Pepper(Capsicum annuum)is one of the most important horticultural crops worldwide,which makes the development of an effective protoplast system for transient gene expression highly significant.Typically,plant protopla...Pepper(Capsicum annuum)is one of the most important horticultural crops worldwide,which makes the development of an effective protoplast system for transient gene expression highly significant.Typically,plant protoplasts are initially isolated through enzymatic digestion and then used for transient transformations mediated by polyethylene glycol(PEG).However,PEG-mediated protoplast transformation suffers from low and inconsistent efficiency,is influenced by various factors,and requires greater operator expertise.Here,we present a simple and efficient protoplast system for transient gene expression in C.annuum and Nicotiana benthamiana,without PEG-mediated transfection.This procedure involved using the first and second fully expanded true leaves of pepper and N.benthamiana plants at the six-leaf stage for Agrobacterium infiltration,followed by enzymatic digestion for protoplast isolation.The resulting protoplast transfections achieved remarkably high efficiencies,facilitating functional analyses such as subcellular localization and protein—protein interaction studies(for example,BiFC,Co-IP,and Split-LUC assays).Thus,we have demonstrated a simplified and highly efficient transient expression system for protoplasts and potential wide-ranging applications in C.annuum and N.benthamiana while bypassing PEG-mediated transfection.展开更多
Metallothionein gene (MT) has been transferred into mushroom protoplasts by electroporation. It is a low molecular weight, cysteine-rich and metal-binding protein. MT can bind metals. Its synthesis is induced by Zn io...Metallothionein gene (MT) has been transferred into mushroom protoplasts by electroporation. It is a low molecular weight, cysteine-rich and metal-binding protein. MT can bind metals. Its synthesis is induced by Zn ion. Thus the expression of MT gene in mushroom can improve the accumulation of Zn in this fungus. This transgenic mushroom, consumed as a kind of vegetable, can supply the necessary Zn to people who are short of the element. When protoplasts were prepared, the concentration (C) of protoplasts is 6.745 x 10(6) /mL. After protoplast electroporation, the transformation rate of protoplasts is 0.01 %. Polymerase chain reaction (PCR) analysis showed that the gene had been integrated into the mushroom chromosome, SDS-PAGE, Western blot analysis indicated that the MT gene had been expressed in the transgenic mushroom. The expressing level, detected by ELISA, is 0.6 % - 0.8 %. Tested for metal resistance, the wild-type mushroom growth was inhibited on die medium containing 1.0 - 1.2 mmoL/L ZnSO4. While the transgenic mushroom was inhibited on the medium containing 1. 5 - 2.0 mmol/L ZnSO4. The mycelium can develop into hymenophore in the medium of rice bran: sawdust = 1: 3, and not in the medium of rice bran: sawdust = 1: 4.展开更多
Protoplasts prepared from tobacco (Nicotiana tabacum L., cultivar BY-2) suspension cells have similar morphological characteristics to those in animal cells. The hallmarks of apoptosis such as condensation and periphe...Protoplasts prepared from tobacco (Nicotiana tabacum L., cultivar BY-2) suspension cells have similar morphological characteristics to those in animal cells. The hallmarks of apoptosis such as condensation and peripheral distribution of nuclei, TUNEL positive reaction, and DNA ladders were observed when tobacco protoplasts were treated with the hydroxyl radical generating system (1.0 mmol/L FeSO4/0.5 mmol/L H2O2). In animals, the loss of transmembrane potential (DeltaPsi(m)) and the exposure of phospholipid phosphatidylserine (PS) are believed to be the main apoptosis events. To test whether these significant processes take place in plants, flow cytometry was used to detect annexin V binding and changes in DeltaPsi(m). Results showed that the PS turned out from inner membrane and DeltaPsi(m) gradually decreased during the apoptosis. All these apoptotic characteristics proved that hydroxyl radicals can cause typical programmed cell death (PCD) in tobacco protoplasts and this design can be served as an effective experiment system to explore the mechanism of plant apoptosis.展开更多
The application of Penman's method of cell fractionation to plant protoplasts leads to our finding of keratin intermediate filament(IF)-like system in maize protoplasts,which was identified by using immunogold lab...The application of Penman's method of cell fractionation to plant protoplasts leads to our finding of keratin intermediate filament(IF)-like system in maize protoplasts,which was identified by using immunogold labelling with monoclonal antibody of cytokeratin from animal cells.Many gold particles were found to be bound on filaments,linked by 3 nm filaments.After further digestion and extraction with DNase I and ammonium sulphate.IF-like framework-lamina-nuclear matrix system was shown under electron microscope.That IF system exists in plant protoplasts just like in animal cells,and their main component is keratin-like protein.展开更多
Protoplasts of embryogenic suspension cells of loblolly pine (Pinus taeda L).were isolated at exponential growth stage.Influences of various concentrations of basal medium,levels of BA,and concentrations of inositol ...Protoplasts of embryogenic suspension cells of loblolly pine (Pinus taeda L).were isolated at exponential growth stage.Influences of various concentrations of basal medium,levels of BA,and concentrations of inositol on the differentiation of embryonal suspensor mass (ESM),early stage somatic embryos (ESE) ,and lae stage somatic embryos (LSE) were investigated .A study of the effect of various concentrations of LP basal medium sowed that the optimal basal medium concentration of ESM,ESE,and LSE differentiation was 1.25 LP medium.The effects of various levels of BA and inositol showed that the optimal concentrations of BA for the formation of ESM,ESE and LSE were 4 mg/L ,2mg/L and 1mg/L,respectively ,and the optimal concentrations of inositol for the ESM ,ESE and LSM formation were 400mg/L,800mg/L and 1,200mg/L,respectively.展开更多
Isolated protoplasts from thalli of Porphyra haitanensis and Porphyra yezoensis were treated with colchicine or irradiated by ultraviolet (UV ). Several types of color variants were observed among the protoplast offsp...Isolated protoplasts from thalli of Porphyra haitanensis and Porphyra yezoensis were treated with colchicine or irradiated by ultraviolet (UV ). Several types of color variants were observed among the protoplast offspring. After treatment with colchicine: (1) 0.04-0.09% of red type variants in P. haitanensis were obtained; (2) The rate of red type variants and the variegated chimeral thalli composed of red type and wild type of sectors were 6.31- 1.11% in P. yezoensis. After irradiation with UV: (1) 3.5- 10.5% of red type variants in P. yezoensis were obtained: (2) 0.5-2-0% of red type variants and the variegated chimeral thalli composed of red type and wild type of sectors were obtained in P. haitanensis. Colchicine and UV’s mutangenic effects on P. yezoensis protoplasts were stronger than those on P. haitanensis protoplasts. The most efficient concentration of colchicine was 0.05%. The optimal length of UV-radiation was 1/2 min (radiation distance 5 cm). The red type variants induced, by colchicine展开更多
Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression sys...Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression system is a powerful tool for gene functional characterization and CRISPR/Cas9 genome editing in higher plants, but it has not been widely used in citrus. In this study, the polyethylene glycol(PEG)-mediated method was optimized for citrus callus protoplast transfection, with an improved transfection efficiency of 68.4%. Consequently, the efficiency of protein subcellular localization assay was increased to 65.8%, through transient expression of the target gene in protoplasts that stably express the fluorescent organelle marker protein. The gene editing frequencies in citrus callus protoplasts reached 14.2% after transient expression of CRISPR/Cas9 constructs. We demonstrated that the intronic polycistronic tRNAgRNA(inPTG) genome editing construct was functional in both the protoplast transient expression system and epicotyl stable transformation system in citrus. With this optimized protoplast transient expression system, we improved the efficiency of protein subcellular localization assay and developed the genome editing system in callus protoplasts, which provides an approach for prompt test of CRISPR vectors.展开更多
基金supported by the National Natural Science Foundation of China(Grant Nos.32472536 and 32302526)the Natural Science Foundation of Anhui Province,China(Grant No.2208085MC64)+2 种基金the Outstanding Innovative Research Team for Molecular Enzymology and Detection in Anhui Provincial Universities(Grant No.2022AH010012)the University Synergy Innovation Program of Anhui Province(Grant No.GXXT-2022-067)the University Natural Science Research Program of Anhui Provincial Education Department(Grant No.KJ2021A0118).
文摘Pepper(Capsicum annuum)is one of the most important horticultural crops worldwide,which makes the development of an effective protoplast system for transient gene expression highly significant.Typically,plant protoplasts are initially isolated through enzymatic digestion and then used for transient transformations mediated by polyethylene glycol(PEG).However,PEG-mediated protoplast transformation suffers from low and inconsistent efficiency,is influenced by various factors,and requires greater operator expertise.Here,we present a simple and efficient protoplast system for transient gene expression in C.annuum and Nicotiana benthamiana,without PEG-mediated transfection.This procedure involved using the first and second fully expanded true leaves of pepper and N.benthamiana plants at the six-leaf stage for Agrobacterium infiltration,followed by enzymatic digestion for protoplast isolation.The resulting protoplast transfections achieved remarkably high efficiencies,facilitating functional analyses such as subcellular localization and protein—protein interaction studies(for example,BiFC,Co-IP,and Split-LUC assays).Thus,we have demonstrated a simplified and highly efficient transient expression system for protoplasts and potential wide-ranging applications in C.annuum and N.benthamiana while bypassing PEG-mediated transfection.
文摘Metallothionein gene (MT) has been transferred into mushroom protoplasts by electroporation. It is a low molecular weight, cysteine-rich and metal-binding protein. MT can bind metals. Its synthesis is induced by Zn ion. Thus the expression of MT gene in mushroom can improve the accumulation of Zn in this fungus. This transgenic mushroom, consumed as a kind of vegetable, can supply the necessary Zn to people who are short of the element. When protoplasts were prepared, the concentration (C) of protoplasts is 6.745 x 10(6) /mL. After protoplast electroporation, the transformation rate of protoplasts is 0.01 %. Polymerase chain reaction (PCR) analysis showed that the gene had been integrated into the mushroom chromosome, SDS-PAGE, Western blot analysis indicated that the MT gene had been expressed in the transgenic mushroom. The expressing level, detected by ELISA, is 0.6 % - 0.8 %. Tested for metal resistance, the wild-type mushroom growth was inhibited on die medium containing 1.0 - 1.2 mmoL/L ZnSO4. While the transgenic mushroom was inhibited on the medium containing 1. 5 - 2.0 mmol/L ZnSO4. The mycelium can develop into hymenophore in the medium of rice bran: sawdust = 1: 3, and not in the medium of rice bran: sawdust = 1: 4.
文摘Protoplasts prepared from tobacco (Nicotiana tabacum L., cultivar BY-2) suspension cells have similar morphological characteristics to those in animal cells. The hallmarks of apoptosis such as condensation and peripheral distribution of nuclei, TUNEL positive reaction, and DNA ladders were observed when tobacco protoplasts were treated with the hydroxyl radical generating system (1.0 mmol/L FeSO4/0.5 mmol/L H2O2). In animals, the loss of transmembrane potential (DeltaPsi(m)) and the exposure of phospholipid phosphatidylserine (PS) are believed to be the main apoptosis events. To test whether these significant processes take place in plants, flow cytometry was used to detect annexin V binding and changes in DeltaPsi(m). Results showed that the PS turned out from inner membrane and DeltaPsi(m) gradually decreased during the apoptosis. All these apoptotic characteristics proved that hydroxyl radicals can cause typical programmed cell death (PCD) in tobacco protoplasts and this design can be served as an effective experiment system to explore the mechanism of plant apoptosis.
文摘The application of Penman's method of cell fractionation to plant protoplasts leads to our finding of keratin intermediate filament(IF)-like system in maize protoplasts,which was identified by using immunogold labelling with monoclonal antibody of cytokeratin from animal cells.Many gold particles were found to be bound on filaments,linked by 3 nm filaments.After further digestion and extraction with DNase I and ammonium sulphate.IF-like framework-lamina-nuclear matrix system was shown under electron microscope.That IF system exists in plant protoplasts just like in animal cells,and their main component is keratin-like protein.
文摘Protoplasts of embryogenic suspension cells of loblolly pine (Pinus taeda L).were isolated at exponential growth stage.Influences of various concentrations of basal medium,levels of BA,and concentrations of inositol on the differentiation of embryonal suspensor mass (ESM),early stage somatic embryos (ESE) ,and lae stage somatic embryos (LSE) were investigated .A study of the effect of various concentrations of LP basal medium sowed that the optimal basal medium concentration of ESM,ESE,and LSE differentiation was 1.25 LP medium.The effects of various levels of BA and inositol showed that the optimal concentrations of BA for the formation of ESM,ESE and LSE were 4 mg/L ,2mg/L and 1mg/L,respectively ,and the optimal concentrations of inositol for the ESM ,ESE and LSM formation were 400mg/L,800mg/L and 1,200mg/L,respectively.
文摘Isolated protoplasts from thalli of Porphyra haitanensis and Porphyra yezoensis were treated with colchicine or irradiated by ultraviolet (UV ). Several types of color variants were observed among the protoplast offspring. After treatment with colchicine: (1) 0.04-0.09% of red type variants in P. haitanensis were obtained; (2) The rate of red type variants and the variegated chimeral thalli composed of red type and wild type of sectors were 6.31- 1.11% in P. yezoensis. After irradiation with UV: (1) 3.5- 10.5% of red type variants in P. yezoensis were obtained: (2) 0.5-2-0% of red type variants and the variegated chimeral thalli composed of red type and wild type of sectors were obtained in P. haitanensis. Colchicine and UV’s mutangenic effects on P. yezoensis protoplasts were stronger than those on P. haitanensis protoplasts. The most efficient concentration of colchicine was 0.05%. The optimal length of UV-radiation was 1/2 min (radiation distance 5 cm). The red type variants induced, by colchicine
基金supported by the National Natural Science Foundation of ChinaChina (Grant Nos. 31872051, 32072528)the Foundation of Hubei Hongshan Laboratory (Grant No.2021hszd009)。
文摘Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression system is a powerful tool for gene functional characterization and CRISPR/Cas9 genome editing in higher plants, but it has not been widely used in citrus. In this study, the polyethylene glycol(PEG)-mediated method was optimized for citrus callus protoplast transfection, with an improved transfection efficiency of 68.4%. Consequently, the efficiency of protein subcellular localization assay was increased to 65.8%, through transient expression of the target gene in protoplasts that stably express the fluorescent organelle marker protein. The gene editing frequencies in citrus callus protoplasts reached 14.2% after transient expression of CRISPR/Cas9 constructs. We demonstrated that the intronic polycistronic tRNAgRNA(inPTG) genome editing construct was functional in both the protoplast transient expression system and epicotyl stable transformation system in citrus. With this optimized protoplast transient expression system, we improved the efficiency of protein subcellular localization assay and developed the genome editing system in callus protoplasts, which provides an approach for prompt test of CRISPR vectors.
