水稻硝转运蛋白基因OsNRT1.1a和OsNRT1.1b的功能研究
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摘要
中国农业已经进入高成本时代,生产资料价格已经普遍上涨,种子、肥料、农药等价格都处于上涨趋势中。氮肥在水稻的生产中起到至关重要的作用,近年来中国的氮肥施用量也是急剧上升。中国水稻种植面积占世界总面积的20%,但氮肥施用量却占到世界总施用量的37%,1995年中国的氮肥生产量和使用量已达到世界第一位,但中国的氮肥使用效率却不高,氮肥施用的同时也加剧了环境的污染,导致生态恶化。因此,在现代化农业建设中,通过生物学手段来提高水稻对氮肥的利用率,减少氮肥污染,保护自然资源,是解决氮肥利用率低的一条比较好的途径。植物对硝酸盐的吸收是通过根系一整套高效吸收转运系统-硝酸盐转运蛋白家族来完成的,主要由高亲和低亲和两套吸收转运系统来完成。通过分子生物学方法,得到水稻两个同源性很高的硝酸盐转运蛋白基因OsNRT1.1a, OsNRT1.1b的克隆。本研究以模式水稻品种日本晴(Nipponbare)为材料,通过RT-PCR、超表达、蛙卵异源表达系统、电生理等方法研究了OsNRT1.1a和OsNRT1.1b的功能和表达调控模式,所获得的主要结果如下：
1.通过对两个基因全序列、氨基酸结构以及跨膜分析,得出他们基因的同源性达到95%,二者仅各有一个内含子的差别。但OsNRT1.1b的开放阅读框(ORF)只有OsNRT1.1a的一半长,为OsNRT1.1a的前半部分。通过跨膜预测分析得出OsNRT1.1b为6次跨膜,而OsNRT1.1a为正常的12次跨膜。通过序列分析表明OsNRT1.1a与OsNRT1.1b在水稻基因组中都是以单拷贝的形式存在,都位于3号染色体上,拥有相同的启动子。
2.通过RT-PCR技术,分析了OsNRT1.1a与OsNRT1.1b在水稻中地上部和地下部的基因表达情况,两个基因对不同处理的表达情况具有一定的相似性,在完全缺氮下这两个基因的表达都相对较弱,当以低浓度N03-和NH4+诱导以后,表达量都相应增加许多,因此这两个基因是受氮诱导的。
3.分别克隆了水稻OsNRT1.1a与OsNRT1.1b两个基因,通过农杆菌介导转到水稻愈伤组织中进行过表达实验。对所得到的OsNRT1.1a与OsNRT1.1b两个转基因品种进行不同氮形态和浓度的水培处理后得到结果为这两个品种都明显比野生型植株长得高大、分蘖多、结实率更高。同样在相同处理的土培条件下也得到类似的结果。同时分析了不同处理下水稻地上部地下部总氮的含量,结果显示,地上部总氮含量转基因植物都高于野生型植株,地下部的总氮含量不固定,不同处理结果趋势不相同。在前期用MS培养基同时进行转基因植株与野生型植株发苗,在发苗一周后转基因植株地上部高度明显高于野生型,而后进行的1/4全营养液培养下每四天记录一次地上部、地下部高度和鲜重后得到结果为：转基因植株的地上部生长速度明显快于野生型,处理20天后平均高于野生型5厘米左右,而地下部的生长转基因水稻与野生型没有明显差异。
4.在对水稻中OsNRT1.1a与OsNRT1.1b两个基因分别进行过表达后,转基因植株和野生型同时饥饿一周后进行根部电生理实验分析得到：OsNRT1.1a过表达后对高低浓度的硝态氮的响应情况与野生型类似,而OsNRT1.1b过表达对低浓度的硝态氮只有微弱响应,而对高浓度的硝态氮几乎不响应。同样饥饿一周,OsNRT1.1a过表达后对高低浓度铵态氮的响应情况也与野生型类似,但OsNRT1.1b对高低浓度的铵态氮都有强烈的响应。在正常营养液培养条件下OsNRT1.1b对高低浓度的硝态氮的响应与野生型相比基本没什么差异。在用低浓度的硝态氮诱导培养条件下,OsNRT1.1a的表现基本与野生型相类似,也体现出了OsNRT1.1a为低亲和硝态氮转运蛋白基因的特性,在高浓度的硝态氮条件下响应强烈。而OsNRT1.1b在高低浓度硝态氮条件下的响应都较野生型平淡。
5.蛙卵异源表达结果显示,当注射OsNRT1.1b基因时,蛙卵对低浓度的NO3-表现出大约10mv的去极化,说明蛙卵相应地对NO3-有一定吸收。所以OsNRT1.1b可能是负责硝酸盐吸收转运的一个基因。
China's agriculture has entered the erea of high-cost times. The seeds, ferilizers, pesticides and other prices have been generally increased and the rural labor force is also greatly reduced. China's agriculture has entered the times of the quality of agricutural products and environmental protection. Nitrogen fertilizer plays a crucial role in the rice production. In recent years, Chinese uses of nitrogen fertilizer is also sharply increased. Rice planting area in China accounts for 20% of the total area of the world, but they account for the world's nitrogen fertilizer application rate of 37% in total. In 1995 China's nitrogen fertilizer production and use have reached first in the world, but China's nitrogen fertilizer use efficiency is low, also aggravated the pollution of the environment, leading to ecological deterioration. Therefore, by biological means to increase the utilization of nitrogen fertilizer in rice, reduce nitrogen pollution, protect natural resources, agricultural production is currently a relatively good way. Plant uptake of nitrate is absorbed through the root system by transport protein system to complete nitrate transporter.There are high-affinity and low affinity uptake system to complete the transportion. Through the rice nitrate transporter gene cloning, we got two highly homologous rice nitrate transporter gene OsNRTl.1a and OsNRT1.1b. In this study, model variety Nipponbare (Nipponbare) were used, the function and expression regulation paterns were studied by RT-PCR, electrophysiological and overexpresstion. The main results obtained are as follows:
1. OsNRT1.1a and OsNRT1.1b obtained 95% homology by anaysised of gene, the amino acid sequence and ransmembrane. Both have only one intron difference. But the ORF longthe of OsNRT1.1a is two times of OsNRT1.1b,and the ORF of OsNRT1.1b is the first part of OsNRT1.1a.OsNRT1.1b is 6 times transmembrane by transmembrane prediction analysis. Sequence analysis showed that OsNRT1.1a and OsNRT1.1b both located on chromosome 3,with the same promoter and a single copy in the rice genome.
2. Analyzes the gene expression of OsNRT1.1a and OsNRT1.1b in rice shoots and underground by RT-PCR technology, The results showed that they are not subject to different nitrogen forms and concentrations. This two genes has some similarities on the expression by different treatments. They don't vary with different nirogen forms and different concentrations.This two genes are relatively weak under full nitrogen. when a low concenration of NO3- or NH4+ induced,the corresponding increased in the expression.So these two genes maybe constitutively expressed induced by nitrigen..
3.OsNRT1.1a and OsNRT1.1b were coloned and overexpressed mediaed by agrobacterium through rice callus. These two transgenic plants were treated by different forms and concentrations,and the results showed that these two transgenic plants are significantly higher than WT plants and taller,more tillers and seed set higher.and with the same result treated in soil culture conditions.we analysis the total N of underground and aboveground parts,and the total N of two transgenic plants of aboveground is higher than WT but underground of the total nitrogen content is not fixed.the transgenic plants and wt were cultured at the same time,and the transgenic plants were significantly higher than wild-type after a week culture.The Length of shoot and root was recored every four days.The results showed that the both transgenic plants grow faster than WT,and final average of about 5 cm higher than WT.but the underground part of transgenic plants and WT did not differ significantly.
4. Electrophysiology analyse showed that after overexpresstion of OsNRT1.1a and OsNRT1.1b in rice,the Overexpression of OsNRT1.1a (OEa) has no singinficent difference to WT treated with High and low concentrations of nitrate. The overexpression of OsNRT1.1b(OEb) on low concentration of nitrate is only a weak response but with no response on high concentration of nitrate. the response of OEa was similar to WT treated with high and low concentrations of ammonium, but OEb has a strong response on high and low concentrations of annonium.Low concentrations of nitrate induced expression OEa basic and similar to wild-type. High concentrations of nitrate in response to strong conditions, reflect the lower affinity of OsNRT1.1a nitrate transporter gene characteristics.
5. Heterologous expression in Xenopus showed that,when inject OsNRT1.1b cRNA,it depolarizated about 10mv to low concentrations of NO3-,So Xenopus may absorb certain NO3-.So OsNRT1.1b may be responsible for a nitrate transporter gene.
1.通过对两个基因全序列、氨基酸结构以及跨膜分析,得出他们基因的同源性达到95%,二者仅各有一个内含子的差别。但OsNRT1.1b的开放阅读框(ORF)只有OsNRT1.1a的一半长,为OsNRT1.1a的前半部分。通过跨膜预测分析得出OsNRT1.1b为6次跨膜,而OsNRT1.1a为正常的12次跨膜。通过序列分析表明OsNRT1.1a与OsNRT1.1b在水稻基因组中都是以单拷贝的形式存在,都位于3号染色体上,拥有相同的启动子。
2.通过RT-PCR技术,分析了OsNRT1.1a与OsNRT1.1b在水稻中地上部和地下部的基因表达情况,两个基因对不同处理的表达情况具有一定的相似性,在完全缺氮下这两个基因的表达都相对较弱,当以低浓度N03-和NH4+诱导以后,表达量都相应增加许多,因此这两个基因是受氮诱导的。
3.分别克隆了水稻OsNRT1.1a与OsNRT1.1b两个基因,通过农杆菌介导转到水稻愈伤组织中进行过表达实验。对所得到的OsNRT1.1a与OsNRT1.1b两个转基因品种进行不同氮形态和浓度的水培处理后得到结果为这两个品种都明显比野生型植株长得高大、分蘖多、结实率更高。同样在相同处理的土培条件下也得到类似的结果。同时分析了不同处理下水稻地上部地下部总氮的含量,结果显示,地上部总氮含量转基因植物都高于野生型植株,地下部的总氮含量不固定,不同处理结果趋势不相同。在前期用MS培养基同时进行转基因植株与野生型植株发苗,在发苗一周后转基因植株地上部高度明显高于野生型,而后进行的1/4全营养液培养下每四天记录一次地上部、地下部高度和鲜重后得到结果为：转基因植株的地上部生长速度明显快于野生型,处理20天后平均高于野生型5厘米左右,而地下部的生长转基因水稻与野生型没有明显差异。
4.在对水稻中OsNRT1.1a与OsNRT1.1b两个基因分别进行过表达后,转基因植株和野生型同时饥饿一周后进行根部电生理实验分析得到：OsNRT1.1a过表达后对高低浓度的硝态氮的响应情况与野生型类似,而OsNRT1.1b过表达对低浓度的硝态氮只有微弱响应,而对高浓度的硝态氮几乎不响应。同样饥饿一周,OsNRT1.1a过表达后对高低浓度铵态氮的响应情况也与野生型类似,但OsNRT1.1b对高低浓度的铵态氮都有强烈的响应。在正常营养液培养条件下OsNRT1.1b对高低浓度的硝态氮的响应与野生型相比基本没什么差异。在用低浓度的硝态氮诱导培养条件下,OsNRT1.1a的表现基本与野生型相类似,也体现出了OsNRT1.1a为低亲和硝态氮转运蛋白基因的特性,在高浓度的硝态氮条件下响应强烈。而OsNRT1.1b在高低浓度硝态氮条件下的响应都较野生型平淡。
5.蛙卵异源表达结果显示,当注射OsNRT1.1b基因时,蛙卵对低浓度的NO3-表现出大约10mv的去极化,说明蛙卵相应地对NO3-有一定吸收。所以OsNRT1.1b可能是负责硝酸盐吸收转运的一个基因。
China's agriculture has entered the erea of high-cost times. The seeds, ferilizers, pesticides and other prices have been generally increased and the rural labor force is also greatly reduced. China's agriculture has entered the times of the quality of agricutural products and environmental protection. Nitrogen fertilizer plays a crucial role in the rice production. In recent years, Chinese uses of nitrogen fertilizer is also sharply increased. Rice planting area in China accounts for 20% of the total area of the world, but they account for the world's nitrogen fertilizer application rate of 37% in total. In 1995 China's nitrogen fertilizer production and use have reached first in the world, but China's nitrogen fertilizer use efficiency is low, also aggravated the pollution of the environment, leading to ecological deterioration. Therefore, by biological means to increase the utilization of nitrogen fertilizer in rice, reduce nitrogen pollution, protect natural resources, agricultural production is currently a relatively good way. Plant uptake of nitrate is absorbed through the root system by transport protein system to complete nitrate transporter.There are high-affinity and low affinity uptake system to complete the transportion. Through the rice nitrate transporter gene cloning, we got two highly homologous rice nitrate transporter gene OsNRTl.1a and OsNRT1.1b. In this study, model variety Nipponbare (Nipponbare) were used, the function and expression regulation paterns were studied by RT-PCR, electrophysiological and overexpresstion. The main results obtained are as follows:
1. OsNRT1.1a and OsNRT1.1b obtained 95% homology by anaysised of gene, the amino acid sequence and ransmembrane. Both have only one intron difference. But the ORF longthe of OsNRT1.1a is two times of OsNRT1.1b,and the ORF of OsNRT1.1b is the first part of OsNRT1.1a.OsNRT1.1b is 6 times transmembrane by transmembrane prediction analysis. Sequence analysis showed that OsNRT1.1a and OsNRT1.1b both located on chromosome 3,with the same promoter and a single copy in the rice genome.
2. Analyzes the gene expression of OsNRT1.1a and OsNRT1.1b in rice shoots and underground by RT-PCR technology, The results showed that they are not subject to different nitrogen forms and concentrations. This two genes has some similarities on the expression by different treatments. They don't vary with different nirogen forms and different concentrations.This two genes are relatively weak under full nitrogen. when a low concenration of NO3- or NH4+ induced,the corresponding increased in the expression.So these two genes maybe constitutively expressed induced by nitrigen..
3.OsNRT1.1a and OsNRT1.1b were coloned and overexpressed mediaed by agrobacterium through rice callus. These two transgenic plants were treated by different forms and concentrations,and the results showed that these two transgenic plants are significantly higher than WT plants and taller,more tillers and seed set higher.and with the same result treated in soil culture conditions.we analysis the total N of underground and aboveground parts,and the total N of two transgenic plants of aboveground is higher than WT but underground of the total nitrogen content is not fixed.the transgenic plants and wt were cultured at the same time,and the transgenic plants were significantly higher than wild-type after a week culture.The Length of shoot and root was recored every four days.The results showed that the both transgenic plants grow faster than WT,and final average of about 5 cm higher than WT.but the underground part of transgenic plants and WT did not differ significantly.
4. Electrophysiology analyse showed that after overexpresstion of OsNRT1.1a and OsNRT1.1b in rice,the Overexpression of OsNRT1.1a (OEa) has no singinficent difference to WT treated with High and low concentrations of nitrate. The overexpression of OsNRT1.1b(OEb) on low concentration of nitrate is only a weak response but with no response on high concentration of nitrate. the response of OEa was similar to WT treated with high and low concentrations of ammonium, but OEb has a strong response on high and low concentrations of annonium.Low concentrations of nitrate induced expression OEa basic and similar to wild-type. High concentrations of nitrate in response to strong conditions, reflect the lower affinity of OsNRT1.1a nitrate transporter gene characteristics.
5. Heterologous expression in Xenopus showed that,when inject OsNRT1.1b cRNA,it depolarizated about 10mv to low concentrations of NO3-,So Xenopus may absorb certain NO3-.So OsNRT1.1b may be responsible for a nitrate transporter gene.
引文
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