The ＇double T-DNA＇ binary vector p13HSR which harbored two independent T-DNAs, containing hygromycin phosphotransferase gene （hpf） in one T-DNA region and three target genes （hLF, SB401, RZ10） in another T-DNA r...The ＇double T-DNA＇ binary vector p13HSR which harbored two independent T-DNAs, containing hygromycin phosphotransferase gene （hpf） in one T-DNA region and three target genes （hLF, SB401, RZ10） in another T-DNA region, was used to generate selectable marker-free transgenic rice by Agrobacterium-mediated transformation. The regenerated plants with both the three target genes and the selectable marker gene hpt were selected for anther culture. RT-PCR analysis indicated that target genes were inserted in rice genomic DNA and successfully transcribed. It took only one year to obtain double haploid selectable marker-free transgenic plants containing the three target genes with co-transformation followed by anther culture technique, and the efficiency was 12.2%. It was also noted that one or two target genes derived from the binary vector were lost in some transgenic rice plants.展开更多
CrylAb gene was transformed into four rice varieties, Zhejing 22, Zhejing 27, Jiahua 1 and Xiushui 63 mediated by Agrobacterium-mixture co-transformation. Rice genotype had an important effect on callus induction and ...CrylAb gene was transformed into four rice varieties, Zhejing 22, Zhejing 27, Jiahua 1 and Xiushui 63 mediated by Agrobacterium-mixture co-transformation. Rice genotype had an important effect on callus induction and transformation efficiency. Different mixtures of Agrobacterium strains （EHA105 and EHA101） contained Hpt and CrylAb genes resulted in different frequencies of resistant calli. There was no correlation between the frequency of transformants with the ratio of the Agrobacterium strain mixture contained Hpt and CrylAb genes. A total of 509 transgenic plants were obtained from the four rice varieties, and 272 T2 progenies were analyzed for CrylAb and Hpt genes. PCR analysis revealed that 412 regenerated plants were Hpt positive （80.94%）, 62 plants were also CrylAb co-transformants （15.05% in total frequency）, and 42 plants among the 272 T2 progenies were CrylAb positive but Hpt negative. This suggests that marker-free transgenic plants could be produced by co-transformation mediated by mixed Agrobacterium strains with the selectable marker gene and target gene Southern blot analysis of five independent marker-free T2 transgenic lines co-transformed from Zhejing 22 showed that CrylAb gene had been inserted into rice genome with a single copy. The transgenic plants showed significantly stronger resistance to lepidopteron than the non-transgenic plants under no application of insecticides against lepidopteron.展开更多
Abiotic stresses such as drought, salinity, and low temperature cause–losses in rice production worldwide. The emergence of transgenic technology has enabled improvements in the drought resistance of rice plants and ...Abiotic stresses such as drought, salinity, and low temperature cause–losses in rice production worldwide. The emergence of transgenic technology has enabled improvements in the drought resistance of rice plants and helped avert crop damage due to drought stress.Selectable marker genes conferring resistance to antibiotics or herbicides have been widely used to identify genetically modified plants. However, the use of such markers has limited the public acceptance of genetically modified organisms. Marker-free materials (i.e., those containing a single foreign gene) may be more easily accepted by the public and more likely to find common use. In the present study, we created marker-free drought-tolerant transgenic rice plants using particle bombardment. Overall, 842 T_0plants overexpressing the rice ascorbate peroxidase-coding gene OsAPX2 were generated. Eight independentmarker-free lines were identified from T_1 seedlings using the polymerase chain reaction.The molecular characteristics of these lines were examined, including the expression level,copy number, and flanking sequences of OsAPX2, in the T_2 progeny. A simulated drought test using polyethylene glycol and a drought-tolerance test of seedlings confirmed that the marker-free lines carrying OsAPX2 showed significantly improved drought tolerance in seedlings. In the field, the yield of the wild-type plant decreased by 60% under drought conditions compared with normal conditions. However, the transgenic line showed a yield loss of approximately 26%. The results demonstrated that marker-free transgenic lines significantly improved grain yield under drought-stressed conditions.展开更多
Antimony(Sb)is a toxic and carcinogenic element that often enters soil in the form of antimony trioxide(Sb_(2)O_(3))and coexists with manganese(Mn)in weakly alkaline conditions.Mn oxides such as birnessite have been f...Antimony(Sb)is a toxic and carcinogenic element that often enters soil in the form of antimony trioxide(Sb_(2)O_(3))and coexists with manganese(Mn)in weakly alkaline conditions.Mn oxides such as birnessite have been found to promote the oxidative dissolution of Sb_(2)O_(3),but few researches concerned the co-transformations of Sb_(2)O_(3) and Mn(II)in environment.This study investigated themutual effect of abiotic oxidation of Mn(II)and the coupled oxidative dissolution of Sb_(2)O_(3).The influencing factors,such as Mn(II)concentrations,pH and oxygen were also discussed.Furthermore,their co-transformed mechanism was also explored based on the analysis of Mn(II)oxidation products with or without Sb_(2)O_(3) using XRD,SEM and XPS.The results showed that the oxidative dissolution of Sb_(2)O_(3) was enhanced under higher pH and higher Mn(II)loadings.With a lower Mn(II)concentration such as 0.01 mmol/L Mn(II)at pH 9.0,the improved dissolution of Sb_(2)O_(3) was attributed to the generation of dissolved intermediate Mn(III)species with strong oxidation capacity.However,under higher Mn(II)concentrations,both amorphous Mn(III)oxides and intermediate Mn(III)species were responsible for promoting the oxidative dissolution of Sb_(2)O_(3).Most released Sb(∼72%)was immobilized by Mn oxides and Sb(V)was dominant in the adsorbed and dissolved total Sb.Meanwhile,the presence of Sb_(2)O_(3) not only inhibited the removal of Mn(II)by reducing Mn(III)to Mn(II)but also affected the final products of Mn oxides.For example,amorphous Mn oxides were formed instead of crystalline Mn(III)oxides,such as MnOOH.Furthermore,rhodochrosite(MnCO_(3))was formed with the high Mn(II)/Sb_(2)O_(3) ratio,but without being observed in the low Mn(II)/Sb_(2)O_(3) ratio.The results of study could help provide more understanding about the fate of Sb in the environment and the redox transformation of Mn.展开更多
Using multi-color fluorescence in situ hybridization (FISH), we localized transferred barnase-psl and pHctinG DNA sequences onto chromosomes of two trans-genie rice plants, named Q12 and Q13, both of which were produc...Using multi-color fluorescence in situ hybridization (FISH), we localized transferred barnase-psl and pHctinG DNA sequences onto chromosomes of two trans-genie rice plants, named Q12 and Q13, both of which were produced by micro-projectile bombardment. In both Q12 and Q13, each detected cell showed 2-3 signal spots on their chromosomes respectively. The signals of both barnase-psl and pHctinG were mostly detected in the adjacent chromosomal sites in which their signals were overlapped and could be recognized by the signal color on the metaphase chromosomes. Fiber FISH further demonstrated that the multiple copies in each of the two DNA sequences distributed adjacently on the DNA fiber in Q13. Combined with the results of Southern hybridization, the possible integration patterns in transgenic rice co-transformed by micro-projectile bombardment have been discussed.展开更多
Gene encoding sulphur amino acid-rich protein (HNP) and rol genes were transferred into Medicago sativa L (alfalfa) mediated by Agrobacterium tumafeciens. Regeneration of trans-genie plants was induced successfully fr...Gene encoding sulphur amino acid-rich protein (HNP) and rol genes were transferred into Medicago sativa L (alfalfa) mediated by Agrobacterium tumafeciens. Regeneration of trans-genie plants was induced successfully from hairy root tissue of cotyledon in alfalfa. Cotyledon tissues were an ideally transformed recipient. There was a negative correlation between age of hairy roots and embryogenesis frequency in alfalfa. Production of co-transformed plants with greater yield and super quality was important for development of new alfalfa varieties.展开更多
For the temporally and spatially regulated expression of the barnase gene in plant, two kinds of plasmids with ere gene and its directly repeat recognition sites lox from bacteriophage P1 were constructed and co-trans...For the temporally and spatially regulated expression of the barnase gene in plant, two kinds of plasmids with ere gene and its directly repeat recognition sites lox from bacteriophage P1 were constructed and co-transformed into tobacco by agrobacterium mediated procedure. The transgenic plants were conformed by PCR analysis. The blocking fragment between the two lox directly repeat sites was excised by Cre protein in the transgenic plant genome. Cloning and seguencing the DNA fragment from the co-transformed plant DNA showed that the precise DNA excision occurred in transgenic tobacco genome directed by Cre//ox site-specif ic recombination .展开更多
文摘The ＇double T-DNA＇ binary vector p13HSR which harbored two independent T-DNAs, containing hygromycin phosphotransferase gene （hpf） in one T-DNA region and three target genes （hLF, SB401, RZ10） in another T-DNA region, was used to generate selectable marker-free transgenic rice by Agrobacterium-mediated transformation. The regenerated plants with both the three target genes and the selectable marker gene hpt were selected for anther culture. RT-PCR analysis indicated that target genes were inserted in rice genomic DNA and successfully transcribed. It took only one year to obtain double haploid selectable marker-free transgenic plants containing the three target genes with co-transformation followed by anther culture technique, and the efficiency was 12.2%. It was also noted that one or two target genes derived from the binary vector were lost in some transgenic rice plants.
基金supported by the Zhejiang Provincial Natural Science Foundation of China (Grant No.Z305650 and No.Y3080361)the Science and Technology Department of Zhejiang Province,China (Grant No.2004C12020)+1 种基金the National Key Technology Research & Development Program,China (Grant No.2006BAD01A01-5 and No.2008ZX08001-001)the 151 Foundation for the Talents of Zhejiang Province,China
文摘CrylAb gene was transformed into four rice varieties, Zhejing 22, Zhejing 27, Jiahua 1 and Xiushui 63 mediated by Agrobacterium-mixture co-transformation. Rice genotype had an important effect on callus induction and transformation efficiency. Different mixtures of Agrobacterium strains （EHA105 and EHA101） contained Hpt and CrylAb genes resulted in different frequencies of resistant calli. There was no correlation between the frequency of transformants with the ratio of the Agrobacterium strain mixture contained Hpt and CrylAb genes. A total of 509 transgenic plants were obtained from the four rice varieties, and 272 T2 progenies were analyzed for CrylAb and Hpt genes. PCR analysis revealed that 412 regenerated plants were Hpt positive （80.94%）, 62 plants were also CrylAb co-transformants （15.05% in total frequency）, and 42 plants among the 272 T2 progenies were CrylAb positive but Hpt negative. This suggests that marker-free transgenic plants could be produced by co-transformation mediated by mixed Agrobacterium strains with the selectable marker gene and target gene Southern blot analysis of five independent marker-free T2 transgenic lines co-transformed from Zhejing 22 showed that CrylAb gene had been inserted into rice genome with a single copy. The transgenic plants showed significantly stronger resistance to lepidopteron than the non-transgenic plants under no application of insecticides against lepidopteron.
基金supported by the National Major Project for Developing New GM Crops (2016ZX08001-003)
文摘Abiotic stresses such as drought, salinity, and low temperature cause–losses in rice production worldwide. The emergence of transgenic technology has enabled improvements in the drought resistance of rice plants and helped avert crop damage due to drought stress.Selectable marker genes conferring resistance to antibiotics or herbicides have been widely used to identify genetically modified plants. However, the use of such markers has limited the public acceptance of genetically modified organisms. Marker-free materials (i.e., those containing a single foreign gene) may be more easily accepted by the public and more likely to find common use. In the present study, we created marker-free drought-tolerant transgenic rice plants using particle bombardment. Overall, 842 T_0plants overexpressing the rice ascorbate peroxidase-coding gene OsAPX2 were generated. Eight independentmarker-free lines were identified from T_1 seedlings using the polymerase chain reaction.The molecular characteristics of these lines were examined, including the expression level,copy number, and flanking sequences of OsAPX2, in the T_2 progeny. A simulated drought test using polyethylene glycol and a drought-tolerance test of seedlings confirmed that the marker-free lines carrying OsAPX2 showed significantly improved drought tolerance in seedlings. In the field, the yield of the wild-type plant decreased by 60% under drought conditions compared with normal conditions. However, the transgenic line showed a yield loss of approximately 26%. The results demonstrated that marker-free transgenic lines significantly improved grain yield under drought-stressed conditions.
基金This work was supported by the National Natural Science Foundation of China(Nos.42077184,41772251 and 41521001)the National Key Research and Development Program(No.2018YFC1801700).
文摘Antimony(Sb)is a toxic and carcinogenic element that often enters soil in the form of antimony trioxide(Sb_(2)O_(3))and coexists with manganese(Mn)in weakly alkaline conditions.Mn oxides such as birnessite have been found to promote the oxidative dissolution of Sb_(2)O_(3),but few researches concerned the co-transformations of Sb_(2)O_(3) and Mn(II)in environment.This study investigated themutual effect of abiotic oxidation of Mn(II)and the coupled oxidative dissolution of Sb_(2)O_(3).The influencing factors,such as Mn(II)concentrations,pH and oxygen were also discussed.Furthermore,their co-transformed mechanism was also explored based on the analysis of Mn(II)oxidation products with or without Sb_(2)O_(3) using XRD,SEM and XPS.The results showed that the oxidative dissolution of Sb_(2)O_(3) was enhanced under higher pH and higher Mn(II)loadings.With a lower Mn(II)concentration such as 0.01 mmol/L Mn(II)at pH 9.0,the improved dissolution of Sb_(2)O_(3) was attributed to the generation of dissolved intermediate Mn(III)species with strong oxidation capacity.However,under higher Mn(II)concentrations,both amorphous Mn(III)oxides and intermediate Mn(III)species were responsible for promoting the oxidative dissolution of Sb_(2)O_(3).Most released Sb(∼72%)was immobilized by Mn oxides and Sb(V)was dominant in the adsorbed and dissolved total Sb.Meanwhile,the presence of Sb_(2)O_(3) not only inhibited the removal of Mn(II)by reducing Mn(III)to Mn(II)but also affected the final products of Mn oxides.For example,amorphous Mn oxides were formed instead of crystalline Mn(III)oxides,such as MnOOH.Furthermore,rhodochrosite(MnCO_(3))was formed with the high Mn(II)/Sb_(2)O_(3) ratio,but without being observed in the low Mn(II)/Sb_(2)O_(3) ratio.The results of study could help provide more understanding about the fate of Sb in the environment and the redox transformation of Mn.
基金This work was supported by the National Natural Science Foundation of China (Grant No. 39900083) the Research Fund for the Doctoral Program of Higher Education (Grant No. 207980112).
文摘Using multi-color fluorescence in situ hybridization (FISH), we localized transferred barnase-psl and pHctinG DNA sequences onto chromosomes of two trans-genie rice plants, named Q12 and Q13, both of which were produced by micro-projectile bombardment. In both Q12 and Q13, each detected cell showed 2-3 signal spots on their chromosomes respectively. The signals of both barnase-psl and pHctinG were mostly detected in the adjacent chromosomal sites in which their signals were overlapped and could be recognized by the signal color on the metaphase chromosomes. Fiber FISH further demonstrated that the multiple copies in each of the two DNA sequences distributed adjacently on the DNA fiber in Q13. Combined with the results of Southern hybridization, the possible integration patterns in transgenic rice co-transformed by micro-projectile bombardment have been discussed.
文摘Gene encoding sulphur amino acid-rich protein (HNP) and rol genes were transferred into Medicago sativa L (alfalfa) mediated by Agrobacterium tumafeciens. Regeneration of trans-genie plants was induced successfully from hairy root tissue of cotyledon in alfalfa. Cotyledon tissues were an ideally transformed recipient. There was a negative correlation between age of hairy roots and embryogenesis frequency in alfalfa. Production of co-transformed plants with greater yield and super quality was important for development of new alfalfa varieties.
文摘For the temporally and spatially regulated expression of the barnase gene in plant, two kinds of plasmids with ere gene and its directly repeat recognition sites lox from bacteriophage P1 were constructed and co-transformed into tobacco by agrobacterium mediated procedure. The transgenic plants were conformed by PCR analysis. The blocking fragment between the two lox directly repeat sites was excised by Cre protein in the transgenic plant genome. Cloning and seguencing the DNA fragment from the co-transformed plant DNA showed that the precise DNA excision occurred in transgenic tobacco genome directed by Cre//ox site-specif ic recombination .
