黄瓜水孔蛋白CsNIP2；1尿素转运功能及其分子机理研究
发布时间：2018-12-14 07:33
【摘要】：氮是植物生长发育过程中必不可少的营养元素之一,而尿素CO(NH2)2不仅是农作物生产中土壤施用氮肥的主要形式,而且是植物体内氮素新陈代谢的主要产物。目前,高等植物从外界环境吸收尿素的过程主要有高亲和性尿素转运蛋白DUR3介导的主动运输途径和低亲和性尿素转运蛋白MIPs介导的被动运输途径。高亲和性尿素转运蛋白DUR3家族相关研究在拟南芥、水稻、玉米等植物中已有详细报道,而水孔蛋白MIPs家族除了具有高效跨膜转运水分子的功能外,还参与其它小分子营养物质如尿素的跨膜运输过程。与高亲和性尿素转运蛋白不同,在植物体内水孔蛋白不仅参与尿素的转运过程,同时也在其它物质的运输途径中发挥重大作用。因此,分离鉴定高等植物体内的水孔蛋白并探究其在尿素等物质转运过程中发挥的生理功能,对探讨农业生产中合理施用尿素、提高氮肥利用效率具有基础性的研究意义。黄瓜是葫芦科重要的双子叶植物代表之一,其产量和品质均依赖于氮素等营养物质的合理施用。本文从植物分子生物学角度系统地对黄瓜水孔蛋白CsNIP2;1在尿素转运功能、转运特性、组织特异性表达、亚细胞定位以及诱导表达模式等方面进行探讨。结论如下：1.同源序列比较分析发现黄瓜基因组中水孔蛋白CsNIP2;1与南瓜尿素转运蛋白CpNIPl的同源性较高,生物信息学分析显示CsNIP2;1长867bp,编码288个氨基酸,拥有6个跨膜区域,两边的N端和C端均位于膜内,在跨膜区域之间存在两个分别位于细胞内和细胞外的NPA/NPA(V)保守区域；2.选用酵母尿素吸收缺陷型突变菌株,其在低浓度尿素作为唯一氮源条件下无法正常生长。CsNIP2;1的异源表达会让酵母突变体恢复正常生长,这一尿素转运过程会受到高pH条件的抑制作用。在渗透条件以及甘油存在的条件下,酵母功能互补检测显示CsNIP2;1不仅具有转运尿素的功能,还具有转运水的功能,但对甘油没有渗透性；3.利用转基因技术获得表达CsNIP2;1启动子的拟南芥植株,对转基因拟南芥幼苗以及成熟期的花、果荚等不同组织进行GUS化学染色,结果显示CsNIP2;1在根部和子叶中的表达量较高,在成熟组织的花萼和果荚中均有表达。采用实时定量PCR技术对黄瓜种子萌发时期CsNIP2;1的转录水平表达进行检测,同时对转化CsNIP2;1启动子的转基因拟南芥植株在种子萌发初期进行GUS组织化学染色分析,结果均显示CsNIP2;1在植株种子萌发初期表达量较高；4.根据蔗糖密度梯度离心原理建立黄瓜原生质体分离提取方法,构建原生质体重组表达载体CsNIP2;1-EGFP,分别分离提取黄瓜原生质体和拟南芥原生质体后,采用PEG转化方法进行基因瞬时表达。荧光显微镜下观察显示CsNIP2;1-EGFP发出的绿光出现在原生质体边缘,并且与细胞膜染料FM4-64发出的红光完全重合,结果证明CsNIP2;1定位于植物细胞膜上；5.采用实时定量PCR技术对不同外源氮源处理下的黄瓜根部进行转录水平上CsNIP2;1的诱导表达分析,同时采用相同的处理方式,对不同外源氮源条件下转化CsNIP2;1启动子的拟南芥植株进行GUS组织化学染色,两者结果均显示黄瓜CsNIP2;1分别会受到缺氮处理、尿素和硝酸盐诱导的上调表达；6.选用野生型拟南芥Wt和拟南芥尿素吸收缺陷型突变体atdur3-3, atdur3-3突变体在低浓度尿素作为唯一氮源生长条件下因无法吸收足够氮营养而出现缺氮表型。构建植物重组过表达载体csNIP2;1-Early102,利用转基因技术将CsNIP2;1分别转入Wt和atdur3-3,筛选获得过表达CsNIP2;1的纯合子转基因拟南芥植株,生长表型结果显示在低浓度尿素作为唯一氮源条件下,CsNIP2;1的表达不仅可以提高野生型拟南芥wt的生长状况,而且还可明显恢复拟南芥尿素吸收缺陷型突变体atdur3-3的缺氮生长表型,从而证明在植物体内CsNIP2;1呈现了尿素转运功能。以上研究结果证明黄瓜水孔蛋白CsNIP2;1是位于植物细胞膜上的尿素转运蛋白,在植物体内尿素循环代谢途径中发挥重要作用,即尿素由外界环境进入根部的吸收转运、种子萌发初期氮素活化和再利用过程中植物内源尿素的运输以及由根部向发育组织转运氮素的过程。
[Abstract]:Nitrogen is one of the essential nutrient elements in the process of plant growth and development, and urea CO (NH2) 2 is not only the main form of soil application in crop production, but also the main product of nitrogen metabolism in plant. At present, the process of absorbing urea from the external environment mainly includes the high-affinity urea transport protein DUR3-mediated active transport route and the passive transport route mediated by the low-affinity urea transporter MIPs. The high-affinity urea transporter DUR3 family-related research has been reported in detail in Arabidopsis, rice, and maize plants, while the aquaporin-MIPs family is involved in the cross-membrane transport process of other small molecular nutrients, such as urea, in addition to the function of high-efficiency transmembrane transport water molecules. in contrast to that high-affinity urea-transport protein, the aquaporin in the plant not only participates in the transport of the urea, but also plays a significant role in the transport route of other substances. Therefore, it is of great significance to separate and identify the aquaporin in higher plants and to explore its physiological function in the process of the transfer of urea and other substances, and to study the rational application of urea in agricultural production and to improve the use efficiency of nitrogen fertilizer. Cucumber is one of the important dicotyledonous plant representatives in the Cucurbitaceae, and its yield and quality depend on the rational application of nutrients such as nitrogen. In this paper, the cucumber aquaporin CsNIP2 is systematically studied from the point of view of the plant molecular biology, and the transport function, the transport characteristic, the tissue-specific expression, the subcellular localization and the induction of the expression pattern are discussed. The conclusion is as follows: 1. The homologous sequence comparison analysis found that the homologies of the aquaporin CsNIP2, 1 and the pumpkin urea transporter CpNIPl were high, the bioinformatics analysis showed that the CsNIP2, the length of 867bp, the encoding 288 amino acids, the six cross-membrane regions, both the N-and C-ends of the two sides were located in the membrane, there are two npa/ npa (v) conserved regions located in and out of the cell, respectively, between the transmembrane regions; 2. the yeast-urea absorption-deficient mutant strain is selected, and the mutant strain can not grow normally under the condition of low-concentration urea as the sole nitrogen source. The isogenic expression of CsNIP2; 1 results in a normal growth of the yeast mutant, which is inhibited by high pH conditions. Under the conditions of penetration and the presence of glycerol, the complementary detection of yeast function shows that CsNIP2; 1 not only has the function of transferring urea, but also has the function of transporting water, but has no permeability to glycerol; 3. The expression of CsNIP2 is obtained by using the transgenic technology, and GUS chemical staining is carried out on the transgenic Arabidopsis seedlings and different tissues such as the flowers and the fruits of the mature period, the results show that the expression amount of the CsNIP2; 1 in the roots and the cotyledons is high, It is expressed in both the flower and fruit of the mature tissue. The expression of CsNIP2, 1 in the seed germination of cucumber was detected by real-time quantitative PCR, and the transformation of CsNIP2, the transgenic Arabidopsis plant of the promoter was analyzed by the histochemical staining of GUS in the early stage of the seed germination, and the results showed that CsNIP2 was found. 1. The expression was higher in the initial stage of the germination of the plant. according to the sucrose density gradient centrifugation principle, the protoplast isolation and extraction method is established, the protoplast recombinant expression vector CsNIP2, 1-EGFP is constructed, the cucumber protoplast and the Arabidopsis protoplast are separately extracted, and the gene transient expression is carried out by adopting a PEG transformation method. Under the fluorescence microscope, CsNIP2 was observed; the green light emitted from 1-EGFP appeared on the edge of the protoplast, and the red light emitted from the cell membrane dye FM4-64 was completely coincident, and the result showed that the CsNIP2; 1 was located on the cell membrane of the plant; and 5. in that method, a real-time quantitative PCR technique is adopt to carry out the induction and expression analysis of CsNIP2; 1 on the root of the cucumber under the treatment of different exogenous nitrogen sources, and the same treatment method is adopted to carry out GUS histochemical staining on the Arabidopsis plant which is transformed with the CsNIP2; 1 promoter under the condition of different exogenous nitrogen sources, The results showed that the cucumber CsNIP2; 1 was induced by nitrogen-deficient treatment, urea and nitrate, and 6. The mutant atdur3-3, atdur3-3, a wild-type arabidopsis thaliana Wt and an Arabidopsis urea-absorption-deficient mutant, was selected to develop a nitrogen-deficient phenotype under the condition of low-concentration urea as the only nitrogen source. constructing a plant recombinant overexpressing vector csNIP2; 1-Early102, transferring the CsNIP2; 1 into the Wt and the atdri3-3 by using the transgenic technology, and screening to obtain a homozygote transgenic Arabidopsis plant expressing the CsNIP2; 1, wherein the growth phenotype results show that the CsNIP2 is under the condition of the low-concentration urea as the sole nitrogen source; the expression of 1 can not only improve the growth condition of the wild type arabidopsis thaliana wt, but also can obviously restore the nitrogen-deficient growth phenotype of the arabidopsis urea absorption-deficient mutant atdur3-3, so that the CsNIP2 is present in the plant body, and the urea transfer function is presented in the plant body. The results of the above research prove that the cucumber aquaporin CsNIP2; 1 is a urea transporter located on the cell membrane of the plant, and plays an important role in the urea cycle metabolic pathway in the plant, that is, the absorption and transfer of the urea from the external environment to the root, During the initial stage of seed germination, the transport of endogenous urea and the transport of nitrogen from the root to the development tissue during the initial stage of nitrogen activation and re-utilization of the seeds.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q946
本文编号：2378198
[Abstract]:Nitrogen is one of the essential nutrient elements in the process of plant growth and development, and urea CO (NH2) 2 is not only the main form of soil application in crop production, but also the main product of nitrogen metabolism in plant. At present, the process of absorbing urea from the external environment mainly includes the high-affinity urea transport protein DUR3-mediated active transport route and the passive transport route mediated by the low-affinity urea transporter MIPs. The high-affinity urea transporter DUR3 family-related research has been reported in detail in Arabidopsis, rice, and maize plants, while the aquaporin-MIPs family is involved in the cross-membrane transport process of other small molecular nutrients, such as urea, in addition to the function of high-efficiency transmembrane transport water molecules. in contrast to that high-affinity urea-transport protein, the aquaporin in the plant not only participates in the transport of the urea, but also plays a significant role in the transport route of other substances. Therefore, it is of great significance to separate and identify the aquaporin in higher plants and to explore its physiological function in the process of the transfer of urea and other substances, and to study the rational application of urea in agricultural production and to improve the use efficiency of nitrogen fertilizer. Cucumber is one of the important dicotyledonous plant representatives in the Cucurbitaceae, and its yield and quality depend on the rational application of nutrients such as nitrogen. In this paper, the cucumber aquaporin CsNIP2 is systematically studied from the point of view of the plant molecular biology, and the transport function, the transport characteristic, the tissue-specific expression, the subcellular localization and the induction of the expression pattern are discussed. The conclusion is as follows: 1. The homologous sequence comparison analysis found that the homologies of the aquaporin CsNIP2, 1 and the pumpkin urea transporter CpNIPl were high, the bioinformatics analysis showed that the CsNIP2, the length of 867bp, the encoding 288 amino acids, the six cross-membrane regions, both the N-and C-ends of the two sides were located in the membrane, there are two npa/ npa (v) conserved regions located in and out of the cell, respectively, between the transmembrane regions; 2. the yeast-urea absorption-deficient mutant strain is selected, and the mutant strain can not grow normally under the condition of low-concentration urea as the sole nitrogen source. The isogenic expression of CsNIP2; 1 results in a normal growth of the yeast mutant, which is inhibited by high pH conditions. Under the conditions of penetration and the presence of glycerol, the complementary detection of yeast function shows that CsNIP2; 1 not only has the function of transferring urea, but also has the function of transporting water, but has no permeability to glycerol; 3. The expression of CsNIP2 is obtained by using the transgenic technology, and GUS chemical staining is carried out on the transgenic Arabidopsis seedlings and different tissues such as the flowers and the fruits of the mature period, the results show that the expression amount of the CsNIP2; 1 in the roots and the cotyledons is high, It is expressed in both the flower and fruit of the mature tissue. The expression of CsNIP2, 1 in the seed germination of cucumber was detected by real-time quantitative PCR, and the transformation of CsNIP2, the transgenic Arabidopsis plant of the promoter was analyzed by the histochemical staining of GUS in the early stage of the seed germination, and the results showed that CsNIP2 was found. 1. The expression was higher in the initial stage of the germination of the plant. according to the sucrose density gradient centrifugation principle, the protoplast isolation and extraction method is established, the protoplast recombinant expression vector CsNIP2, 1-EGFP is constructed, the cucumber protoplast and the Arabidopsis protoplast are separately extracted, and the gene transient expression is carried out by adopting a PEG transformation method. Under the fluorescence microscope, CsNIP2 was observed; the green light emitted from 1-EGFP appeared on the edge of the protoplast, and the red light emitted from the cell membrane dye FM4-64 was completely coincident, and the result showed that the CsNIP2; 1 was located on the cell membrane of the plant; and 5. in that method, a real-time quantitative PCR technique is adopt to carry out the induction and expression analysis of CsNIP2; 1 on the root of the cucumber under the treatment of different exogenous nitrogen sources, and the same treatment method is adopted to carry out GUS histochemical staining on the Arabidopsis plant which is transformed with the CsNIP2; 1 promoter under the condition of different exogenous nitrogen sources, The results showed that the cucumber CsNIP2; 1 was induced by nitrogen-deficient treatment, urea and nitrate, and 6. The mutant atdur3-3, atdur3-3, a wild-type arabidopsis thaliana Wt and an Arabidopsis urea-absorption-deficient mutant, was selected to develop a nitrogen-deficient phenotype under the condition of low-concentration urea as the only nitrogen source. constructing a plant recombinant overexpressing vector csNIP2; 1-Early102, transferring the CsNIP2; 1 into the Wt and the atdri3-3 by using the transgenic technology, and screening to obtain a homozygote transgenic Arabidopsis plant expressing the CsNIP2; 1, wherein the growth phenotype results show that the CsNIP2 is under the condition of the low-concentration urea as the sole nitrogen source; the expression of 1 can not only improve the growth condition of the wild type arabidopsis thaliana wt, but also can obviously restore the nitrogen-deficient growth phenotype of the arabidopsis urea absorption-deficient mutant atdur3-3, so that the CsNIP2 is present in the plant body, and the urea transfer function is presented in the plant body. The results of the above research prove that the cucumber aquaporin CsNIP2; 1 is a urea transporter located on the cell membrane of the plant, and plays an important role in the urea cycle metabolic pathway in the plant, that is, the absorption and transfer of the urea from the external environment to the root, During the initial stage of seed germination, the transport of endogenous urea and the transport of nitrogen from the root to the development tissue during the initial stage of nitrogen activation and re-utilization of the seeds.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q946
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1 张璐;黄瓜水孔蛋白CsNIP2；1尿素转运功能及其分子机理研究[D];中国农业大学;2016年
，本文编号：2378198
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