灰绿藜液泡膜焦磷酸酶V-PPase基因增强烟草和拟南芥耐盐性的研究
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摘要
土壤盐渍化严重影响植物的生长与发育,土壤中高浓度的盐分会造成植物体内离子失衡、氧化伤害、水分亏缺、营养缺乏并导致生物大分子破坏、生长迟缓、甚至植株死亡,从而使作物减产或绝收。而植物响应盐胁迫的最终结果就是降低胞质内的Na~+浓度并维持K~+、Na~+平衡。
在盐胁迫的影响下,大量Na~+流入细胞,植物会将Na~+区隔到液泡中,即离子区隔化,这样不仅能够消除Na~+对胞质的毒害并维持较高的K~+/ Na~+比,而且还能使细胞利用累积在液泡中的大量无机离子进行渗透调节。因此,离子区隔化机制对植物细胞适应盐生境十分必要。Na~+区隔化主要是液泡膜上Na~+/H~+逆向转运蛋白利用液泡膜质子泵( V-H~+-ATPase,V-H~+-PPase)提供的质子驱动力将胞质中的Na~+逆浓度梯度运入液泡内积累。这就意味着可以通过增大液泡膜质子泵基因表达来增大质子驱动力,从而使细胞质中更多的Na~+区隔到液泡中以增强植物的耐盐性。在拟南芥、番茄、水稻中过表达液泡膜焦磷酸酶H~+-PPase基因,可使转基因植株在较高盐浓度下生长,说明液泡膜H~+-PPase在植物耐盐方面起重要作用。
灰绿藜(Chenopodium glaucum L.)是新疆广泛分布的一种藜科耐盐植物,它可以在100~600 mmol/L NaCl甚至更高盐浓度的环境中生长。从形态学上看,它既无肉质茎也无变态叶,与甜土植物更为接近,因此,研究灰绿藜的耐盐机制将对提高甜土植物的耐盐性很有帮助。本研究将实验室已克隆获得的灰绿藜液泡膜焦磷酸酶H~+-PPase基因(CgVP1)构建到植物表达载体pCAMBIA1301.1上,通过根癌农杆菌介导转化植物,并通过对转基因植株的分子生物学及生理生化检测,对灰绿藜液泡膜H~+-PPase基因进行了功能鉴定,主要结果如下:
1.通过根癌农杆菌浸染法转化拟南芥并经潮霉素筛选,获得T3代纯合子转基因拟南芥,PCR及RT-PCR检测表明CgVP1已整合到拟南芥基因组中,并在转录水平有所表达,这使得转基因拟南芥表现出比野生型更强的耐盐性。在150mmol/L NaCl胁迫下,野生型拟南芥生长受到了明显抑制,种子萌发率明显下降,当NaCl浓度分别为100mmol/L、150 mmol/L、175 mmol/L时,转基因植株的萌发率分别为91.7%、89.2%和84.2%,而野生型萌发率仅为67.5%、53.3%和35.8%。而且在175mmol/L NaCl培养基上,尽管二者生长均受到明显抑制,但转基因植株净生长量明显大于野生型。对Na~+、K~+含量测定表明:在盐胁迫下,转基因拟南芥将Na~+区隔到根部的细胞中减少Na~+向地上部的运输,从而保持叶中相对较高的K~+浓度;相对含水量和叶绿素测定结果显示:在盐胁迫下转基因拟南芥还能够使机体保持相对较高的相对含水量,确保叶片中叶绿素含量较高。以上实验结果初步显示,CgVP1的过表达提高了转基因拟南芥的耐盐能力。
2.通过根癌农杆菌介导转化烟草并经潮霉素筛选,获得T0代转基因烟草。PCR及RT-PCR检测结果表明CgVP1已整合到烟草基因组中,并在转录水平有所表达。在盐胁迫下,转基因烟草和野生型烟草的生长都受到了轻微抑制。400mmol/L NaCl胁迫30d后,转基因烟草的K~+ /Na~+比率较野生型高,并且转基因烟草能够使机体保持较高的相对含水量以及较低的电解液渗漏率。通过检测转基因烟草的耐旱性,结果发现转基因烟草在干旱胁迫45d后,仍然生长良好,只是轻微受到干旱伤害,而野生型烟草失水十分严重,并且不能恢复。实验结果初步显示,CgVP1的过表达对转基因烟草的耐盐和耐旱能力有所提高。
Salt stress is the primary effect that limits the growth and development of plant, the higher salt concentration in the soil what can arouse many damages for the plant, for example, the loss of ion balance, oxidation damage, the deficiency of water, the lack of nutrition, which lead to the destroy of biology molecule, the slower of growth and the faster death of plant.Ultimately, those could result in the fall of plant yield or on harvest.That is why plant has to reduce cellular Na~+ and keep K~+/Na~+ balance when exposed to salt stress.
Under salt stress, Na~+ influx into cells occurs via Na~+ permeable transporters which in turn elevates the cytoplasm sodium concentration and causes toxicity.Na~+ sequestration into vacuoles was not only avoid to destroy the cytoplasm, keep a higher K~+/Na~+ balance in cytoplasm, but also can be used as an osmolyte in the vacuole to help to achieve osmotic homeostasis.So, it is necessary to adapt the salt stress for plant, Na?~+ compartmentation means
by the energy provided by transporting proton along its concentration gradient,vacuolar-type Na~+/H~+ antiporters deliver Na~+ into the vacuoles against the electrochemical gradient generated by vacuolar H~+-ATPase and H~+-PPase.The salt tolerance of plant can be advanced by accumulate to a higher level Na~+ in the cytoplasm through enhance the over expressing of vacuolar H~+-PPase.The enhanced salt tolerance of transgenic Arabidopsis, tomato, rice by overexpressing vacuolar H~+-PPase genes indicated their important roles in plant salt tolerance. Chenopodium glaucum L. is a Chenopodiacea halophytic plant species, which widely distributes in Xinjiang.It is the typical salt-tolerant plant, and it can grow normally under 100~600mmol/L NaCl treatment.The appearance of the whole plant of C.glaucum has neither chylocaulous stem, nor also no any specialized leaf, relatively closer to that of glycophyte.Therefore, it is more valuable to study the mechanism of salt tolerance and for applying on glycophyte to enhance the salt tolerance.Based on all of these, in this thesis, a binary expression vector pCAMBIA1301.1-CgVP1 which harbored CgVP1 gene was constructed and introduced into Arabidopsis thaliana and Nicotiana tabaccum plant by Agrobacterium-mediated method.The function analyses of CgVP1 transgenic plants have been done by molecular identification and some physiological indexes assay.The key results were as follows:
1. The binary expression vector pCAMBIA1301.1-CgVP1 was introduced into A. thaliana by Agrobacterium-mediated method.T3 homogenous plantlets were obtained by screening hygromycin-resistant seedlings.PCR and RT-PCR analysis indicated that CgVP1 have been integrated into the genome of A.thaliana and expressed at transcriptional level.Overexpression of CgVP1 gene in A. thaliana led to enhanced tolerance to salt stress.The relative growth rate of wild-type was restrained markedly under 150mmol/L NaCl treatment.The germination rate of transgenic plants was 91.7%, 89.2%, 84.2%, repectively, while the wild-type was 67.5%, 53.3%, 35.8%, respectively when exposed to 100 mmol/L, 150 mmol/L, 175 mmol/L NaCl.The result of the Na~+, K~+ determination suggested that sodium was compartmentated in the root in order to decrease the sodium concentration of the leaf in transgenic plants under salt stress.Moreover, the relative water and chlorophyll content assay indicated that the relative water content of transgenic plants was higher than that of wild-type,and a higher chlorophyll content was also detected in transgenic plants under salt stress.Our data showed that overexpression of CgVP1 gene could enhance the capacity of salt-tolerance in transgenic A.thaliana.
2. The binary expression vector pCAMBIA1301.1-CgVP1 was introduced into tobacco by Agrobacterium-mediated method.T0 plantlets were obtained by screening the hygromycin- resistant seedlings.PCR and RT-PCR analysis indicated that CgVP1 gene have been integrated into the genome of tobacco and expressed at transcriptional level.The relative growth rate of both wild-type and transgenic plants were restrained lightly under salt treatment.The K~+ /Na~+ rate and the relative water content of transgenic plants were higher than that of wild-type, and the electrolyte leakage rate was lower than that of wild-type after being exposed to 400 mmol/L NaCl for 30 days.The drought tolerance assay indicated that transgenic plants performed better than that of wild-type, such as growth status, almost no hurt after being drought stress for 45 days.But the water loss was too serious in wild-type to be restored.Our data showed that overexpression CgVP1 gene could enhance the capacity both in salt and drought tolerance in transgenic tobacco.
在盐胁迫的影响下,大量Na~+流入细胞,植物会将Na~+区隔到液泡中,即离子区隔化,这样不仅能够消除Na~+对胞质的毒害并维持较高的K~+/ Na~+比,而且还能使细胞利用累积在液泡中的大量无机离子进行渗透调节。因此,离子区隔化机制对植物细胞适应盐生境十分必要。Na~+区隔化主要是液泡膜上Na~+/H~+逆向转运蛋白利用液泡膜质子泵( V-H~+-ATPase,V-H~+-PPase)提供的质子驱动力将胞质中的Na~+逆浓度梯度运入液泡内积累。这就意味着可以通过增大液泡膜质子泵基因表达来增大质子驱动力,从而使细胞质中更多的Na~+区隔到液泡中以增强植物的耐盐性。在拟南芥、番茄、水稻中过表达液泡膜焦磷酸酶H~+-PPase基因,可使转基因植株在较高盐浓度下生长,说明液泡膜H~+-PPase在植物耐盐方面起重要作用。
灰绿藜(Chenopodium glaucum L.)是新疆广泛分布的一种藜科耐盐植物,它可以在100~600 mmol/L NaCl甚至更高盐浓度的环境中生长。从形态学上看,它既无肉质茎也无变态叶,与甜土植物更为接近,因此,研究灰绿藜的耐盐机制将对提高甜土植物的耐盐性很有帮助。本研究将实验室已克隆获得的灰绿藜液泡膜焦磷酸酶H~+-PPase基因(CgVP1)构建到植物表达载体pCAMBIA1301.1上,通过根癌农杆菌介导转化植物,并通过对转基因植株的分子生物学及生理生化检测,对灰绿藜液泡膜H~+-PPase基因进行了功能鉴定,主要结果如下:
1.通过根癌农杆菌浸染法转化拟南芥并经潮霉素筛选,获得T3代纯合子转基因拟南芥,PCR及RT-PCR检测表明CgVP1已整合到拟南芥基因组中,并在转录水平有所表达,这使得转基因拟南芥表现出比野生型更强的耐盐性。在150mmol/L NaCl胁迫下,野生型拟南芥生长受到了明显抑制,种子萌发率明显下降,当NaCl浓度分别为100mmol/L、150 mmol/L、175 mmol/L时,转基因植株的萌发率分别为91.7%、89.2%和84.2%,而野生型萌发率仅为67.5%、53.3%和35.8%。而且在175mmol/L NaCl培养基上,尽管二者生长均受到明显抑制,但转基因植株净生长量明显大于野生型。对Na~+、K~+含量测定表明:在盐胁迫下,转基因拟南芥将Na~+区隔到根部的细胞中减少Na~+向地上部的运输,从而保持叶中相对较高的K~+浓度;相对含水量和叶绿素测定结果显示:在盐胁迫下转基因拟南芥还能够使机体保持相对较高的相对含水量,确保叶片中叶绿素含量较高。以上实验结果初步显示,CgVP1的过表达提高了转基因拟南芥的耐盐能力。
2.通过根癌农杆菌介导转化烟草并经潮霉素筛选,获得T0代转基因烟草。PCR及RT-PCR检测结果表明CgVP1已整合到烟草基因组中,并在转录水平有所表达。在盐胁迫下,转基因烟草和野生型烟草的生长都受到了轻微抑制。400mmol/L NaCl胁迫30d后,转基因烟草的K~+ /Na~+比率较野生型高,并且转基因烟草能够使机体保持较高的相对含水量以及较低的电解液渗漏率。通过检测转基因烟草的耐旱性,结果发现转基因烟草在干旱胁迫45d后,仍然生长良好,只是轻微受到干旱伤害,而野生型烟草失水十分严重,并且不能恢复。实验结果初步显示,CgVP1的过表达对转基因烟草的耐盐和耐旱能力有所提高。
Salt stress is the primary effect that limits the growth and development of plant, the higher salt concentration in the soil what can arouse many damages for the plant, for example, the loss of ion balance, oxidation damage, the deficiency of water, the lack of nutrition, which lead to the destroy of biology molecule, the slower of growth and the faster death of plant.Ultimately, those could result in the fall of plant yield or on harvest.That is why plant has to reduce cellular Na~+ and keep K~+/Na~+ balance when exposed to salt stress.
Under salt stress, Na~+ influx into cells occurs via Na~+ permeable transporters which in turn elevates the cytoplasm sodium concentration and causes toxicity.Na~+ sequestration into vacuoles was not only avoid to destroy the cytoplasm, keep a higher K~+/Na~+ balance in cytoplasm, but also can be used as an osmolyte in the vacuole to help to achieve osmotic homeostasis.So, it is necessary to adapt the salt stress for plant, Na?~+ compartmentation means
by the energy provided by transporting proton along its concentration gradient,vacuolar-type Na~+/H~+ antiporters deliver Na~+ into the vacuoles against the electrochemical gradient generated by vacuolar H~+-ATPase and H~+-PPase.The salt tolerance of plant can be advanced by accumulate to a higher level Na~+ in the cytoplasm through enhance the over expressing of vacuolar H~+-PPase.The enhanced salt tolerance of transgenic Arabidopsis, tomato, rice by overexpressing vacuolar H~+-PPase genes indicated their important roles in plant salt tolerance. Chenopodium glaucum L. is a Chenopodiacea halophytic plant species, which widely distributes in Xinjiang.It is the typical salt-tolerant plant, and it can grow normally under 100~600mmol/L NaCl treatment.The appearance of the whole plant of C.glaucum has neither chylocaulous stem, nor also no any specialized leaf, relatively closer to that of glycophyte.Therefore, it is more valuable to study the mechanism of salt tolerance and for applying on glycophyte to enhance the salt tolerance.Based on all of these, in this thesis, a binary expression vector pCAMBIA1301.1-CgVP1 which harbored CgVP1 gene was constructed and introduced into Arabidopsis thaliana and Nicotiana tabaccum plant by Agrobacterium-mediated method.The function analyses of CgVP1 transgenic plants have been done by molecular identification and some physiological indexes assay.The key results were as follows:
1. The binary expression vector pCAMBIA1301.1-CgVP1 was introduced into A. thaliana by Agrobacterium-mediated method.T3 homogenous plantlets were obtained by screening hygromycin-resistant seedlings.PCR and RT-PCR analysis indicated that CgVP1 have been integrated into the genome of A.thaliana and expressed at transcriptional level.Overexpression of CgVP1 gene in A. thaliana led to enhanced tolerance to salt stress.The relative growth rate of wild-type was restrained markedly under 150mmol/L NaCl treatment.The germination rate of transgenic plants was 91.7%, 89.2%, 84.2%, repectively, while the wild-type was 67.5%, 53.3%, 35.8%, respectively when exposed to 100 mmol/L, 150 mmol/L, 175 mmol/L NaCl.The result of the Na~+, K~+ determination suggested that sodium was compartmentated in the root in order to decrease the sodium concentration of the leaf in transgenic plants under salt stress.Moreover, the relative water and chlorophyll content assay indicated that the relative water content of transgenic plants was higher than that of wild-type,and a higher chlorophyll content was also detected in transgenic plants under salt stress.Our data showed that overexpression of CgVP1 gene could enhance the capacity of salt-tolerance in transgenic A.thaliana.
2. The binary expression vector pCAMBIA1301.1-CgVP1 was introduced into tobacco by Agrobacterium-mediated method.T0 plantlets were obtained by screening the hygromycin- resistant seedlings.PCR and RT-PCR analysis indicated that CgVP1 gene have been integrated into the genome of tobacco and expressed at transcriptional level.The relative growth rate of both wild-type and transgenic plants were restrained lightly under salt treatment.The K~+ /Na~+ rate and the relative water content of transgenic plants were higher than that of wild-type, and the electrolyte leakage rate was lower than that of wild-type after being exposed to 400 mmol/L NaCl for 30 days.The drought tolerance assay indicated that transgenic plants performed better than that of wild-type, such as growth status, almost no hurt after being drought stress for 45 days.But the water loss was too serious in wild-type to be restored.Our data showed that overexpression CgVP1 gene could enhance the capacity both in salt and drought tolerance in transgenic tobacco.
引文
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