Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacillus thuringiensis（Bt） cry gene and epsps（5-enolpyruvylshikimat...Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacillus thuringiensis（Bt） cry gene and epsps（5-enolpyruvylshikimate-3-phosphate synthase） gene are two important genes for culturing pest-resistant and glyphosate-tolerant crops. We used linker peptide LP4/2A to connect the Bt cry1 Ah gene with the 2m G2-epsps gene and combined the wide-used man A gene as a selective marker to construct one coordinated expression vector called p2 EPUHLAGN. The expression vector was transferred into maize by Agrobacterium tumefaciens-mediated transformation, and 60 plants were obtained, 40% of which were positive transformants. Molecular detection demonstrated that the two genes in the fusion vector were expressed simultaneously and spliced correctly in translation processing; meanwhile bioassay detection proved the transgenic maize had preferable pest resistance and glyphosate tolerance. Therefore, linker peptide LP4/2A provided a simple and reliable strategy for producing gene stacking in maize and the result showed that the fusion gene transformation system of LP4/2A was feasible in monocot plants.展开更多
Herbicide tolerance has been the dominant trait introduced during the global commercialization of genetically modified(GM)crops.Herbicide-tolerant crops,especially glyphosate-resistant crops,offer great advantages for...Herbicide tolerance has been the dominant trait introduced during the global commercialization of genetically modified(GM)crops.Herbicide-tolerant crops,especially glyphosate-resistant crops,offer great advantages for weed management;however,despite these benefits,glyphosate-resistant maize(Zea mays L.)has not yet been commercially deployed in China.To develop a new bio-breeding resource for glyphosate-resistant maize,we introduced a codon-optimized glyphosate N-acetyltransferase gene,gat,and the enolpyruvyl-shikimate-3-phosphate synthase gene,gr79-epsps,into the maize variety B104.We selected a genetically stable high glyphosate resistance(GR)transgenic event,designated GG2,from the transgenic maize population through screening with high doses of glyphosate.A molecular analysis demonstrated that single copy of gat and gr79-epsps were integrated into the maize genome,and these two genes were stably transcribed and translated.Field trials showed that the transgenic event GG2 could tolerate 9000 g acid equivalent(a.e.)glyphosate per ha with no effect on phenotype or yield.A gas chromatography-mass spectrometry(GC–MS)analysis revealed that,shortly after glyphosate application,the glyphosate(PMG)and aminomethylphosphonic acid(AMPA)residues in GG2 leaves decreased by more than 90%compared to their levels in HGK60 transgenic plants,which only harbored the epsps gene.Additionally,PMG and its metabolic residues(AMPA and N-acetyl-PMG)were not detected in the silage or seeds of GG2,even when far more than the recommended agricultural dose of glyphosate was applied.The co-expression of gat and gr79-epsps,therefore,confers GG2 with high GR and a low risk of herbicide residue accumulation,making this germplasm a valuable GR event in herbicide-tolerant maize breeding.展开更多
基金support of the National Natural Science Foundation of China(30771383)the Genetically Modified Organisms Breeding Major Projects, China(2013ZX08003-001)the National Basic Research Program of China (2009CB118902)
文摘Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacillus thuringiensis（Bt） cry gene and epsps（5-enolpyruvylshikimate-3-phosphate synthase） gene are two important genes for culturing pest-resistant and glyphosate-tolerant crops. We used linker peptide LP4/2A to connect the Bt cry1 Ah gene with the 2m G2-epsps gene and combined the wide-used man A gene as a selective marker to construct one coordinated expression vector called p2 EPUHLAGN. The expression vector was transferred into maize by Agrobacterium tumefaciens-mediated transformation, and 60 plants were obtained, 40% of which were positive transformants. Molecular detection demonstrated that the two genes in the fusion vector were expressed simultaneously and spliced correctly in translation processing; meanwhile bioassay detection proved the transgenic maize had preferable pest resistance and glyphosate tolerance. Therefore, linker peptide LP4/2A provided a simple and reliable strategy for producing gene stacking in maize and the result showed that the fusion gene transformation system of LP4/2A was feasible in monocot plants.
基金supported by the National Transgenic Major Program of China(2016ZX08003001),。
文摘Herbicide tolerance has been the dominant trait introduced during the global commercialization of genetically modified(GM)crops.Herbicide-tolerant crops,especially glyphosate-resistant crops,offer great advantages for weed management;however,despite these benefits,glyphosate-resistant maize(Zea mays L.)has not yet been commercially deployed in China.To develop a new bio-breeding resource for glyphosate-resistant maize,we introduced a codon-optimized glyphosate N-acetyltransferase gene,gat,and the enolpyruvyl-shikimate-3-phosphate synthase gene,gr79-epsps,into the maize variety B104.We selected a genetically stable high glyphosate resistance(GR)transgenic event,designated GG2,from the transgenic maize population through screening with high doses of glyphosate.A molecular analysis demonstrated that single copy of gat and gr79-epsps were integrated into the maize genome,and these two genes were stably transcribed and translated.Field trials showed that the transgenic event GG2 could tolerate 9000 g acid equivalent(a.e.)glyphosate per ha with no effect on phenotype or yield.A gas chromatography-mass spectrometry(GC–MS)analysis revealed that,shortly after glyphosate application,the glyphosate(PMG)and aminomethylphosphonic acid(AMPA)residues in GG2 leaves decreased by more than 90%compared to their levels in HGK60 transgenic plants,which only harbored the epsps gene.Additionally,PMG and its metabolic residues(AMPA and N-acetyl-PMG)were not detected in the silage or seeds of GG2,even when far more than the recommended agricultural dose of glyphosate was applied.The co-expression of gat and gr79-epsps,therefore,confers GG2 with high GR and a low risk of herbicide residue accumulation,making this germplasm a valuable GR event in herbicide-tolerant maize breeding.
