[Objective] Genetic Engineering technology was used to regulate the expression of PttGA20-oxidase gene thus restrained plant height growth and internode elongation for cultivating dwarfed plant.[Method] Based on the R...[Objective] Genetic Engineering technology was used to regulate the expression of PttGA20-oxidase gene thus restrained plant height growth and internode elongation for cultivating dwarfed plant.[Method] Based on the RNAi principle,the gene specific sequences of PttGA20-oxidase in the antisense and sense orientations interrupted by a gene sequence from GUS were cloned into a binary vector pBI121.The selection marker gene npt Ⅱ was replaced with bar gene to RNAi plasmid.[Result] After undergone different endonuclease restrictions,the constructed constraint vector released different segments whose sizes were similar to that of target segment,which demonstrated that the RNAi plasmid of PttGA20-oxidase gene was successfully constructed.[Conclusion] The experiment provided a new way for culturing dwarfed plant.展开更多
Gibberellin （GA） 2-oxidase plays a key role in the GA catabolic pathway through 2β-hydroxylation.In the present study,we isolated a CaMV 35S-enhancer activation tagged mutant,H032.This mutant exhibited a dominant d...Gibberellin （GA） 2-oxidase plays a key role in the GA catabolic pathway through 2β-hydroxylation.In the present study,we isolated a CaMV 35S-enhancer activation tagged mutant,H032.This mutant exhibited a dominant dwarf and GA-deficient phenotype,with a final stature that was less than half of its wild-type counterpart.The endogenous bioactive GAs are markedly decreased in the H032 mutant,and application of bioactive GAs （GA3 or GA4） can reverse the dwarf phenotype.The integrated T-DNA was detected 12.8 kb upstream of the OsGA2ox6 in the H032 genome by TAIL-PCR.An increased level of OsGA2ox6 mRNA was detected at a high level in the H032 mutant,which might be due to the enhancer role of the CaMV 35S promoter.RNAi and ectopic expression analysis of OsGA2ox6 indicated that the dwarf trait and the decreased levels of bioactive GAs in the H032 mutant were a result of the up-regulation of the OsGA2ox6 gene.BLASTP analysis revealed that OsGA2ox6 belongs to the class III of GA 2-oxidases,which is a novel type of GA2ox that uses C20-GAs （GA12 and/or GA53） as the substrates.Interestingly,we found that a GA biosynthesis inhibitor,paclobutrazol,positively regulated the OsGA2ox6 gene.Unlike the over-expression of OsGA2ox1,which led to a high rate of seed abortion,the H032 mutant retained normal flowering and seed production.These results indicate that OsGA2ox6 mainly affects plant stature,and the dominant dwarf trait of the H032 mutant can be used as an efficient dwarf resource in rice breeding.展开更多
Senescence is the process of programmed degradation. The G2 line of pea exhibits apical senescence-delaying phenotype under short-day （SD） conditions, but the mechanism regulating the apical senescence is still larg...Senescence is the process of programmed degradation. The G2 line of pea exhibits apical senescence-delaying phenotype under short-day （SD） conditions, but the mechanism regulating the apical senescence is still largely unknown. Gibberellin （GA） was proved to be able to delay this apical senescence phenotype in G2 pea grown under long-day （LD） conditions. Here we show that the initiation of cell death signals in the terminal floral meristem was involved in the regulation of apical senescence in pea plants. SD signals prevented the formation of the cell death region in the apical mersitem. Moreover, GA3 treatment could effectively inhibit the occurrence of cell death-mediated apical senescence in LD-grown apical buds. Therefore, our data suggest that the prevention of apical senescence in SD-grown G2 pea through GA3 treatment may be largely responsible for the regulation of occurrence of the DNA fragmentation in apical meristem.展开更多
Alfalfa(Medicago sativa L.)is a nutritious forage crop with wide ecological adaptability.The molecular breeding of alfalfa is restricted by its heterozygous tetraploid genome and the difficult genetic manipulation pro...Alfalfa(Medicago sativa L.)is a nutritious forage crop with wide ecological adaptability.The molecular breeding of alfalfa is restricted by its heterozygous tetraploid genome and the difficult genetic manipulation process.Under time and resource constraints,we applied a more convenient approach.We investigated two MtGA3ox genes,MtGA3ox1 and MtGA3ox2,of Medicago truncatula,a diploid legume model species,finding that MtGA3ox1 plays a major role in GA-regulated plant architecture.Mutation of neither gene affected nitrogenase activity.These results suggest that MtGA3ox1 can be used in semidwarf and prostrate alfalfa breeding.Based on the M.truncatula MtGA3ox1 sequence,MsGA3ox1 was cloned from alfalfa,and two knockout targets were designed.An efficient CRISPR/Cas9-based genome editing protocol was used to generate msga3ox1 mutants in alfalfa.We obtained three lines that carried mutations in all four alleles in the T0 generation.Fifteen clonal plants were vegetatively propagated from each transgenic line using shoot cuttings.The plant height and internode length of msga3ox1 null mutants were significantly decreased.The number of total lateral branches,leaf/stem ratio and crude protein content of aerial plant parts of msga3ox1 mutants were significantly increased.Thus,we obtained semi-dwarf and prostrate alfalfa by gene editing.展开更多
The phytohormones gibberellic acid （GA） and abscisic acid （ABA） antagonistically control seed germina- tion. High levels of GA favor seed germination, whereas high levels of ABA hinder this process. The direct rel...The phytohormones gibberellic acid （GA） and abscisic acid （ABA） antagonistically control seed germina- tion. High levels of GA favor seed germination, whereas high levels of ABA hinder this process. The direct relationship between GA biosynthesis and seed germina- tion ability need further investigation. Here, we identified the ABA-insensitive gain-of-function mutant germination insensitive to ABA mutant 2 （gim2） by screening a population of XVE T-DNA-tagged mutant lines. Based on two loss-of-function gim2-ko mutant lines, the disruption of GIM2 function caused a delay in seed germination. By contrast, upregulation of GIM2 accelerated seed germina- tion, as observed in transgenic lines overexpressing GIM2 （OE）. We detected a reduction in endogenous bioactive GA levels and an increase in endogenous ABA levels in the gim2-ko mutants compared to wild type. Conversely, the OE lines had increased endogenous bioactive GA levels and decreased endogenous ABA levels. The expression levels of a set of GA- and]or ABA-related genes were altered in both the gim2-ko mutants and the OE lines. We confirmed that GIM2 has dioxygenase activity using an in vitro enzyme assay, observing that GIM2 can oxidize GA^2. Hence, our characterization of GIM2 demonstrates that it plays a role in seed germination by affecting the GA metabolic pathway in Arabidopsis.展开更多
文摘[Objective] Genetic Engineering technology was used to regulate the expression of PttGA20-oxidase gene thus restrained plant height growth and internode elongation for cultivating dwarfed plant.[Method] Based on the RNAi principle,the gene specific sequences of PttGA20-oxidase in the antisense and sense orientations interrupted by a gene sequence from GUS were cloned into a binary vector pBI121.The selection marker gene npt Ⅱ was replaced with bar gene to RNAi plasmid.[Result] After undergone different endonuclease restrictions,the constructed constraint vector released different segments whose sizes were similar to that of target segment,which demonstrated that the RNAi plasmid of PttGA20-oxidase gene was successfully constructed.[Conclusion] The experiment provided a new way for culturing dwarfed plant.
基金supported by grants from the Ministry of Sciences and Technology of China (No. 2005CB120805 and 2006AA10A101)the National Natural Science Foundation of China (No. 30621001 and 30871512)
文摘Gibberellin （GA） 2-oxidase plays a key role in the GA catabolic pathway through 2β-hydroxylation.In the present study,we isolated a CaMV 35S-enhancer activation tagged mutant,H032.This mutant exhibited a dominant dwarf and GA-deficient phenotype,with a final stature that was less than half of its wild-type counterpart.The endogenous bioactive GAs are markedly decreased in the H032 mutant,and application of bioactive GAs （GA3 or GA4） can reverse the dwarf phenotype.The integrated T-DNA was detected 12.8 kb upstream of the OsGA2ox6 in the H032 genome by TAIL-PCR.An increased level of OsGA2ox6 mRNA was detected at a high level in the H032 mutant,which might be due to the enhancer role of the CaMV 35S promoter.RNAi and ectopic expression analysis of OsGA2ox6 indicated that the dwarf trait and the decreased levels of bioactive GAs in the H032 mutant were a result of the up-regulation of the OsGA2ox6 gene.BLASTP analysis revealed that OsGA2ox6 belongs to the class III of GA 2-oxidases,which is a novel type of GA2ox that uses C20-GAs （GA12 and/or GA53） as the substrates.Interestingly,we found that a GA biosynthesis inhibitor,paclobutrazol,positively regulated the OsGA2ox6 gene.Unlike the over-expression of OsGA2ox1,which led to a high rate of seed abortion,the H032 mutant retained normal flowering and seed production.These results indicate that OsGA2ox6 mainly affects plant stature,and the dominant dwarf trait of the H032 mutant can be used as an efficient dwarf resource in rice breeding.
基金Supported by the Southeast University Foundation for Excellent Young Scholars(4023001013).
文摘Senescence is the process of programmed degradation. The G2 line of pea exhibits apical senescence-delaying phenotype under short-day （SD） conditions, but the mechanism regulating the apical senescence is still largely unknown. Gibberellin （GA） was proved to be able to delay this apical senescence phenotype in G2 pea grown under long-day （LD） conditions. Here we show that the initiation of cell death signals in the terminal floral meristem was involved in the regulation of apical senescence in pea plants. SD signals prevented the formation of the cell death region in the apical mersitem. Moreover, GA3 treatment could effectively inhibit the occurrence of cell death-mediated apical senescence in LD-grown apical buds. Therefore, our data suggest that the prevention of apical senescence in SD-grown G2 pea through GA3 treatment may be largely responsible for the regulation of occurrence of the DNA fragmentation in apical meristem.
基金supported by the Agricultural Variety Improvement Project of Shandong Province(2019LZGC010)the National Key Research and Development Program of China(2019YFD1002701).
文摘Alfalfa(Medicago sativa L.)is a nutritious forage crop with wide ecological adaptability.The molecular breeding of alfalfa is restricted by its heterozygous tetraploid genome and the difficult genetic manipulation process.Under time and resource constraints,we applied a more convenient approach.We investigated two MtGA3ox genes,MtGA3ox1 and MtGA3ox2,of Medicago truncatula,a diploid legume model species,finding that MtGA3ox1 plays a major role in GA-regulated plant architecture.Mutation of neither gene affected nitrogenase activity.These results suggest that MtGA3ox1 can be used in semidwarf and prostrate alfalfa breeding.Based on the M.truncatula MtGA3ox1 sequence,MsGA3ox1 was cloned from alfalfa,and two knockout targets were designed.An efficient CRISPR/Cas9-based genome editing protocol was used to generate msga3ox1 mutants in alfalfa.We obtained three lines that carried mutations in all four alleles in the T0 generation.Fifteen clonal plants were vegetatively propagated from each transgenic line using shoot cuttings.The plant height and internode length of msga3ox1 null mutants were significantly decreased.The number of total lateral branches,leaf/stem ratio and crude protein content of aerial plant parts of msga3ox1 mutants were significantly increased.Thus,we obtained semi-dwarf and prostrate alfalfa by gene editing.
基金the ‘Large-scale Instrument and Equipment Sharing Foundation of Wuhan University’ for supporting the use of the instruments in the College of Life Sciences in Wuhan Universitysupported by grants to Y.W. from Nation Natural Science Foundation of China (31270333 and 90817013)the Major State Basic Research Program from the Ministry of Science and Technology of China (2013CB126900)
文摘The phytohormones gibberellic acid （GA） and abscisic acid （ABA） antagonistically control seed germina- tion. High levels of GA favor seed germination, whereas high levels of ABA hinder this process. The direct relationship between GA biosynthesis and seed germina- tion ability need further investigation. Here, we identified the ABA-insensitive gain-of-function mutant germination insensitive to ABA mutant 2 （gim2） by screening a population of XVE T-DNA-tagged mutant lines. Based on two loss-of-function gim2-ko mutant lines, the disruption of GIM2 function caused a delay in seed germination. By contrast, upregulation of GIM2 accelerated seed germina- tion, as observed in transgenic lines overexpressing GIM2 （OE）. We detected a reduction in endogenous bioactive GA levels and an increase in endogenous ABA levels in the gim2-ko mutants compared to wild type. Conversely, the OE lines had increased endogenous bioactive GA levels and decreased endogenous ABA levels. The expression levels of a set of GA- and]or ABA-related genes were altered in both the gim2-ko mutants and the OE lines. We confirmed that GIM2 has dioxygenase activity using an in vitro enzyme assay, observing that GIM2 can oxidize GA^2. Hence, our characterization of GIM2 demonstrates that it plays a role in seed germination by affecting the GA metabolic pathway in Arabidopsis.
