黃瓜水孔蛋白CsNIP2；1尿素轉(zhuǎn)運(yùn)功能及其分子機(jī)理研究
發(fā)布時(shí)間：2018-12-14 07:33
【摘要】：氮是植物生長(zhǎng)發(fā)育過程中必不可少的營(yíng)養(yǎng)元素之一,而尿素CO(NH2)2不僅是農(nóng)作物生產(chǎn)中土壤施用氮肥的主要形式,而且是植物體內(nèi)氮素新陳代謝的主要產(chǎn)物。目前,高等植物從外界環(huán)境吸收尿素的過程主要有高親和性尿素轉(zhuǎn)運(yùn)蛋白DUR3介導(dǎo)的主動(dòng)運(yùn)輸途徑和低親和性尿素轉(zhuǎn)運(yùn)蛋白MIPs介導(dǎo)的被動(dòng)運(yùn)輸途徑。高親和性尿素轉(zhuǎn)運(yùn)蛋白DUR3家族相關(guān)研究在擬南芥、水稻、玉米等植物中已有詳細(xì)報(bào)道,而水孔蛋白MIPs家族除了具有高效跨膜轉(zhuǎn)運(yùn)水分子的功能外,還參與其它小分子營(yíng)養(yǎng)物質(zhì)如尿素的跨膜運(yùn)輸過程。與高親和性尿素轉(zhuǎn)運(yùn)蛋白不同,在植物體內(nèi)水孔蛋白不僅參與尿素的轉(zhuǎn)運(yùn)過程,同時(shí)也在其它物質(zhì)的運(yùn)輸途徑中發(fā)揮重大作用。因此,分離鑒定高等植物體內(nèi)的水孔蛋白并探究其在尿素等物質(zhì)轉(zhuǎn)運(yùn)過程中發(fā)揮的生理功能,對(duì)探討農(nóng)業(yè)生產(chǎn)中合理施用尿素、提高氮肥利用效率具有基礎(chǔ)性的研究意義。黃瓜是葫蘆科重要的雙子葉植物代表之一,其產(chǎn)量和品質(zhì)均依賴于氮素等營(yíng)養(yǎng)物質(zhì)的合理施用。本文從植物分子生物學(xué)角度系統(tǒng)地對(duì)黃瓜水孔蛋白CsNIP2;1在尿素轉(zhuǎn)運(yùn)功能、轉(zhuǎn)運(yùn)特性、組織特異性表達(dá)、亞細(xì)胞定位以及誘導(dǎo)表達(dá)模式等方面進(jìn)行探討。結(jié)論如下：1.同源序列比較分析發(fā)現(xiàn)黃瓜基因組中水孔蛋白CsNIP2;1與南瓜尿素轉(zhuǎn)運(yùn)蛋白CpNIPl的同源性較高,生物信息學(xué)分析顯示CsNIP2;1長(zhǎng)867bp,編碼288個(gè)氨基酸,擁有6個(gè)跨膜區(qū)域,兩邊的N端和C端均位于膜內(nèi),在跨膜區(qū)域之間存在兩個(gè)分別位于細(xì)胞內(nèi)和細(xì)胞外的NPA/NPA(V)保守區(qū)域；2.選用酵母尿素吸收缺陷型突變菌株,其在低濃度尿素作為唯一氮源條件下無法正常生長(zhǎng)。CsNIP2;1的異源表達(dá)會(huì)讓酵母突變體恢復(fù)正常生長(zhǎng),這一尿素轉(zhuǎn)運(yùn)過程會(huì)受到高pH條件的抑制作用。在滲透條件以及甘油存在的條件下,酵母功能互補(bǔ)檢測(cè)顯示CsNIP2;1不僅具有轉(zhuǎn)運(yùn)尿素的功能,還具有轉(zhuǎn)運(yùn)水的功能,但對(duì)甘油沒有滲透性；3.利用轉(zhuǎn)基因技術(shù)獲得表達(dá)CsNIP2;1啟動(dòng)子的擬南芥植株,對(duì)轉(zhuǎn)基因擬南芥幼苗以及成熟期的花、果莢等不同組織進(jìn)行GUS化學(xué)染色,結(jié)果顯示CsNIP2;1在根部和子葉中的表達(dá)量較高,在成熟組織的花萼和果莢中均有表達(dá)。采用實(shí)時(shí)定量PCR技術(shù)對(duì)黃瓜種子萌發(fā)時(shí)期CsNIP2;1的轉(zhuǎn)錄水平表達(dá)進(jìn)行檢測(cè),同時(shí)對(duì)轉(zhuǎn)化CsNIP2;1啟動(dòng)子的轉(zhuǎn)基因擬南芥植株在種子萌發(fā)初期進(jìn)行GUS組織化學(xué)染色分析,結(jié)果均顯示CsNIP2;1在植株種子萌發(fā)初期表達(dá)量較高；4.根據(jù)蔗糖密度梯度離心原理建立黃瓜原生質(zhì)體分離提取方法,構(gòu)建原生質(zhì)體重組表達(dá)載體CsNIP2;1-EGFP,分別分離提取黃瓜原生質(zhì)體和擬南芥原生質(zhì)體后,采用PEG轉(zhuǎn)化方法進(jìn)行基因瞬時(shí)表達(dá)。熒光顯微鏡下觀察顯示CsNIP2;1-EGFP發(fā)出的綠光出現(xiàn)在原生質(zhì)體邊緣,并且與細(xì)胞膜染料FM4-64發(fā)出的紅光完全重合,結(jié)果證明CsNIP2;1定位于植物細(xì)胞膜上；5.采用實(shí)時(shí)定量PCR技術(shù)對(duì)不同外源氮源處理下的黃瓜根部進(jìn)行轉(zhuǎn)錄水平上CsNIP2;1的誘導(dǎo)表達(dá)分析,同時(shí)采用相同的處理方式,對(duì)不同外源氮源條件下轉(zhuǎn)化CsNIP2;1啟動(dòng)子的擬南芥植株進(jìn)行GUS組織化學(xué)染色,兩者結(jié)果均顯示黃瓜CsNIP2;1分別會(huì)受到缺氮處理、尿素和硝酸鹽誘導(dǎo)的上調(diào)表達(dá)；6.選用野生型擬南芥Wt和擬南芥尿素吸收缺陷型突變體atdur3-3, atdur3-3突變體在低濃度尿素作為唯一氮源生長(zhǎng)條件下因無法吸收足夠氮營(yíng)養(yǎng)而出現(xiàn)缺氮表型。構(gòu)建植物重組過表達(dá)載體csNIP2;1-Early102,利用轉(zhuǎn)基因技術(shù)將CsNIP2;1分別轉(zhuǎn)入Wt和atdur3-3,篩選獲得過表達(dá)CsNIP2;1的純合子轉(zhuǎn)基因擬南芥植株,生長(zhǎng)表型結(jié)果顯示在低濃度尿素作為唯一氮源條件下,CsNIP2;1的表達(dá)不僅可以提高野生型擬南芥wt的生長(zhǎng)狀況,而且還可明顯恢復(fù)擬南芥尿素吸收缺陷型突變體atdur3-3的缺氮生長(zhǎng)表型,從而證明在植物體內(nèi)CsNIP2;1呈現(xiàn)了尿素轉(zhuǎn)運(yùn)功能。以上研究結(jié)果證明黃瓜水孔蛋白CsNIP2;1是位于植物細(xì)胞膜上的尿素轉(zhuǎn)運(yùn)蛋白,在植物體內(nèi)尿素循環(huán)代謝途徑中發(fā)揮重要作用,即尿素由外界環(huán)境進(jìn)入根部的吸收轉(zhuǎn)運(yùn)、種子萌發(fā)初期氮素活化和再利用過程中植物內(nèi)源尿素的運(yùn)輸以及由根部向發(fā)育組織轉(zhuǎn)運(yùn)氮素的過程。
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q946
本文編號(hào)：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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q946
【相似文獻(xiàn)】
相關(guān)博士學(xué)位論文 前1條
1 張璐;黃瓜水孔蛋白CsNIP2；1尿素轉(zhuǎn)運(yùn)功能及其分子機(jī)理研究[D];中國(guó)農(nóng)業(yè)大學(xué);2016年
，本文編號(hào)：2378198
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