盐角草SeCMO、SePEAMT基因在大肠杆菌中的表达及转双基因烟草耐旱性研究
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摘要
在干旱、盐碱等逆境条件下植物体内可积累大量的甜菜碱,甜菜碱的积累对改善渗透调节、保护细胞膜具有重要作用,因此甜菜碱被认为是很有效的渗调剂之一。胆碱单加氧酶(CMO)和磷酸乙醇胺N-甲基转移酶(PEAMT)是高等植物甜菜碱合成途径中的两个关键酶。利用基因工程技术,把与甜菜碱合成相关的基因导入植物,使其积累甜菜碱,将有可能达到增强植物抗旱性的目的。
为了制备CMO、PEAMT抗体,我们将盐角草SeCMO、SePEAMT基因分别连接到表达质粒pGEX-6p1中,得到重组质粒pGEX6p1-SeCMO和pGEX6p1-SePEAMT,转化大肠杆菌BL21(DE3),经IPTG诱导,SDS-PAGE分析,显示SeCMO、SePEAMT基因能够在大肠杆菌中大量表达,通过GST亲和层析纯化,获得了较纯的融合蛋白。
为评价转基因烟草的耐旱性,以野生型烟草、转SeCMO烟草及转SePEAMT+SeCMO烟草为材料,进行自然停水干旱处理,测定甜菜碱含量、相对含水量、光合参数、质膜损伤、活性氧代谢、主要渗透调节物质含量等生理生化指标。结果显示:干旱胁迫后,转基因烟草甜菜碱含量显著高于野生型烟草,并能维持较高的相对含水量。干旱胁迫后,所有株系的净光合速率、气孔导度均下降,转双基因株系PC26、PC45仍具有显著高于野生型烟草的净光合速率,且胞间CO_2浓度低于对照,表明其光合系统损伤较轻。此外,转双基因株系的细胞膜离子渗透率和丙二醛含量显著低于野生型,且SOD活性、POD活性、可溶性糖含量、游离脯氨酸含量、可溶性蛋白含量普遍高于野生型烟草。以上结果表明:转基因烟草中高水平甜菜碱的积累,能通过渗透调节、保护细胞膜及代谢酶类稳定性的作用,提高烟草的耐旱性。
Betaine will be accumulated in plants in response to drought and salinity stress.The betaine accumulation plays an important role in improving osmotic adjustment and protecting cell membrane,therefore,betaine is a very effective osmoprotectant.Choline monooxygenase(CMO) and Phosphoethanolamine N-methyltransferase(PEAMT) are the key enzyme in the pathways of GlyBet synthesis in plants.The genes related to glycine betaine synthesis are transformed into plants through genetic engineering,which could enhance the drought tolerance of plants.
The SeCMO and SePEAMT genes were subcloned into the expression vector pGEX-6p1, recombinant plasmids pGEX6p1-SeCMO and pGEX6p1-SePEAMT were obtained and transformed into Escherichia coli BL21(DE3).Expression induced by IPTG and SDS-PAGE analysis showed that the SeCMO and SePEAMT gene could express in E.Coli.The fusion proteins were purified with affinity chromatography of GST.
To evaluate the drought tolerance of the transgenic tobacco,the wild type(WT) and transgenic tobacco were exposed to drought stress by withholding water.During the stress period,the Glycinebetaine(GB) content and other physiological parameters including relative water content(RWC),photosynthesis,cell membrane stability,active oxygen metabolism and osmoprotectants content of tobacco plants were measured.The transgenic tobacco plants accumulated significantly higher levels of GB than WT tobacco plants.Under the water stress, the transgenic lines had higher RWC than WT tobacco plants,and the net photosynthesis rate (Pn) and stomatal conductance(Gs) of all plants were reduced,while PC26,PC45 had significantly higher Pn than WT plants and lower substomatal CO_2 concentration(Ci) than non-stress plants,which demonstrating that the photosynthetic system damaged slightly.The percentage of ion leakage and malondialdehyde(MDA) content of transgenic plants were lower than those of the WT plants,while the SOD,POD activity and content of proline, soluble sugar,soluble protein of transgenic plants were generally higher than those of WT plants.Results convinced that the higher level GB content of transgenic tobacco may play an important role in osmotic adjustment and protecting membranes and enzymes from damage, thus the drought tolerance of transgenic tobacco is enhanced.
为了制备CMO、PEAMT抗体,我们将盐角草SeCMO、SePEAMT基因分别连接到表达质粒pGEX-6p1中,得到重组质粒pGEX6p1-SeCMO和pGEX6p1-SePEAMT,转化大肠杆菌BL21(DE3),经IPTG诱导,SDS-PAGE分析,显示SeCMO、SePEAMT基因能够在大肠杆菌中大量表达,通过GST亲和层析纯化,获得了较纯的融合蛋白。
为评价转基因烟草的耐旱性,以野生型烟草、转SeCMO烟草及转SePEAMT+SeCMO烟草为材料,进行自然停水干旱处理,测定甜菜碱含量、相对含水量、光合参数、质膜损伤、活性氧代谢、主要渗透调节物质含量等生理生化指标。结果显示:干旱胁迫后,转基因烟草甜菜碱含量显著高于野生型烟草,并能维持较高的相对含水量。干旱胁迫后,所有株系的净光合速率、气孔导度均下降,转双基因株系PC26、PC45仍具有显著高于野生型烟草的净光合速率,且胞间CO_2浓度低于对照,表明其光合系统损伤较轻。此外,转双基因株系的细胞膜离子渗透率和丙二醛含量显著低于野生型,且SOD活性、POD活性、可溶性糖含量、游离脯氨酸含量、可溶性蛋白含量普遍高于野生型烟草。以上结果表明:转基因烟草中高水平甜菜碱的积累,能通过渗透调节、保护细胞膜及代谢酶类稳定性的作用,提高烟草的耐旱性。
Betaine will be accumulated in plants in response to drought and salinity stress.The betaine accumulation plays an important role in improving osmotic adjustment and protecting cell membrane,therefore,betaine is a very effective osmoprotectant.Choline monooxygenase(CMO) and Phosphoethanolamine N-methyltransferase(PEAMT) are the key enzyme in the pathways of GlyBet synthesis in plants.The genes related to glycine betaine synthesis are transformed into plants through genetic engineering,which could enhance the drought tolerance of plants.
The SeCMO and SePEAMT genes were subcloned into the expression vector pGEX-6p1, recombinant plasmids pGEX6p1-SeCMO and pGEX6p1-SePEAMT were obtained and transformed into Escherichia coli BL21(DE3).Expression induced by IPTG and SDS-PAGE analysis showed that the SeCMO and SePEAMT gene could express in E.Coli.The fusion proteins were purified with affinity chromatography of GST.
To evaluate the drought tolerance of the transgenic tobacco,the wild type(WT) and transgenic tobacco were exposed to drought stress by withholding water.During the stress period,the Glycinebetaine(GB) content and other physiological parameters including relative water content(RWC),photosynthesis,cell membrane stability,active oxygen metabolism and osmoprotectants content of tobacco plants were measured.The transgenic tobacco plants accumulated significantly higher levels of GB than WT tobacco plants.Under the water stress, the transgenic lines had higher RWC than WT tobacco plants,and the net photosynthesis rate (Pn) and stomatal conductance(Gs) of all plants were reduced,while PC26,PC45 had significantly higher Pn than WT plants and lower substomatal CO_2 concentration(Ci) than non-stress plants,which demonstrating that the photosynthetic system damaged slightly.The percentage of ion leakage and malondialdehyde(MDA) content of transgenic plants were lower than those of the WT plants,while the SOD,POD activity and content of proline, soluble sugar,soluble protein of transgenic plants were generally higher than those of WT plants.Results convinced that the higher level GB content of transgenic tobacco may play an important role in osmotic adjustment and protecting membranes and enzymes from damage, thus the drought tolerance of transgenic tobacco is enhanced.
引文
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