Acidic digestion is an important digestive process of marine fish.In fish stomach,two enzymes are involved in the secretion of hydrochloric acid(HCl)and proteomic digestion:H^(+)/K^(+)-ATPase and pepsinogen.However,th...Acidic digestion is an important digestive process of marine fish.In fish stomach,two enzymes are involved in the secretion of hydrochloric acid(HCl)and proteomic digestion:H^(+)/K^(+)-ATPase and pepsinogen.However,the starting of digestive function in fish is still unclear.To reveal the details of acidic digestion of turbot Scophthalmus maximus in early development,a 40 day of turbot larvae culture was conducted.The H^(+)/K^(+)-ATPase gene from the turbot S.maximus(smH^(+)/K^(+)-ATPase)was identified and characterized.Based on our previous discription on pepsinogen of turbot S.maximus,we combined pepsinogen and H^(+)/K^(+)-ATPase and analyzed the mechanism of acidic digestion in turbot.Results show that the spatial and temporal expression profiles of H^(+)/K^(+)-ATPase agreed with pepsinogen A and C in turbot,indicating a synergetic action between H^(+)/K^(+)-ATPase and pepsinogen during the acidic digestion process.In addition,the turbot juveniles showed a faster growth after the expressions of H^(+)/K^(+)-ATPase gene and pepsinogen gene,demonstrating that pepsin had a higher digestive efficiency,for which a compound diet should be provided to the fish from Day 22 onward.This study provided a reference for biology research and aquaculture of turbot and other marine fishes.展开更多
本文克隆了RIN4(RPM1-interacting protein 4)在胡杨中的同源基因PeRIN4,并在拟南芥中进行过表达,通过研究转基因株系的耐盐表型、质膜H^+-ATPsae活性及H^+、Na^+、K^+等的动态离子流,揭示了PeRIN4基因在植物响应和适应盐胁迫环境中的...本文克隆了RIN4(RPM1-interacting protein 4)在胡杨中的同源基因PeRIN4,并在拟南芥中进行过表达,通过研究转基因株系的耐盐表型、质膜H^+-ATPsae活性及H^+、Na^+、K^+等的动态离子流,揭示了PeRIN4基因在植物响应和适应盐胁迫环境中的作用。利用定位载体p Green0029-PeRIN4-GFP瞬时转化拟南芥叶肉细胞原生质体的方法,对胡杨PeRIN4蛋白进行亚细胞定位,发现该蛋白定位在细胞的胞质中。耐盐表型实验结果显示,在100 mmol/L NaCl处理下,拟南芥PeRIN4过表达株系(OE1和OE8)的生存率和根长均明显高于野生型(WT)和转空载体拟南芥(VC),说明PeRIN4基因能够提高拟南芥的耐盐性。与WT和VC相比,拟南芥PeRIN4过表达株系质膜H^+-ATPsae的活性较高。动态离子流数据显示,在盐胁迫下,PeRIN4过表达株系外排H^+和Na^+离子的能力强于野生型和转空载体拟南芥,然而K+的外流却弱于WT和VC。因此,PeRIN4蛋白具有调节质膜H^+-ATPsae活性的功能。拟南芥质膜H^+-ATPsae活性的提高主要有两方面的作用:一是可以增强H+泵的质子动力势,驱动Na^+/H^+逆向转运蛋白,提高Na^+外排的能力;二是抑制质膜的去极化,减少K+离子通过去极化激活的外向型K^+通道(DA-KORCs)和非选择性阳离子通道(DA-NSCCs)外流,维持了K^+/Na^+平衡,从而提高PeRIN4转基因拟南芥的耐盐性。展开更多
Tonoplast-enriched vesicles were prepared from suspension-cultured Populus euphratica Oliv. cells by differential centrifugation and discontinuous sucrose density gradient centrifugation. The properties of the proton ...Tonoplast-enriched vesicles were prepared from suspension-cultured Populus euphratica Oliv. cells by differential centrifugation and discontinuous sucrose density gradient centrifugation. The properties of the proton pumping activity of H+-ATPases in tonoplast vesicles were studied by acridine orange fluorescent quenching measured at 22 degreesC. The proton pumping activity of ATPase was ATP-dependent with apparent Michaelis-Menten Constant (K-m) for ATP about 0.65 mmol/L. The optimal pH for H+-ATPases activity was 7.5. The proton pumping activity of H+-ATPase could be initiated by some divalent cations, Mg2+ being highly efficient, much more than Fe2+; and Ca2+, Cu2+ and Zn2+ were inefficient under the experimental condition. The proton translocation could be stimulated by halide anions, with potencies decreasing in the order Cl- > Br- > I- > F-. The proton pumping activity was greatly inhibited by N-ethylmaleimide (NEM), N, N'-dicyclohexylcarbodiimide (DCCD), NO3- and Bafilomycin A(1), but not by orthovanadate and azide. These results demonstrated that the H+-ATPase in the tonoplast of Populus euphratica belonged to vacuolar type ATPase. This work was the first time that tonoplast-enriched vesicles were isolated from Populus euphratica cells.展开更多
Under NaCl stress for 2 d, H+-ATPase activity increased, and H+-PPase activity decreased in the tonoplast of salt-tolerant barley ( Hordeum vulgare L. cv. 'Tanyin 2') roots. La3+ (1 mmol/L), an inhibitor of Ca...Under NaCl stress for 2 d, H+-ATPase activity increased, and H+-PPase activity decreased in the tonoplast of salt-tolerant barley ( Hordeum vulgare L. cv. 'Tanyin 2') roots. La3+ (1 mmol/L), an inhibitor of Ca2+ channel in plasma membrane, and EGTA (5 mmol/L), a Ca2+ chelator, inhibited this NaCl-induced increase in H+-ATPase activity but stimulated the H+-PPase activity. Treatment of barley roots with CaM antagonist (trifluoperazine, TFP, 20 mumol/L) also diminished the increase of H+-ATPase activity induced by NaCl. La3+, TFP or La3+ + TFP increased Na+ uptake and decreased K+ and Ca2+ uptake in barley roots under NaCl stress. These results suggested that the activation of tonoplast H+-ATPase and the regulation of Na+ and K+ uptake under NaCl stress may be related to Ca2+-CaM system.展开更多
基金Supported by the National Key Research and Development Program(No.2018YFD0901204)the Key Special Project for Introduced Talents Team of Southern Marine Science and Engineering Guangdong Laboratory(Guangzhou)(No.GML2019ZD0402)+3 种基金the Major Agricultural Application Technology Innovation Project of Shandong Province(No.SD2019YY011)the Qingdao National Laboratory for Marine Science and Technology(No.2018SDKJ0502-2)the China Agriculture Research System(No.CARS-47),the Major Science and Technology for Scientific and Technological Innovation Projects(Shandong)(No.2019JZZY020710)the STS Project(Nos.KFZD-SW-106,ZSSD-019,2017T3017,2019T3022)。
文摘Acidic digestion is an important digestive process of marine fish.In fish stomach,two enzymes are involved in the secretion of hydrochloric acid(HCl)and proteomic digestion:H^(+)/K^(+)-ATPase and pepsinogen.However,the starting of digestive function in fish is still unclear.To reveal the details of acidic digestion of turbot Scophthalmus maximus in early development,a 40 day of turbot larvae culture was conducted.The H^(+)/K^(+)-ATPase gene from the turbot S.maximus(smH^(+)/K^(+)-ATPase)was identified and characterized.Based on our previous discription on pepsinogen of turbot S.maximus,we combined pepsinogen and H^(+)/K^(+)-ATPase and analyzed the mechanism of acidic digestion in turbot.Results show that the spatial and temporal expression profiles of H^(+)/K^(+)-ATPase agreed with pepsinogen A and C in turbot,indicating a synergetic action between H^(+)/K^(+)-ATPase and pepsinogen during the acidic digestion process.In addition,the turbot juveniles showed a faster growth after the expressions of H^(+)/K^(+)-ATPase gene and pepsinogen gene,demonstrating that pepsin had a higher digestive efficiency,for which a compound diet should be provided to the fish from Day 22 onward.This study provided a reference for biology research and aquaculture of turbot and other marine fishes.
文摘Tonoplast-enriched vesicles were prepared from suspension-cultured Populus euphratica Oliv. cells by differential centrifugation and discontinuous sucrose density gradient centrifugation. The properties of the proton pumping activity of H+-ATPases in tonoplast vesicles were studied by acridine orange fluorescent quenching measured at 22 degreesC. The proton pumping activity of ATPase was ATP-dependent with apparent Michaelis-Menten Constant (K-m) for ATP about 0.65 mmol/L. The optimal pH for H+-ATPases activity was 7.5. The proton pumping activity of H+-ATPase could be initiated by some divalent cations, Mg2+ being highly efficient, much more than Fe2+; and Ca2+, Cu2+ and Zn2+ were inefficient under the experimental condition. The proton translocation could be stimulated by halide anions, with potencies decreasing in the order Cl- > Br- > I- > F-. The proton pumping activity was greatly inhibited by N-ethylmaleimide (NEM), N, N'-dicyclohexylcarbodiimide (DCCD), NO3- and Bafilomycin A(1), but not by orthovanadate and azide. These results demonstrated that the H+-ATPase in the tonoplast of Populus euphratica belonged to vacuolar type ATPase. This work was the first time that tonoplast-enriched vesicles were isolated from Populus euphratica cells.
文摘Under NaCl stress for 2 d, H+-ATPase activity increased, and H+-PPase activity decreased in the tonoplast of salt-tolerant barley ( Hordeum vulgare L. cv. 'Tanyin 2') roots. La3+ (1 mmol/L), an inhibitor of Ca2+ channel in plasma membrane, and EGTA (5 mmol/L), a Ca2+ chelator, inhibited this NaCl-induced increase in H+-ATPase activity but stimulated the H+-PPase activity. Treatment of barley roots with CaM antagonist (trifluoperazine, TFP, 20 mumol/L) also diminished the increase of H+-ATPase activity induced by NaCl. La3+, TFP or La3+ + TFP increased Na+ uptake and decreased K+ and Ca2+ uptake in barley roots under NaCl stress. These results suggested that the activation of tonoplast H+-ATPase and the regulation of Na+ and K+ uptake under NaCl stress may be related to Ca2+-CaM system.
