The phosphomannose isomerase （PMI） gene from Saccharomyces cerevisiae acted as selectable marker and mannose acted as selective agent for the production of transgenic plants of rice （Oryza sativa L.） via Agrobacte...The phosphomannose isomerase （PMI） gene from Saccharomyces cerevisiae acted as selectable marker and mannose acted as selective agent for the production of transgenic plants of rice （Oryza sativa L.） via Agrobacterium-mediated transformation. The concentration of mannose during the selection was stepwise increased, 5 g L-1 mannose combined with 15 g L-1 sucrose and 500 mg L-1 cefotaxime was used in the initial selection stage, then the concentration of mannose was increased to 11 g L-1, the highest transformation rate was 20.0%. The integration of PMI gene was confirmed by PCR, and the result of RT-PCR assay proved that the intron of PMI gene can be excised correctly during RNA splicing. 13- Glucuronidase （GUS） activity analysis confirmed the expression of GUS gene. All those means the PMI gene from yeast can be used as a selectable marker in rice transformation.展开更多
Establishing a transgenic plant largely relies on a selectable marker gene that can confer antibiotic or herbicide resistance to plant cells.The existence of such selectable marker genes in genetically modified foods ...Establishing a transgenic plant largely relies on a selectable marker gene that can confer antibiotic or herbicide resistance to plant cells.The existence of such selectable marker genes in genetically modified foods has long been criticized.Plant cells generally exhibit too low an activity of phosphomannose isomerase(PMI)to grow with mannose as a sole carbon source.In this study,we characterized PMI from the green microalga Chlorococcum sp.and assessed its feasibility as a selectable marker for plant biotechnology.Chlorococcum sp.PMI(ChlPMI)was shown to be closely related to higher plants but more distant to bacterial counterparts.Overexpression of ChlPMI in tomato induced callus and shoot formation in media containing mannose(6 g/L)and had an average transformation rate of 3.9%.Based on this transformation system,a polycistronic gene cluster containing crtB,HpBHY,CrBKT and SlLCYB(BBBB)was co-expressed in a different tomato cultivar.Six putative transformants were achieved with a transformation rate of 1.4%,which produced significant amounts of astaxanthin due to the expression of the BBBB genes.Taken together,these findings indicate that we have established an additional tool for plant biotechnology that may be suitable for genetically modifying foods safely.展开更多
Phosphomannose isomerase （PMI） encoding gene manA is a desirable selective marker in transgenic research. Under- standing of its expression patterns in transgenic plant and establishing highly sensitive detection me...Phosphomannose isomerase （PMI） encoding gene manA is a desirable selective marker in transgenic research. Under- standing of its expression patterns in transgenic plant and establishing highly sensitive detection method based on immunoassay have great impacts on the application of PMI. In this study, PMI-specific monoclonal antibodies were generated using recombinant protein as immunogen, and could be used in Western blot to detect as little as 0.5 ng His-tagged PMI protein or rice expressed PMI protein in sample accounted for 0.4% of single rice grain （about 0.08 mg）. PMI protein driven by CaMV-35S promoter was detected in dozens of tested tissues, including root, stem, leaf, panicle, and seed at all developmental stages during rice growing, and PMI protein accounted for about 0.036% of total protein in the leaves at seedling stage. The established method potentially can be used to monitor PMI protein in rice grains.展开更多
基金the Genetically Modified Organisms Breeding Major Projects, China (2011ZX08003-003)
文摘The phosphomannose isomerase （PMI） gene from Saccharomyces cerevisiae acted as selectable marker and mannose acted as selective agent for the production of transgenic plants of rice （Oryza sativa L.） via Agrobacterium-mediated transformation. The concentration of mannose during the selection was stepwise increased, 5 g L-1 mannose combined with 15 g L-1 sucrose and 500 mg L-1 cefotaxime was used in the initial selection stage, then the concentration of mannose was increased to 11 g L-1, the highest transformation rate was 20.0%. The integration of PMI gene was confirmed by PCR, and the result of RT-PCR assay proved that the intron of PMI gene can be excised correctly during RNA splicing. 13- Glucuronidase （GUS） activity analysis confirmed the expression of GUS gene. All those means the PMI gene from yeast can be used as a selectable marker in rice transformation.
基金supported by a grant from Yunnan high talents program(Y33D331),Yunnan Province,China.
文摘Establishing a transgenic plant largely relies on a selectable marker gene that can confer antibiotic or herbicide resistance to plant cells.The existence of such selectable marker genes in genetically modified foods has long been criticized.Plant cells generally exhibit too low an activity of phosphomannose isomerase(PMI)to grow with mannose as a sole carbon source.In this study,we characterized PMI from the green microalga Chlorococcum sp.and assessed its feasibility as a selectable marker for plant biotechnology.Chlorococcum sp.PMI(ChlPMI)was shown to be closely related to higher plants but more distant to bacterial counterparts.Overexpression of ChlPMI in tomato induced callus and shoot formation in media containing mannose(6 g/L)and had an average transformation rate of 3.9%.Based on this transformation system,a polycistronic gene cluster containing crtB,HpBHY,CrBKT and SlLCYB(BBBB)was co-expressed in a different tomato cultivar.Six putative transformants were achieved with a transformation rate of 1.4%,which produced significant amounts of astaxanthin due to the expression of the BBBB genes.Taken together,these findings indicate that we have established an additional tool for plant biotechnology that may be suitable for genetically modifying foods safely.
基金supported in part by the Natural Science Foundation of Beijing, China (5121001)the Cultivate New Varieties of Genetically Modified Organisms Technology Major Projects, the Ministry of Science and Technology of China (2009ZX08012-006B)
文摘Phosphomannose isomerase （PMI） encoding gene manA is a desirable selective marker in transgenic research. Under- standing of its expression patterns in transgenic plant and establishing highly sensitive detection method based on immunoassay have great impacts on the application of PMI. In this study, PMI-specific monoclonal antibodies were generated using recombinant protein as immunogen, and could be used in Western blot to detect as little as 0.5 ng His-tagged PMI protein or rice expressed PMI protein in sample accounted for 0.4% of single rice grain （about 0.08 mg）. PMI protein driven by CaMV-35S promoter was detected in dozens of tested tissues, including root, stem, leaf, panicle, and seed at all developmental stages during rice growing, and PMI protein accounted for about 0.036% of total protein in the leaves at seedling stage. The established method potentially can be used to monitor PMI protein in rice grains.
