In order to learn the expression pattern of GRP1 8(glycine rich protein) gene promoter in transgenic plants and to explore its potential application in plant genetic engineering for vascular specific expression of...In order to learn the expression pattern of GRP1 8(glycine rich protein) gene promoter in transgenic plants and to explore its potential application in plant genetic engineering for vascular specific expression of interested genes, GRP 1 8 promoter was amplified by PCR from Chinese bean genomic DNA. The intermediate vector was constructed by inserting vascular specific expression promoter of GRP 1 8 gene in vector pBI 101. The regenerated tobacco plants obtained were analyzed by PCR to select the putative transgenic plants. The histochemical localization of GUS( β D glucosidase) activity indicates that as for that of GRP 1 8 promoter we can confer the vascular specific expression of GUS gene.展开更多
There was a bi-directional promoter between gene 38 kd phosphorylated protein (pp38) gene; 1.8-kb mRNA transcript gene family in the genome of Marek's disease virus (MDV). In this study, enhanced green fluorescenc...There was a bi-directional promoter between gene 38 kd phosphorylated protein (pp38) gene; 1.8-kb mRNA transcript gene family in the genome of Marek's disease virus (MDV). In this study, enhanced green fluorescence protein (EGFP) reporter plamids, pP(pp38)-EGFP; pP(1.8-kb)-EGFP, were constructed under this bi-directional promoter in two directions. The two plasmids were transfected into uninfected chicken embryo fibroblast (CEF), MDV clone rMd5 infected CEF (rMd5-CEF); pp38-deleted derivative rMd5Δpp38 infected CEF (rMd5Δpp38-CEF) respectively. Transfection analysis showed that EGFP was only expressed in rMd5-CEF,; no EGFP could be detected in uninfected CEF or rMd5Δpp38-CEF, implying that pp38 was a factor influencing the activity of the promoter. The pp38-expressing recombinant plasmid pcDNA-pp38 was constructed to co-transfect CEF or rMd5Δpp38-CEF with pP(pp38)-EGFP or pP(1.8-kb)-EGFP. In this case, EGFP could be detected only in rMd5Δpp38-CEF but still not in uninfected CEF, implying that pp38 needs other protein(s) to work together for the complete trans-acting activity. Another MDV gene, 24 kd phosphorylated protein pp24 gene was cloned into pcDNA3.1 as a pp24-expressing recombinant plasmid pcDNA-pp24. When uninfected CEF was co-transfected with pcDNA-pp38, pcDNA-pp24; EGFP expressing plasmids pP(pp38)-EGFP or pP(1.8-kb)-EGFP, the EGFP could be detected. These results indicated that pp38; pp24 could enhance the activity of the promoter when they worked together. DNA mobility shift assay showed that pp38 would bind to the bi-directional promoter with the co-existing of pp24, although neither of them alone influenced mobility of the promoter DNA. All the above suggested that MDV pp38 could transactivate the bi-directional promoter when combined with pp24. The results also indicated that the activity of the promoter in the direction of 1.8-kb mRNA was significantly stronger than that of pp38 direction.展开更多
基金Supported by the National Natural Science Foundation of China(No.39730 35 0 ) .
文摘In order to learn the expression pattern of GRP1 8(glycine rich protein) gene promoter in transgenic plants and to explore its potential application in plant genetic engineering for vascular specific expression of interested genes, GRP 1 8 promoter was amplified by PCR from Chinese bean genomic DNA. The intermediate vector was constructed by inserting vascular specific expression promoter of GRP 1 8 gene in vector pBI 101. The regenerated tobacco plants obtained were analyzed by PCR to select the putative transgenic plants. The histochemical localization of GUS( β D glucosidase) activity indicates that as for that of GRP 1 8 promoter we can confer the vascular specific expression of GUS gene.
基金supported by the National Natural Science Foundation of China(Grant Nos.30300450&30070544).
文摘There was a bi-directional promoter between gene 38 kd phosphorylated protein (pp38) gene; 1.8-kb mRNA transcript gene family in the genome of Marek's disease virus (MDV). In this study, enhanced green fluorescence protein (EGFP) reporter plamids, pP(pp38)-EGFP; pP(1.8-kb)-EGFP, were constructed under this bi-directional promoter in two directions. The two plasmids were transfected into uninfected chicken embryo fibroblast (CEF), MDV clone rMd5 infected CEF (rMd5-CEF); pp38-deleted derivative rMd5Δpp38 infected CEF (rMd5Δpp38-CEF) respectively. Transfection analysis showed that EGFP was only expressed in rMd5-CEF,; no EGFP could be detected in uninfected CEF or rMd5Δpp38-CEF, implying that pp38 was a factor influencing the activity of the promoter. The pp38-expressing recombinant plasmid pcDNA-pp38 was constructed to co-transfect CEF or rMd5Δpp38-CEF with pP(pp38)-EGFP or pP(1.8-kb)-EGFP. In this case, EGFP could be detected only in rMd5Δpp38-CEF but still not in uninfected CEF, implying that pp38 needs other protein(s) to work together for the complete trans-acting activity. Another MDV gene, 24 kd phosphorylated protein pp24 gene was cloned into pcDNA3.1 as a pp24-expressing recombinant plasmid pcDNA-pp24. When uninfected CEF was co-transfected with pcDNA-pp38, pcDNA-pp24; EGFP expressing plasmids pP(pp38)-EGFP or pP(1.8-kb)-EGFP, the EGFP could be detected. These results indicated that pp38; pp24 could enhance the activity of the promoter when they worked together. DNA mobility shift assay showed that pp38 would bind to the bi-directional promoter with the co-existing of pp24, although neither of them alone influenced mobility of the promoter DNA. All the above suggested that MDV pp38 could transactivate the bi-directional promoter when combined with pp24. The results also indicated that the activity of the promoter in the direction of 1.8-kb mRNA was significantly stronger than that of pp38 direction.
