Improved chilling tolerance is important for maize production. Previous efforts in transgenics and marker-assisted breeding have not achieved practical results. In this study, the antifreeze protein(AnAFP) from the su...Improved chilling tolerance is important for maize production. Previous efforts in transgenics and marker-assisted breeding have not achieved practical results. In this study, the antifreeze protein(AnAFP) from the super-xerophyte Ammopiptanthus nanus was aligned to KnS-type dehydrins.Phosphorylation in vitro and subcellular localization showed that AnAFP was phosphorylated by maize casein kinase II and translocated from nucleus to cytoplasm under chilling stress. AnAFP also increased lactate dehydrogenase activity. A parent line of commercial maize hybrids was transformed with the AnAFP gene. Based on thermal asymmetric interlaced PCR, one hemizygous and two homozygous integration sites were identified in one T_(1) line. Ectopic expression of AnAFP in transgenic lines was confirmed by quantitative real-time PCR, RNA-seq, and Western blotting. After chilling treatment, the transgenic lines showed significantly improved phenotype, higher kernel production, survival rate and biomass, and lower relative electrolyte leakage and superoxide dismutation than the untransformed line. Thus, ectopic expression of AnAFP gene improved chilling tolerance in the transgenic lines, which could be used to apply for further safety assessment for commercial breeding.展开更多
Antifreeze proteins (AFPs) enable organisms to survive under cold conditions, and have great potential in improving cold tolerance of cold-sensitive plants, In order to determine whether expression of the carrot 36 ...Antifreeze proteins (AFPs) enable organisms to survive under cold conditions, and have great potential in improving cold tolerance of cold-sensitive plants, In order to determine whether expression of the carrot 36 kD antifreeze protein gene confers improved cold-resistant properties to plant tissues, we tried to obtain transgenic tobacco plants which expressed the antifreeze protein. Cold, salt, and drought induced promoter Prd29A was cloned using PCR from Arabidopsis. Two plant expression vectors based on pBI121 were constructed with CaMV35S:AFP and Prd29A:AFP. Tobacco plantlets were transformed by Agrobacterium-medicated transformation. PCR and Southern blotting demonstrated that the carrot 36 kD afp gene was successfully integrated into the genomes of transformed plantlets. The expression of the afp gene in transgenic plants led to improved tolerance to cold stress. However, the use of the strong constitutive 35S cauliflower mosaic virus (CaMV) promoter to drive expression of afp also resulted in growth retardation under normal growing conditions. In contrast, the expression of afp driven by the stress-inducible Prd29A promoter from Arabidopsis gave rise to minimal effects on plant growth while providing an increased tolerance to cold stress condition (2℃). The results demonstrated the prospect of using Prd29A-AFP transgenic plants in cold-stressed conditions that will in turn benefit agriculture.展开更多
Antifreeze proteins(AFP) are produced by certain plants, animals, fungi and bacteria that enable them to survive upon extremely low temperature. Perennial rye grass, Lolium perenne, was reported to possess AFP which...Antifreeze proteins(AFP) are produced by certain plants, animals, fungi and bacteria that enable them to survive upon extremely low temperature. Perennial rye grass, Lolium perenne, was reported to possess AFP which protects them from cold environments. In the present investigation, we isolated AFP gene from L. perenne and expressed it in tomato plants to elucidate its role upon chilling stress. The T1 transgenic tomato lines were selected and subjected to molecular, biochemical and physiological analyses. Stable integration and transcription of Lp AFP in transgenic tomato plants was confirmed by Southern blot hybridization and RT-PCR, respectively. Physiological analyses under chilling conditions showed that the chilling stress induced physiological damage in wild type(WT) plants, while the transgenic plants remained healthy. Total sugar content increased gradually in both WT and transgenic plants throughout the chilling treatment. Interestingly, transgenic plants exhibited remarkable alterations in terms of relative water content(RWC) and electrolyte leakage index(ELI) than those of WT. RWC increased significantly by 3-fold and the electrolyte leakage was reduced by 2.6-fold in transgenic plants comparing with WT. Overall, this report proved that Lp AFP gene confers chilling tolerance in transgenic tomato plants and it could be a potential candidate to extrapolate the chilling tolerance on other chilling-sensitive food crops.展开更多
The recombinant expression vector pET43.1b-AFP, which contains full encoding region of a carrot 36 kD antifreeze protein (AFP) gene was constructed. The recombinant was transformed into expression host carrying T7 RNA...The recombinant expression vector pET43.1b-AFP, which contains full encoding region of a carrot 36 kD antifreeze protein (AFP) gene was constructed. The recombinant was transformed into expression host carrying T7 RNA polymerase gene (DE3 lysogen) and induced by 1 mmol稬-1 IPTG (isopropyl--D-thiogalactoside) to express 110 kD polypeptide of AFP fusion protein. The analysis of product solubility revealed that pET43.1b-AFP was predominately soluble, and the expressed amount reached the maximum after the IPTG treatment for 3 h.展开更多
A statistical thermodynamic theory of linear protein solutions was proposed with the aid of a lattice model and applied to type Ⅰ antifreeze protein(AFPI) solutions.The numerical results for several AFPI solutions ...A statistical thermodynamic theory of linear protein solutions was proposed with the aid of a lattice model and applied to type Ⅰ antifreeze protein(AFPI) solutions.The numerical results for several AFPI solutions show that the Gibbs function of the solution has a minimum at a certain protein concentration,but the protein chemical potential increases with increasing the concentration.The influences of temperature and protein chain length on the AFPI chemical potential were also discussed.The evaluation for the colligative depression of the freezing point confirms that the antifreeze action should be recognized as non-colligative.The theoretical deduction for the concentration dependence of the thermal hysteresis activity coincides qualitatively with the previous experimental and theoretical results.展开更多
The Pseudopleuronectes americanus antifreeze protein gene was synthesized and control sequences were added such as 35S promoter and nos terminator that can facilitate the transcription and Ω sequence and Kozak sequen...The Pseudopleuronectes americanus antifreeze protein gene was synthesized and control sequences were added such as 35S promoter and nos terminator that can facilitate the transcription and Ω sequence and Kozak sequence that can improve the expression in translation level, the high expression cassette of antifreeze protein was constructed. This cassette was connected to pBI121.1 and finally got the high expression vector pBRTSAFP introduced into the maize callus. The expression of gus gene that linked to the antifreeze protein gene was detected, and the results was that the gus gene can express strongly and instantaneously.展开更多
Low temperature is one of the major limiting environmental factors which constitutes the growth, development, productivity and distribution of plants. Over the past several years, the proteins and genes associated wi...Low temperature is one of the major limiting environmental factors which constitutes the growth, development, productivity and distribution of plants. Over the past several years, the proteins and genes associated with freezing resistance of plants have been widely studied. The recent progress of domestic and foreign research on plant antifreeze proteins and the identifica- tion and characterization of plant antifreeze protein genes, especially on expression regulatory mechanism of plant antifreeze proteins are reviewed in this paper. Finally, some unsolved problems and the trend of research in physiological functions and gene expression regulatory mechanism of plant antifreeze proteins are discussed.展开更多
基金supported by National Key Science and Technology Special Project(2016ZX08003-004)Sichuan Science and Technology Program(2018JY0470)。
文摘Improved chilling tolerance is important for maize production. Previous efforts in transgenics and marker-assisted breeding have not achieved practical results. In this study, the antifreeze protein(AnAFP) from the super-xerophyte Ammopiptanthus nanus was aligned to KnS-type dehydrins.Phosphorylation in vitro and subcellular localization showed that AnAFP was phosphorylated by maize casein kinase II and translocated from nucleus to cytoplasm under chilling stress. AnAFP also increased lactate dehydrogenase activity. A parent line of commercial maize hybrids was transformed with the AnAFP gene. Based on thermal asymmetric interlaced PCR, one hemizygous and two homozygous integration sites were identified in one T_(1) line. Ectopic expression of AnAFP in transgenic lines was confirmed by quantitative real-time PCR, RNA-seq, and Western blotting. After chilling treatment, the transgenic lines showed significantly improved phenotype, higher kernel production, survival rate and biomass, and lower relative electrolyte leakage and superoxide dismutation than the untransformed line. Thus, ectopic expression of AnAFP gene improved chilling tolerance in the transgenic lines, which could be used to apply for further safety assessment for commercial breeding.
基金Supported by the National Natural Science Foundation of China (Grant No. 30271067), Fok Ying Tung Education Foundation (Grant No. 71030), Key Teach-ers Foundation of the Educational Ministry of China, and Graduate Training Grant of Beijing Forestry Uni
文摘Antifreeze proteins (AFPs) enable organisms to survive under cold conditions, and have great potential in improving cold tolerance of cold-sensitive plants, In order to determine whether expression of the carrot 36 kD antifreeze protein gene confers improved cold-resistant properties to plant tissues, we tried to obtain transgenic tobacco plants which expressed the antifreeze protein. Cold, salt, and drought induced promoter Prd29A was cloned using PCR from Arabidopsis. Two plant expression vectors based on pBI121 were constructed with CaMV35S:AFP and Prd29A:AFP. Tobacco plantlets were transformed by Agrobacterium-medicated transformation. PCR and Southern blotting demonstrated that the carrot 36 kD afp gene was successfully integrated into the genomes of transformed plantlets. The expression of the afp gene in transgenic plants led to improved tolerance to cold stress. However, the use of the strong constitutive 35S cauliflower mosaic virus (CaMV) promoter to drive expression of afp also resulted in growth retardation under normal growing conditions. In contrast, the expression of afp driven by the stress-inducible Prd29A promoter from Arabidopsis gave rise to minimal effects on plant growth while providing an increased tolerance to cold stress condition (2℃). The results demonstrated the prospect of using Prd29A-AFP transgenic plants in cold-stressed conditions that will in turn benefit agriculture.
基金supported by the Senior Research Fellowship from the Council of Scientific and Industrial Research-Human Resource Development Group (CSIRHRDG), New Delhi, India (09/472(0164)/2012-EMR-I)the funds from the University Grants Commission-Special Assistance Programme (UGC-SAP)the Department of Science and Technology-Fund for Improvement of S&T Infrastructure (DST-FIST), Bharathiar University, Tamil Nadu, India
文摘Antifreeze proteins(AFP) are produced by certain plants, animals, fungi and bacteria that enable them to survive upon extremely low temperature. Perennial rye grass, Lolium perenne, was reported to possess AFP which protects them from cold environments. In the present investigation, we isolated AFP gene from L. perenne and expressed it in tomato plants to elucidate its role upon chilling stress. The T1 transgenic tomato lines were selected and subjected to molecular, biochemical and physiological analyses. Stable integration and transcription of Lp AFP in transgenic tomato plants was confirmed by Southern blot hybridization and RT-PCR, respectively. Physiological analyses under chilling conditions showed that the chilling stress induced physiological damage in wild type(WT) plants, while the transgenic plants remained healthy. Total sugar content increased gradually in both WT and transgenic plants throughout the chilling treatment. Interestingly, transgenic plants exhibited remarkable alterations in terms of relative water content(RWC) and electrolyte leakage index(ELI) than those of WT. RWC increased significantly by 3-fold and the electrolyte leakage was reduced by 2.6-fold in transgenic plants comparing with WT. Overall, this report proved that Lp AFP gene confers chilling tolerance in transgenic tomato plants and it could be a potential candidate to extrapolate the chilling tolerance on other chilling-sensitive food crops.
基金the National Natural Science Foundation of China (Grant No.3980011830271067)Fok Ying Tung Education Foundation (71030) and the Key Teachers Foundation of the Education Ministry of China
文摘The recombinant expression vector pET43.1b-AFP, which contains full encoding region of a carrot 36 kD antifreeze protein (AFP) gene was constructed. The recombinant was transformed into expression host carrying T7 RNA polymerase gene (DE3 lysogen) and induced by 1 mmol稬-1 IPTG (isopropyl--D-thiogalactoside) to express 110 kD polypeptide of AFP fusion protein. The analysis of product solubility revealed that pET43.1b-AFP was predominately soluble, and the expressed amount reached the maximum after the IPTG treatment for 3 h.
基金Supported by the National Natural Science Foundation of China(Nos.10764003,30560039)the Special Fund for Basic Scientific Research of Central Colleges,North China Institute of Science and Technology for Nationalities(No.JCB1201A)
文摘A statistical thermodynamic theory of linear protein solutions was proposed with the aid of a lattice model and applied to type Ⅰ antifreeze protein(AFPI) solutions.The numerical results for several AFPI solutions show that the Gibbs function of the solution has a minimum at a certain protein concentration,but the protein chemical potential increases with increasing the concentration.The influences of temperature and protein chain length on the AFPI chemical potential were also discussed.The evaluation for the colligative depression of the freezing point confirms that the antifreeze action should be recognized as non-colligative.The theoretical deduction for the concentration dependence of the thermal hysteresis activity coincides qualitatively with the previous experimental and theoretical results.
基金Supported by National Transgenic Plant Research and.Industrialization Foundation(J00-B-003-04)
文摘The Pseudopleuronectes americanus antifreeze protein gene was synthesized and control sequences were added such as 35S promoter and nos terminator that can facilitate the transcription and Ω sequence and Kozak sequence that can improve the expression in translation level, the high expression cassette of antifreeze protein was constructed. This cassette was connected to pBI121.1 and finally got the high expression vector pBRTSAFP introduced into the maize callus. The expression of gus gene that linked to the antifreeze protein gene was detected, and the results was that the gus gene can express strongly and instantaneously.
基金Supported by the National Natural Science Foundation of China (Grant No. 30271093)
文摘Low temperature is one of the major limiting environmental factors which constitutes the growth, development, productivity and distribution of plants. Over the past several years, the proteins and genes associated with freezing resistance of plants have been widely studied. The recent progress of domestic and foreign research on plant antifreeze proteins and the identifica- tion and characterization of plant antifreeze protein genes, especially on expression regulatory mechanism of plant antifreeze proteins are reviewed in this paper. Finally, some unsolved problems and the trend of research in physiological functions and gene expression regulatory mechanism of plant antifreeze proteins are discussed.