盐芥钠磷转运体基因在转基因烟草中的功能鉴定
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摘要
磷是植物生长和发育不可缺少的大量营养元素之一,是植物体内重要的组成元素,同时又以多种方式参与植物体内的各种生理生化过程。因此,植物对磷元素的吸收以及磷元素在植物体内的代谢成为植物科学研究的重点课题之一。
本实验室从盐生植物盐芥中克隆出一个Na~+/pi转运体基因,并在酵母突变体中进行了功能验证和编码产物磷转运活性的研究,确定了该基因编码的转运体是一种高亲和Pi转运体,其Km分别为7.4μM(pH=6.0)和8.7μM(pH=7.5)。该基因主要在盐芥的叶中进行表达,通过GFP融合蛋白将该蛋白定位于叶绿体上。在高等植物中,有关Na~+/Pi转运体基因的克隆及功能鉴定鲜有报道,其生物学功能有待研究。
本工作通过PCR技术获得了盐芥Na~+/Pi转运体基因全长序列,经分析发现该基因含有8个内含子、9个外显子,编码一条多肽。对盐芥基因组进行Southernblotting分析,发现Na~+/pi转运体基因为单拷贝基因。将基因切除N-端信号肽编码序列后与gfp融合,转入酵母中进行表达并检测荧光信号,发现融合基因产物定位于酵母细胞膜上,为该基因能够成功修复酵母突变体的磷转运功能提供了细胞学证据。
将Na~+/pi转运体基因转入烟草,PCR检测和Southern杂交结果表明,该基因已成功整合到转基因烟草的基因组中。采用实时荧光定量RT-PCR分析转基因烟草中Na~+/pi转运体基因的表达强度,以烟草内源actin的表达强度为参照,结果表明Na~+/pi转运体基因在转基因植株中有较高强度的表达。从转基因烟草植物分离叶绿体,测定Na~+/Pi转运体蛋白的磷转运活性,结果表明转基因植株的磷转运活性与对照植株相比提高了10%-20%,不同株系的磷转运活性提高幅度有差异。
在低磷培养基上,转基因烟草比对照烟草生长快,小苗根系较长,叶片数多于同期的对照植株,表明该基因在磷胁迫环境中有助于植物对磷的利用和维持较高的生长速率。利用溶液培养的方法研究低磷条件下不同烟草植株的生理变化,发现在低磷处理14天后,转基因植株的根冠比明显高于对照植株的。光合作用测定结果表明转基因烟草的光合速率比对照植株有提高。Na~+/Pi转运体蛋白定位于叶绿体,低磷条件下转基因植株离体叶绿体表现出提高的磷转运活性可能直接或间接影响光合速率,从而增加细胞磷效率。这些结果表明过表达Na~+/Pi转运体蛋白可以提高植物对低磷环境的适应性。
本工作还根据Na~+/Pi转运体基因序列设计并构建出RNAi干扰体,并用以转化拟南芥和盐芥,为深入了解该基因的生物学功能完成了部分基础工作。
Phosphorous (Pi) is an essential macronutrient for plants growth and development. It serves various basic biological functions as a structural element in nucleic acids and phospholipids, in energy metabolism, and in the activation of metabolic intermediates, as a component in signal transduction cascades, and the regulation of enzymes. So it has become the focus on the absorption and metabolism of Pi in the plant science.
Na~+/Pi transporter gene was cloned from T.halophila in our lab. Its function and activity of Pi transport has been studied in yeast mutant PAM2. The results showed that the gene encodes a high affinity Pi transporter. The Km of Na~+/Pi transporter under pH6.0 and pH7.5 is 7.4μM and 8.7μM, respectively. This gene was mainly expressed in leaves. And the protein was located in chloroplast by expressing N terminus GFP fusion protein in tobacco cells. But in higher plants, the reports on studying about Na~+/Pi transporter gene is few and its concrete function still needs further study.
The whole gene DNA sequence of Na~+/Pi transporter gene was cloned by PCR in this work. Sequence analysis showed that this gene had 8 introns and 9 exons. And it encoded a peptide. We found that there was only one copy of Na~+/Pi transporter gene in T.halophila by Southern blotting analysis. Excising its signal peptide and heterogeneously expressed in yeast, Na~+/Pi transporter was located in the membrane of yeast cell. The result provided cytologic evidence for Pi transport in functional complementarity to mutant yeast.
Na~+/Pi transporter gene was transformed into tobacco by Agrobacterium mediated method. By PCR and Southern blotting analysis, a number of transgenic lines were identified. And the expression of Na~+/Pi transporter gene was analyzed by quantitative fluorescent RT-PCR. The results showed that Na~+/Pi transporter gene presented high expression in transgenic tobacco lines. Intact chloroplasts were isolated to determinate the Pi transport activity of Na~+/Pi transporter. The results showed that the Pi transport activity of chloroplasts in transgenic tobacco lines was 10-20% higher than that in wild tobacco line.
In low-Pi medium, the transgenic tobacco lines showed higher growth rates compared with wild tobacco lines and the number of leaf was more than that of wild type tobacco lines at the same stages. This presented that the transgenic tobacco lines increased the absorption of Pi and kept high growth rate. By nutrient solution culturing method, the physiological characteristics of transgenic and wild tobacco lines under lower Pi conditions were studied. The results showed that after 14 days treatment, the rate of root to shoot of transgenic tobacco lines was higher than that of wild lines. The results showed that the gene could increase the response of transgenic tobacco to lower Pi conditions. Further more the photosynthetic characteristics was analyzed, showing that the rate of photosynthesis of transgenic tobacco lines was higher than that of wild lines. The Pi transport activity of chloroplasts of transgenic tobacco lines was higher than that of wild tobacco lines. The gene could increase the usage of Pi of transgenic tobacco. So it may increase the rate of photosynthesis of transgenic tobacco directly or indirectly.
In addition, the RNAi vector was constructed according to the sequence of the gene and planned to transform T.halophila or Arabidopsis thaliana for further analysis. It could be said that the partly job in the process of studying the function of Na/Pi transporter has been done.
本实验室从盐生植物盐芥中克隆出一个Na~+/pi转运体基因,并在酵母突变体中进行了功能验证和编码产物磷转运活性的研究,确定了该基因编码的转运体是一种高亲和Pi转运体,其Km分别为7.4μM(pH=6.0)和8.7μM(pH=7.5)。该基因主要在盐芥的叶中进行表达,通过GFP融合蛋白将该蛋白定位于叶绿体上。在高等植物中,有关Na~+/Pi转运体基因的克隆及功能鉴定鲜有报道,其生物学功能有待研究。
本工作通过PCR技术获得了盐芥Na~+/Pi转运体基因全长序列,经分析发现该基因含有8个内含子、9个外显子,编码一条多肽。对盐芥基因组进行Southernblotting分析,发现Na~+/pi转运体基因为单拷贝基因。将基因切除N-端信号肽编码序列后与gfp融合,转入酵母中进行表达并检测荧光信号,发现融合基因产物定位于酵母细胞膜上,为该基因能够成功修复酵母突变体的磷转运功能提供了细胞学证据。
将Na~+/pi转运体基因转入烟草,PCR检测和Southern杂交结果表明,该基因已成功整合到转基因烟草的基因组中。采用实时荧光定量RT-PCR分析转基因烟草中Na~+/pi转运体基因的表达强度,以烟草内源actin的表达强度为参照,结果表明Na~+/pi转运体基因在转基因植株中有较高强度的表达。从转基因烟草植物分离叶绿体,测定Na~+/Pi转运体蛋白的磷转运活性,结果表明转基因植株的磷转运活性与对照植株相比提高了10%-20%,不同株系的磷转运活性提高幅度有差异。
在低磷培养基上,转基因烟草比对照烟草生长快,小苗根系较长,叶片数多于同期的对照植株,表明该基因在磷胁迫环境中有助于植物对磷的利用和维持较高的生长速率。利用溶液培养的方法研究低磷条件下不同烟草植株的生理变化,发现在低磷处理14天后,转基因植株的根冠比明显高于对照植株的。光合作用测定结果表明转基因烟草的光合速率比对照植株有提高。Na~+/Pi转运体蛋白定位于叶绿体,低磷条件下转基因植株离体叶绿体表现出提高的磷转运活性可能直接或间接影响光合速率,从而增加细胞磷效率。这些结果表明过表达Na~+/Pi转运体蛋白可以提高植物对低磷环境的适应性。
本工作还根据Na~+/Pi转运体基因序列设计并构建出RNAi干扰体,并用以转化拟南芥和盐芥,为深入了解该基因的生物学功能完成了部分基础工作。
Phosphorous (Pi) is an essential macronutrient for plants growth and development. It serves various basic biological functions as a structural element in nucleic acids and phospholipids, in energy metabolism, and in the activation of metabolic intermediates, as a component in signal transduction cascades, and the regulation of enzymes. So it has become the focus on the absorption and metabolism of Pi in the plant science.
Na~+/Pi transporter gene was cloned from T.halophila in our lab. Its function and activity of Pi transport has been studied in yeast mutant PAM2. The results showed that the gene encodes a high affinity Pi transporter. The Km of Na~+/Pi transporter under pH6.0 and pH7.5 is 7.4μM and 8.7μM, respectively. This gene was mainly expressed in leaves. And the protein was located in chloroplast by expressing N terminus GFP fusion protein in tobacco cells. But in higher plants, the reports on studying about Na~+/Pi transporter gene is few and its concrete function still needs further study.
The whole gene DNA sequence of Na~+/Pi transporter gene was cloned by PCR in this work. Sequence analysis showed that this gene had 8 introns and 9 exons. And it encoded a peptide. We found that there was only one copy of Na~+/Pi transporter gene in T.halophila by Southern blotting analysis. Excising its signal peptide and heterogeneously expressed in yeast, Na~+/Pi transporter was located in the membrane of yeast cell. The result provided cytologic evidence for Pi transport in functional complementarity to mutant yeast.
Na~+/Pi transporter gene was transformed into tobacco by Agrobacterium mediated method. By PCR and Southern blotting analysis, a number of transgenic lines were identified. And the expression of Na~+/Pi transporter gene was analyzed by quantitative fluorescent RT-PCR. The results showed that Na~+/Pi transporter gene presented high expression in transgenic tobacco lines. Intact chloroplasts were isolated to determinate the Pi transport activity of Na~+/Pi transporter. The results showed that the Pi transport activity of chloroplasts in transgenic tobacco lines was 10-20% higher than that in wild tobacco line.
In low-Pi medium, the transgenic tobacco lines showed higher growth rates compared with wild tobacco lines and the number of leaf was more than that of wild type tobacco lines at the same stages. This presented that the transgenic tobacco lines increased the absorption of Pi and kept high growth rate. By nutrient solution culturing method, the physiological characteristics of transgenic and wild tobacco lines under lower Pi conditions were studied. The results showed that after 14 days treatment, the rate of root to shoot of transgenic tobacco lines was higher than that of wild lines. The results showed that the gene could increase the response of transgenic tobacco to lower Pi conditions. Further more the photosynthetic characteristics was analyzed, showing that the rate of photosynthesis of transgenic tobacco lines was higher than that of wild lines. The Pi transport activity of chloroplasts of transgenic tobacco lines was higher than that of wild tobacco lines. The gene could increase the usage of Pi of transgenic tobacco. So it may increase the rate of photosynthesis of transgenic tobacco directly or indirectly.
In addition, the RNAi vector was constructed according to the sequence of the gene and planned to transform T.halophila or Arabidopsis thaliana for further analysis. It could be said that the partly job in the process of studying the function of Na/Pi transporter has been done.
引文
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73. Heungsop Shinl, Hwa-Soo Shinl, Gary R. Dewbrel and Maria J. Harrison2, Phosphate transport in Arabidopsis: Pht1;1 and Pht1;4 play a major role in phosphate acquisition from both low- and high-phosphate environments. The Plant Journal (2004) 39,629-642
74. Catarecha~1, M~a Dolores Segura~1, Jose Manuel Franco-Zorrilla, Berenice Garcia-Ponce~2, Monica Lanza, Roberto Solano, Javier Paz-Ares and Antonio Leyva~3 A Mutant of the Arabidopsis Phosphate Transporter PHT1;1 Displays Enhanced Arsenic Accumulation. The Plant Cell 19:1123-1133 (2007)
75. Daram P, Brunner S, Rausch C, Steiner C, Amrhein N, Bucher M (1999) Pht2;1 encodes a low-affinity phosphate transporter from Arabidopsis. Plant Cell 11:2153-2166
76. Martinez P, Persson B (1998) Identification, cloning and characterization of a derepressible Na~+-coupled phosphate transporter in Saccharomyces cerevisiae. Mol Gen Genet, 258:628-638.
77. Versaw WK (1995) A phosphate-repressible, high-affmity phosphate permease is encoded by the pho-5+ gene of Neurospora crassa. Gene, 153:135-139
78. Kavanaugh M, Miller D, Zhang W, Law W, Kozak S, Kabat D, Miller A (1994) Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodiumdependent phosphate symporters. Proc Natl Acad Sci USA 91:7071-7075
79. Daram P, Brunner S, Rausch C, Steiner C, Amrhein N, Bucher M(1999) Pht2; 1 encodes a low-affinity phosphate transporter from Arabidopsis. Plant Cell, 11:2153-2166.
80. Versaw WK, Harrison MJ (2002) A chloroplast phosphate transporter PHT2; 1 influences allocation of phosphate within the plant and phosphate-starvation responses. Plant Cell , 14:1751-1766.
81. Ferro M, Salvi D, Riviere-Rolland H, Vermat T, Seigneurin-Berny D, Grunwald D, Garin J, Joyard J, Rolland N (2002) Integral membrane proteins of the chloroplast envelope: identification and subcellular localization of new transporters. Proc Natl Acad Sci U S A, 99:11487-11492
82. Kiiskinen M, Korhonen M, Kangasjarvi J (1997) Isolation and characterization of cDNA for a plant mitochondrial phosphate translocator (Mpt1): ozone stress induces Mpt1 mRNA accumulation in birch (Betula pendula Roth). Plant Mol. Biol., 35:271-279.
83. Yves P, Marcel B (2002) Phosphate transport and homeostasis in Arabidopsis. The Arabidopsis Book (Washington D.C.: American Society of Plant Biologists): 1-35
84. Knappe S, Flugge U, Fischer K (2003) Analysis of the Plastidic phosphate translocator Gene Family in Arabidopsis and Identification of New phosphate translocator- Homologous Transporters , Classified by Their Putative Substrate-Binding Site. Plant Physiol, 131:1178-1190
85. Flulugge UI (1999) Phosphate translocators in plastids. Annu Rev Plant Physiol Plant Mol Biol, 50:27-45.
86. Rausch C, Bucher M (2002) Molecular mechanisms of phosphate transport in plants. Planta, 216:23-37
87. Stitt M (1997) The flux of carbon between the chloroplast and the cytoplasm. Plant metabolism. Longman Press, 382-400
88. Murer H, Hernando N, Forster I, Biber J (2000) Proximal tubular phosphate reabsorption:molecular mechanisms. Physiol Rev 2000;80: 1373-1409.
89. Zhao N, Tenenhouse HS (2000): Npt2 gene disruption confers resistance to the inhibitory action of parathyroid hormone on renal sodium-phosphate cotransport. Endocrinology; 141: 2159-2165.
90. Murer H, Forster I, Biber J (2004)The sodium-phosphate cotransporter family SLC34. Pflugers Arch; 447: 763-767.
91. Tenenhouse HS (2007) : Phosphate transport: molecular basis, regulation and pathophysiology. J Steroid Biochem Mol Biol; 103: 572-577.
92. Collins JF, Bai L, Ghishan FK (2004) The SLC20 family of proteins: dual functions as sodium-phosphate cotransporters and viral receptors. Pflugers Arch; 447: 647-652.
93. Kavanaugh MP, Miller DG, Zhang W, Law W, Kozak SL, Kabat D, Miller AD ( 1994 ) Cell-surface receptors for gibbon ape leukemia virusand amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters. Proc Natl Acad Sci USA; 91:7071-7075.
94. Hellebust JA (1978) Uptake of organic substrates by Cyclotella cryptica (Bacillariophyceae): effects of ions, ionophores and metabolic and transport inhibitors. J. Phycol, 14:79-83.
95. Rees TA, Cressewell RC, Syrett PJ (1980) Sodium-dependent uptake of nitrate and urea by a marine diatom. BBA, 596: 141-144.
96. Versaw WK (1995) A phosphate-repressible, high-affinity phosphate permease is encoded by the pho-5+ gene ofNeurospora crassa.Gene,153:135-139.
97.Martinez P,Persson B(1998)Identification,cloning and characterization of a derepressible Na~+-coupled phosphate transporter in Saccharomyces cerevisiae.Mol Gen Genet,258:628-638.
98.Zvyagilskaya R,Parchomenko O,Abramova N,Allard P,Panaretakis T,Pattison-Granberg J,Persson BL(2001)Protonand sodium-coupled phosphate transport systems and energy status of Yarrowia lipolytica cells in acidic and alkaline conditions.J Membrane Biol.,183:39-50.
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100.Reid RJ,Mimura T,Ohsumi Y,Walker NA,Smith FA(2000)Phosphate uptake in Chara:membrane transport via Na~+/Pi cotransport.Plant Cell Environ,23:223-228.
101.Li QY,Gao XS,Sun Y,Zhang QQ,Son RT,Xu ZK(2006)Isolation and characterization of a sodium-dependent phosphate transporter gene in Dunaliella viridis.Biochem and Biophy Res Comm,340:95-104.
102.Rubio L,Linares-Rueda A,García-Sáinchez MJ,Fernández JA(2005)Physiological evidence for a sodium-dependent high-affinity phosphate and nitrate transport at the plasma membrane of leaf and root cells of Zostera marina L.J Exp Bot,56(412):613-622.
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104.Livak KJ,Schmittgen TD(2001)Analysis of relative gene expression data using real-time quantitative PCR and the 2~(-△△C)_T method.Methods,25:402-408.
105.Clark MS(1998)顾红雅,翟礼嘉主译,《植物分子生物学实验手册》北京:高等教育出版社
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63. Martinez P, Persson B (1998) Identification, cloning and characterization of a derepressible Na~+-coupled phosphate transporter in Saccharomyces cerevisiae. Mol Gen Genet 258:628-638
64. Daram P, Brunner S, Persson BL, Amrhein N, Bucher M (1998) Functional analysis and cell-specific expression of a phosphate transporter from tomato. Planta 206:225-233
65. Mitsukawa N, Okumura S, Shirano Y, Sato S, Kato T, Harashima S, Shibata D (1997) Over expression of an Arabidopsis thaliana high-affinity phosphate transporter gene in tobacco cultured cells enhances cell growth under phosphate-limited conditions. Proc Natl Acad Sci USA 94:7098-7102
66. Bucher M, Rausch C, Daram P (2001) Molecular and biochemical mechanisms of phosphorus uptake into plants. J Plant Nutr Soil Sci 164:209-217
67. Daram P, Brunner S, Persson BL, Amrhein N, Bucher M (1998) Functional analysis and cell-specific expression of a phosphate transporter from tomato. Planta 206:225-233
68. Liu C, Muchhal US, Uthappa M, Kononowicz AK, Raghothama KG (1998a) Tomato phosphate transporter genes are differentially regulated in plant tissues by phosphorus. Plant Physiol 116:91-99
69. Muchhal US, Raghothama KG (1999) Transcriptional regulation of plant phosphate transporters. Proc Natl Acad Sci USA 96:5868-5872
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71. Bucher M, Rausch C, Daram P (2001) Molecular and biochemical mechanisms of phosphorus uptake into plants. J Plant Nutr Soil Sci 164:209-217
72. Liu C, Muchhal US, Uthappa M, Kononowicz AK, Raghothama KG (1998a) Tomato phosphate transporter genes are differentially regulated in plant tissues by phosphorus. Plant Physiol 116:91-99
73. Heungsop Shinl, Hwa-Soo Shinl, Gary R. Dewbrel and Maria J. Harrison2, Phosphate transport in Arabidopsis: Pht1;1 and Pht1;4 play a major role in phosphate acquisition from both low- and high-phosphate environments. The Plant Journal (2004) 39,629-642
74. Catarecha~1, M~a Dolores Segura~1, Jose Manuel Franco-Zorrilla, Berenice Garcia-Ponce~2, Monica Lanza, Roberto Solano, Javier Paz-Ares and Antonio Leyva~3 A Mutant of the Arabidopsis Phosphate Transporter PHT1;1 Displays Enhanced Arsenic Accumulation. The Plant Cell 19:1123-1133 (2007)
75. Daram P, Brunner S, Rausch C, Steiner C, Amrhein N, Bucher M (1999) Pht2;1 encodes a low-affinity phosphate transporter from Arabidopsis. Plant Cell 11:2153-2166
76. Martinez P, Persson B (1998) Identification, cloning and characterization of a derepressible Na~+-coupled phosphate transporter in Saccharomyces cerevisiae. Mol Gen Genet, 258:628-638.
77. Versaw WK (1995) A phosphate-repressible, high-affmity phosphate permease is encoded by the pho-5+ gene of Neurospora crassa. Gene, 153:135-139
78. Kavanaugh M, Miller D, Zhang W, Law W, Kozak S, Kabat D, Miller A (1994) Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodiumdependent phosphate symporters. Proc Natl Acad Sci USA 91:7071-7075
79. Daram P, Brunner S, Rausch C, Steiner C, Amrhein N, Bucher M(1999) Pht2; 1 encodes a low-affinity phosphate transporter from Arabidopsis. Plant Cell, 11:2153-2166.
80. Versaw WK, Harrison MJ (2002) A chloroplast phosphate transporter PHT2; 1 influences allocation of phosphate within the plant and phosphate-starvation responses. Plant Cell , 14:1751-1766.
81. Ferro M, Salvi D, Riviere-Rolland H, Vermat T, Seigneurin-Berny D, Grunwald D, Garin J, Joyard J, Rolland N (2002) Integral membrane proteins of the chloroplast envelope: identification and subcellular localization of new transporters. Proc Natl Acad Sci U S A, 99:11487-11492
82. Kiiskinen M, Korhonen M, Kangasjarvi J (1997) Isolation and characterization of cDNA for a plant mitochondrial phosphate translocator (Mpt1): ozone stress induces Mpt1 mRNA accumulation in birch (Betula pendula Roth). Plant Mol. Biol., 35:271-279.
83. Yves P, Marcel B (2002) Phosphate transport and homeostasis in Arabidopsis. The Arabidopsis Book (Washington D.C.: American Society of Plant Biologists): 1-35
84. Knappe S, Flugge U, Fischer K (2003) Analysis of the Plastidic phosphate translocator Gene Family in Arabidopsis and Identification of New phosphate translocator- Homologous Transporters , Classified by Their Putative Substrate-Binding Site. Plant Physiol, 131:1178-1190
85. Flulugge UI (1999) Phosphate translocators in plastids. Annu Rev Plant Physiol Plant Mol Biol, 50:27-45.
86. Rausch C, Bucher M (2002) Molecular mechanisms of phosphate transport in plants. Planta, 216:23-37
87. Stitt M (1997) The flux of carbon between the chloroplast and the cytoplasm. Plant metabolism. Longman Press, 382-400
88. Murer H, Hernando N, Forster I, Biber J (2000) Proximal tubular phosphate reabsorption:molecular mechanisms. Physiol Rev 2000;80: 1373-1409.
89. Zhao N, Tenenhouse HS (2000): Npt2 gene disruption confers resistance to the inhibitory action of parathyroid hormone on renal sodium-phosphate cotransport. Endocrinology; 141: 2159-2165.
90. Murer H, Forster I, Biber J (2004)The sodium-phosphate cotransporter family SLC34. Pflugers Arch; 447: 763-767.
91. Tenenhouse HS (2007) : Phosphate transport: molecular basis, regulation and pathophysiology. J Steroid Biochem Mol Biol; 103: 572-577.
92. Collins JF, Bai L, Ghishan FK (2004) The SLC20 family of proteins: dual functions as sodium-phosphate cotransporters and viral receptors. Pflugers Arch; 447: 647-652.
93. Kavanaugh MP, Miller DG, Zhang W, Law W, Kozak SL, Kabat D, Miller AD ( 1994 ) Cell-surface receptors for gibbon ape leukemia virusand amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters. Proc Natl Acad Sci USA; 91:7071-7075.
94. Hellebust JA (1978) Uptake of organic substrates by Cyclotella cryptica (Bacillariophyceae): effects of ions, ionophores and metabolic and transport inhibitors. J. Phycol, 14:79-83.
95. Rees TA, Cressewell RC, Syrett PJ (1980) Sodium-dependent uptake of nitrate and urea by a marine diatom. BBA, 596: 141-144.
96. Versaw WK (1995) A phosphate-repressible, high-affinity phosphate permease is encoded by the pho-5+ gene ofNeurospora crassa.Gene,153:135-139.
97.Martinez P,Persson B(1998)Identification,cloning and characterization of a derepressible Na~+-coupled phosphate transporter in Saccharomyces cerevisiae.Mol Gen Genet,258:628-638.
98.Zvyagilskaya R,Parchomenko O,Abramova N,Allard P,Panaretakis T,Pattison-Granberg J,Persson BL(2001)Protonand sodium-coupled phosphate transport systems and energy status of Yarrowia lipolytica cells in acidic and alkaline conditions.J Membrane Biol.,183:39-50.
99.Uilrich WR,Glaser E(1982)Sodium-phosphate cotransport in the green alga Ankistrodesmus braunii.Plant Sci Let,27:155-1162.
100.Reid RJ,Mimura T,Ohsumi Y,Walker NA,Smith FA(2000)Phosphate uptake in Chara:membrane transport via Na~+/Pi cotransport.Plant Cell Environ,23:223-228.
101.Li QY,Gao XS,Sun Y,Zhang QQ,Son RT,Xu ZK(2006)Isolation and characterization of a sodium-dependent phosphate transporter gene in Dunaliella viridis.Biochem and Biophy Res Comm,340:95-104.
102.Rubio L,Linares-Rueda A,García-Sáinchez MJ,Fernández JA(2005)Physiological evidence for a sodium-dependent high-affinity phosphate and nitrate transport at the plasma membrane of leaf and root cells of Zostera marina L.J Exp Bot,56(412):613-622.
103.García-Sánchez MJ,Jaime MP,Ramos A,Sanders D,Fernández JA(2000)Sodium-dependent nitrate transport at the plasma membrane of leaf cells of the marine higher plant Zostera marina L.Plant Physiol,122:879-885.
104.Livak KJ,Schmittgen TD(2001)Analysis of relative gene expression data using real-time quantitative PCR and the 2~(-△△C)_T method.Methods,25:402-408.
105.Clark MS(1998)顾红雅,翟礼嘉主译,《植物分子生物学实验手册》北京:高等教育出版社