently in plants. At present, the constitutive promoter of cauliflower Mosaic virus CaMV35S promoter and CsVMV promoter isolated fro m Cassava vein mosaic virus are used generally to generate transgenic plants. H owever, multiple genes might are introduce into plant to produce new character, which require more promoters to control the gene expression. Therefore, more effective constitutive promoters have to be found to achieve the artificial control of complex characters in transgenic plants. In addition,achieving exogenous gene expression in certain parts or organs in plant depend on tissue or organ specific promoters. These promoters can not only make the expression products accumul ates in the certain tissue or organs, but also can avoid an adverse effect on plant growth and development by preventing excessive material and energy con-suming in cells.In view of the above problem, this study a constitutive promoter Gmubi fro m soybean and a stem/tuber-specific promoter Patatin2from potato were clonedThe Gmubi and Patatin2promoters were fused with Gus gene respectively to generate plant expression vectors pCABarGmubiGus and pCABarPatatin2Gus.The pC ABar GmubiGus and pCABarPatatin2Gus expression vectors were introduced into "Yu-Feng"mustard by Agrobacterium-mediated method.The aim is to investi gate the expression pattern of Gmubi and Patatin2promoters in heterogenous p1ants, which will contribute to gene function analysis and development of transge nic plants in future. The main results are as follows:1. The cloning of Gmubi promoter and functional analysis vectors constructioBased on the promoter sequence (EU310508.1) of soybean polyubiquitin (ubi) gene published NCBI, A pair specific primers were used to amplify the0.9kb fragment from soybean DNA, the fragment was cloned to pBluescript ve-ctor and sequenced, sequence analysis showed the fragment was917bp,7differ ent nucleotides and99%sequence homology compared with the EU310508.1se quence.917bp Gmubi promoter was digested by HindⅢ and NcoI enzymes to s ubstitute the35S promoter in PCABarGus vector to generate PCABarGmubiGus expression vector2. The cloning of Patatin2promoter and functional analysis vectors constructio n construction of promoterBased on the promoter sequence (X60398.1) of potato Patatin Class Ⅱ ge ne published NCBI, A pair specific primers were used to amplify the0.7kb fragment from potato DNA, the fragment was cloned to pBluescript vector and sequenced, sequence analysis showed the fragment was669bp,4different nucle otides and99%sequencehomology compared with the X60398.1sequence.669b p Patatin2promoter was digested by HindⅢ and Ncol enzymes to substitute th e35S promoter in PCA-BarGus vector to generate PCABarGmubiGus expressio n vector3. Generation of transgenic mustard plants by Agrobacterium-mediated transform ationThe pCABarGmubiGus、pCABarPatatin2Gus and Positive control pCABar35S Gus expression vectors was transformed into"Yu-Feng"mustard by Agrobacterium-mediated hypocotyl transformation, after several rounds selection using herbicide phosphinothricin (PPT), mustard transgenic plants were produced.4. Molecular detection of the transgenic mustard plantsThe DNA was extracted from resistant mustard plants for PCR amplificatio n by corresponding primers. There were6plants that the Gmubi promoter wer e detected from10pCABarGmubiGus transgenic plants, and5plants were det ected containing the Patatin2promoter from10pC ABarPatatin2Gus transgenic plants.5. The GUS histochemical analysis of the pCABarGmubiGus and pCABarPatati n2Gus transgenic plantsThe GUS histochemical analysis results indicated there were intensive GUS expression in leaves, stems, flowers, seeds, roots of the pCABar35SGus transgen ic control, as expected, no GUS expression were detected in leaves, stems, flow ers, seeds, roots of wild-type mustard plants. All pCABarGmubiGus transgenic showed strong expression in leaves, stems, flowers, seeds, roots, and the express ion quantity was comparable with the pCABar35SGus transgenic control. Howev er, no GUS expression were detected in leaves, stems, flowers, seeds, roots of all pCABarPatatin2Gus transgenic plants, the results demonstrated the stem/tu ber-specific promoter Patatin2from potato can not drive the exogenous gene ex pression in mustard.