Cloning and Expression Analysis of <i>MeNRT2.5</i> Gene in Cassava

REN Ning; CHEN Xiuzhen; XIA Youquan; BAI Xueyang; JIANG Xingyu; ZHOU Yang

doi:10.15886/j.cnki.rdswxb.2019.02.003

Message Board

Respected readers, authors and reviewers, you can add comments to this page on any questions about the contribution, review, editing and publication of this journal. We will give you an answer as soon as possible. Thank you for your support!

Name

E-mail

Phone

Title

Content

Verification Code

Volume 10 Issue 2

Jun. 2019

Turn off MathJax

Article Contents

Article Navigation > Journal of Tropical Biology > 2019 > 10(2): 111-118

REN Ning, CHEN Xiuzhen, XIA Youquan, BAI Xueyang, JIANG Xingyu, ZHOU Yang. Cloning and Expression Analysis of MeNRT2.5 Gene in Cassava[J]. Journal of Tropical Biology, 2019, 10(2): 111-118. doi: 10.15886/j.cnki.rdswxb.2019.02.003

Citation:

REN Ning, CHEN Xiuzhen, XIA Youquan, BAI Xueyang, JIANG Xingyu, ZHOU Yang. Cloning and Expression Analysis of MeNRT2.5 Gene in Cassava[J]. Journal of Tropical Biology, 2019, 10(2): 111-118. doi: 10.15886/j.cnki.rdswxb.2019.02.003

Cloning and Expression Analysis of MeNRT2.5 Gene in Cassava

doi: 10.15886/j.cnki.rdswxb.2019.02.003

Institute of Tropical Agriculture and Forestry, Hainan University/Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources, Haikou, Hainan 570228, China

Received Date: 2018-03-16
Rev Recd Date: 2019-03-19

Abstract

Cassava（Manihot esculenta Crantz） is characteristic of high yield, drought tolerance and poor soil tolerance. To probe into the molecular basis for tolerance of poor soils and enhance the utilization rate of nitrogen in cassava, two years old mature plants and 1 month-old tissue cultured plants of cassava cultivar "Huanan 8" were collected and cultured for 30 days in a medium as the experimental material, from which a high-affinity nitrate transporter gene NRT2 was isolated by using homologous cloning technique. Sequence analysis revealed that the open reading frame of this gene called MeNRT2.5 is 1 479 bp and encodes 492 amino acids. Bioinformatics analyses showed that the MeNRT2.5 protein had 10 transmembrane regions, and had a a high homology with the NRT2.5 protein of Hevea brasiliensis, Jatropha curcas and,Theobroma cacao, with its amino acid sequences being 94%, 89.84% and 87.4% in similarity, respectively. Semi-Quantitative real-time PCR analysis showed that the MeNRT2.5 gene was expressed in root, stem, leaf, flower and other organs, with its expression being higher in the roots of mature cassava plants and in the leaves of tissue cultured plants. Quantitative real-time PCR analysis showed that transcript of the MeNRT2.5 in root had a peak expression at 6 h under the treatment with low concentration NO₃^-(0.3 mmol·L^-1) but its expression did not change significantly in the root, stem and leaf after the treatment with high concentration of NO₃^-(3 mmol·L^-1), indicating the transcript of the MeNRT2.5 gene is inhibited by high concentrations of NO₃^-. All these results provide a theoretical foundation for further analysis of the functional verification of the MeNRT2.5 gene in cassava.
- Cassava,
- high affinity nitrate transporter,
- gene cloning,
- expression analysis

References

[1]	黄洁, 李开绵, 叶剑秋, 等.中国木薯产业化的发展研究与对策[J].中国农业通报, 2006, 22 (5):421-426.
[2]	李建勇, 龚继明.植物硝酸根信号感受与传导途径[J].植物生理学报, 2011, 47:111-118.
[3]	朱兆良.农田中氮肥的损失与对策[J].土壤与环境, 2000, 9 (1):1-6.
[4]	郭敏, 韩鹏飞.农业面源污染的成因及控制对策[J].河北农业科学, 2009, 13 (4):93-96.
[5]	CRAWFORD N M, FORDE B G.Molecular and developmental biology of inorganic nitrogen nutrition[J].American Society of Plants Biologists, 2002:e0011.
[6]	FORDE B G.Nitrate transporters in plants:structure, function and regulation[J].Biochimica et Biophysica Acta, 2000, 1465 (12):219-235.
[7]	WILLIAMS L E, MILLER A J.Transporters responsible for the uptake and partitioning of nitrogenous solutes[J].Annual Review of Plant Physiology and Plant Molecular Biology, 2001, 52 (1):659-688.
[8]	CRAWFORD N M, GLASS A D M.Molecular and physiological aspects of nitrate uptake in plants[J].Trends in Plant Science, 1998, 3 (10):389.
[9]	CHAPMAN N, MILLER T.Nitrate transporters and root architecture//GEISLER M, VENEMA K.Transporters and pumps in plant signaling[M].Berlin:Springer, 201l:165-190.
[10]	LEE R B, CLARKSON D T.Nitrogen-13 studies of nitrate fluxes in barley roots.Compartmental analysis from measurements of 13N efflux [J].The EMBO Journal, 1986 (5):1753-1767.
[11]	TSAY Y F, CHU C C, TSAI C B, et al.Nitrate transporters and peptide transporters [J].FEBS Letters, 2001, 581:2290-2300.
[12]	PAO S S, PAULSEN I T, SAIER M H.Major facilitator superfamily[J].Microbiologr and Molecular Biology Review, 1998, 62 (1):1-34.
[13]	GLAVAN A, FERNANDEZ E.Eukaryotic nitrate and nitrite transporters[J].Cellular and Molecular Life Sciences, 2001, 58:225-233.
[14]	TRUEMAN L J, RICHARDSON A, FORDE B G.Molecular cloning of higher plant homologues of the high-affinity nitrate transporters of Chlamydomonas reinhardtii[J].Gene, 1996, 175 (1/2):223-231.
[15]	YAN M, FAN X R, FENG H M, et al.Rice OsNAR2.1 intersects with OsNRT2.1, OsNRT2.2 and OsNRT2.3 a nitrate transporters to provide uptake over high and low concentration ranges [J].Plant Cell and Environment, 2011, 34 (8):1360-1372.
[16]	XU G, FAN X, MILLER A J.Plant nitrogen assimilation and use efficiency [J].Annual Review of Plant Biology, 2012, 63 (1):153-182.
[17]	TANG Z, FAN X R, LI Q, et al.Knock down of a rice stellar nitrate transporter alters long distance translocation but not root influx [J].Plant Physiology, 2012, 160 (4):2052-2063.
[18]	CRAWFORD N M, GLASS A D M.Molecular and physiological aspects of nitrate in plants[J].Trends in Plant Science, 1998, 3 (10):389-395.
[19]	QUAGGIOTTI S, RUPERTI B, BORSA P, et al.Expression of a putative high-affinity NO3-transporter and of an H⁺-ATPase in relation to whole plant nitrate transport physiology in two maize genotypes differently responsive to low nitrogen availability[J].Journal of Experiment Botany, 2003, 54:1023-1031.
[20]	AMARASINGHE B H, DE BRUXELLES G L, BRADDON M, et al.Regulation of Gmnrt2 expression and nitrate transport activity in roots of soybean (Glycine max) [J].Planta, 1998, 206 (1):44-52.
[21]	赵学强, 李玉京, 刘建中, 等.小麦NO3-转运蛋白基因TaNRT2.3的克隆和表达分析[J].植物学报, 2004, 46 (3):347-354.
[22]	NAZOA P, VIDMAR J J, TRANBARGER T J, et al.Regulation of the nitrate transporter gene AtNRT2.1 in Arabidopsis thaliana:responses to nitrate, amino acids and developmental stage[J].Plant Molecular Biology, 2003, 52:689-703.
[23]	TODD C D, ZENG P, HUETE A M P, et al.Transcripts of MYB-like genes respond to phosphorous and nitrogen deprivation in Arabidopsis[J].Planta, 2004, 219:1003-1009.
[24]	冯素花, 王丽鸳, 陈常颂, 等.茶树硝酸根转运蛋白基因NRT2.5的克隆及表达分析[J].茶业科学, 2014, 34 (4):364-370.
[25]	王新亮.果树根系硝态氮信号响应关键基因的克隆与功能分析[D].泰安:山东农业大学, 2012.
[26]	CAI C, ZHAO X Q, ZHU Y G, et al.Regulation of the high-affinity nitrate transport system in wheat roots by exogenous abscisic acid and glutamine[J].Journal of Integrative Plant Biology, 2007 (49):1719-1725.
[27]	ARAKI R, HASEGAWA H.Expression of rice (Oryza sativa L.) genes involved in high-affinity nitrate transport during the period of nitrate induction[J].Breeding Science, 2006, 56:295-302.
[28]	缪其松.水稻硝转运蛋白基因OsNRT1.1a和OsNRT1.1b的功能研究[D].南京:南京农业大学, 2011.
[29]	胡春吉, 邹良平, 彭明.木薯MeNRT2.1基因的克隆及表达分析[J].热带作物学报, 2016, 37 (1):117-124.
[30]	金玲.小白菜水培营养液配方筛选 [J].河南农业科学, 2007 (9):82-85.
[31]	MO C, WAN S, XIA X, et al.Expression patterns and identified protein-protein interactions suggest that cassava CBL-CIPK signal networks function in response to abiotic stresses[J].Frontiers in Plant Science, 2018, 9:269.
[32]	KIVAK K J, SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J].Methods, 2001, 25 (4):402-408.
[33]	LINA L, TAKATOSHI K, ANA-BELEN F B, et al.The Arabidopsis nitrate transporter NRT2.5 plays a role in nitrate acquisition and remobilization in nitrogen-starved plants [J].The Plant Journal, 2014, 80:230-241.
[34]	LITTLE D Y, RAO H, OLIVA S, et al.The putative high-affinity nitrate transporter NRT2.1 represses lateral root initiation in response to nutritional cues[J].Proceedings of the National Academy of Science of United States of America, 2005, 102:13693-13698.
[35]	孔敏, 杨学东, 侯喜林, 等.白菜NRT2基因的克隆及表达模式分析[J].园艺学报, 2011, 38 (12):2309-2316.
[36]	ORSEL M, KRAPP A, DANIEL V.F.Analysis of the NRT2 nitrate transporter family in Arabidopsis:Structure and gene expression[J].Plant Physiology, 2002, 129 (2):886-896.
[37]	MAYA K, GUILHEM D, WAFAA R, et al.The NRT2.5 and NRT2.6 genes are involved in growth promotion of Arabidopsis by the plant growth-promoting rhizobacterium (PGPR) strain Phyllobacterium brassicacearum STM196 [J].New Phytologist, 2013, 198:514-524.
[38]	ZHOU J J, FERNÁNDEZ E, GALVÁN A, et al.A high affinity nitrate transport system from Chlamydomonas requires two gene products [J].FEBS Letter, 2000, 466 (2/3):225-227.
[39]	TONG Y P, ZHOU J J, LI Z S, et al.A two-component high-affinity nitrate uptake system in barley[J].The Plant Journal, 2005, 41 (3):442-450.
[40]	KOTUR Z, GLASS A D M.A 150 kDa plasma membrane complex of AtNRT2.5 and AtNAR2.1 is the major contributor to constitutive high-affinity nitrate influx in Arabidopsis thaliana[J].Plant, Cell and Environment, 2015, 38:1490-1502.

Proportional views

通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Get Citation

PDF

XML

Article views(31) PDF downloads(0) Cited by()

Proportional views

Cloning and Expression Analysis of MeNRT2.5 Gene in Cassava

Institute of Tropical Agriculture and Forestry, Hainan University/Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources, Haikou, Hainan 570228, China

Received Date: 2018-03-16

Rev Recd Date: 2019-03-19

Key words:

Abstract: Cassava（Manihot esculenta Crantz） is characteristic of high yield, drought tolerance and poor soil tolerance. To probe into the molecular basis for tolerance of poor soils and enhance the utilization rate of nitrogen in cassava, two years old mature plants and 1 month-old tissue cultured plants of cassava cultivar "Huanan 8" were collected and cultured for 30 days in a medium as the experimental material, from which a high-affinity nitrate transporter gene NRT2 was isolated by using homologous cloning technique. Sequence analysis revealed that the open reading frame of this gene called MeNRT2.5 is 1 479 bp and encodes 492 amino acids. Bioinformatics analyses showed that the MeNRT2.5 protein had 10 transmembrane regions, and had a a high homology with the NRT2.5 protein of Hevea brasiliensis, Jatropha curcas and,Theobroma cacao, with its amino acid sequences being 94%, 89.84% and 87.4% in similarity, respectively. Semi-Quantitative real-time PCR analysis showed that the MeNRT2.5 gene was expressed in root, stem, leaf, flower and other organs, with its expression being higher in the roots of mature cassava plants and in the leaves of tissue cultured plants. Quantitative real-time PCR analysis showed that transcript of the MeNRT2.5 in root had a peak expression at 6 h under the treatment with low concentration NO₃^-(0.3 mmol·L^-1) but its expression did not change significantly in the root, stem and leaf after the treatment with high concentration of NO₃^-(3 mmol·L^-1), indicating the transcript of the MeNRT2.5 gene is inhibited by high concentrations of NO₃^-. All these results provide a theoretical foundation for further analysis of the functional verification of the MeNRT2.5 gene in cassava.

HTML

Reference (40)

[1]	黄洁, 李开绵, 叶剑秋, 等.中国木薯产业化的发展研究与对策[J].中国农业通报, 2006, 22 (5):421-426.
[2]	李建勇, 龚继明.植物硝酸根信号感受与传导途径[J].植物生理学报, 2011, 47:111-118.
[3]	朱兆良.农田中氮肥的损失与对策[J].土壤与环境, 2000, 9 (1):1-6.
[4]	郭敏, 韩鹏飞.农业面源污染的成因及控制对策[J].河北农业科学, 2009, 13 (4):93-96.
[5]	CRAWFORD N M, FORDE B G.Molecular and developmental biology of inorganic nitrogen nutrition[J].American Society of Plants Biologists, 2002:e0011.
[6]	FORDE B G.Nitrate transporters in plants:structure, function and regulation[J].Biochimica et Biophysica Acta, 2000, 1465 (12):219-235.
[7]	WILLIAMS L E, MILLER A J.Transporters responsible for the uptake and partitioning of nitrogenous solutes[J].Annual Review of Plant Physiology and Plant Molecular Biology, 2001, 52 (1):659-688.
[8]	CRAWFORD N M, GLASS A D M.Molecular and physiological aspects of nitrate uptake in plants[J].Trends in Plant Science, 1998, 3 (10):389.
[9]	CHAPMAN N, MILLER T.Nitrate transporters and root architecture//GEISLER M, VENEMA K.Transporters and pumps in plant signaling[M].Berlin:Springer, 201l:165-190.
[10]	LEE R B, CLARKSON D T.Nitrogen-13 studies of nitrate fluxes in barley roots.Compartmental analysis from measurements of 13N efflux [J].The EMBO Journal, 1986 (5):1753-1767.
[11]	TSAY Y F, CHU C C, TSAI C B, et al.Nitrate transporters and peptide transporters [J].FEBS Letters, 2001, 581:2290-2300.
[12]	PAO S S, PAULSEN I T, SAIER M H.Major facilitator superfamily[J].Microbiologr and Molecular Biology Review, 1998, 62 (1):1-34.
[13]	GLAVAN A, FERNANDEZ E.Eukaryotic nitrate and nitrite transporters[J].Cellular and Molecular Life Sciences, 2001, 58:225-233.
[14]	TRUEMAN L J, RICHARDSON A, FORDE B G.Molecular cloning of higher plant homologues of the high-affinity nitrate transporters of Chlamydomonas reinhardtii[J].Gene, 1996, 175 (1/2):223-231.
[15]	YAN M, FAN X R, FENG H M, et al.Rice OsNAR2.1 intersects with OsNRT2.1, OsNRT2.2 and OsNRT2.3 a nitrate transporters to provide uptake over high and low concentration ranges [J].Plant Cell and Environment, 2011, 34 (8):1360-1372.
[16]	XU G, FAN X, MILLER A J.Plant nitrogen assimilation and use efficiency [J].Annual Review of Plant Biology, 2012, 63 (1):153-182.
[17]	TANG Z, FAN X R, LI Q, et al.Knock down of a rice stellar nitrate transporter alters long distance translocation but not root influx [J].Plant Physiology, 2012, 160 (4):2052-2063.
[18]	CRAWFORD N M, GLASS A D M.Molecular and physiological aspects of nitrate in plants[J].Trends in Plant Science, 1998, 3 (10):389-395.
[19]	QUAGGIOTTI S, RUPERTI B, BORSA P, et al.Expression of a putative high-affinity NO3-transporter and of an H⁺-ATPase in relation to whole plant nitrate transport physiology in two maize genotypes differently responsive to low nitrogen availability[J].Journal of Experiment Botany, 2003, 54:1023-1031.
[20]	AMARASINGHE B H, DE BRUXELLES G L, BRADDON M, et al.Regulation of Gmnrt2 expression and nitrate transport activity in roots of soybean (Glycine max) [J].Planta, 1998, 206 (1):44-52.
[21]	赵学强, 李玉京, 刘建中, 等.小麦NO3-转运蛋白基因TaNRT2.3的克隆和表达分析[J].植物学报, 2004, 46 (3):347-354.
[22]	NAZOA P, VIDMAR J J, TRANBARGER T J, et al.Regulation of the nitrate transporter gene AtNRT2.1 in Arabidopsis thaliana:responses to nitrate, amino acids and developmental stage[J].Plant Molecular Biology, 2003, 52:689-703.
[23]	TODD C D, ZENG P, HUETE A M P, et al.Transcripts of MYB-like genes respond to phosphorous and nitrogen deprivation in Arabidopsis[J].Planta, 2004, 219:1003-1009.
[24]	冯素花, 王丽鸳, 陈常颂, 等.茶树硝酸根转运蛋白基因NRT2.5的克隆及表达分析[J].茶业科学, 2014, 34 (4):364-370.
[25]	王新亮.果树根系硝态氮信号响应关键基因的克隆与功能分析[D].泰安:山东农业大学, 2012.
[26]	CAI C, ZHAO X Q, ZHU Y G, et al.Regulation of the high-affinity nitrate transport system in wheat roots by exogenous abscisic acid and glutamine[J].Journal of Integrative Plant Biology, 2007 (49):1719-1725.
[27]	ARAKI R, HASEGAWA H.Expression of rice (Oryza sativa L.) genes involved in high-affinity nitrate transport during the period of nitrate induction[J].Breeding Science, 2006, 56:295-302.
[28]	缪其松.水稻硝转运蛋白基因OsNRT1.1a和OsNRT1.1b的功能研究[D].南京:南京农业大学, 2011.
[29]	胡春吉, 邹良平, 彭明.木薯MeNRT2.1基因的克隆及表达分析[J].热带作物学报, 2016, 37 (1):117-124.
[30]	金玲.小白菜水培营养液配方筛选 [J].河南农业科学, 2007 (9):82-85.
[31]	MO C, WAN S, XIA X, et al.Expression patterns and identified protein-protein interactions suggest that cassava CBL-CIPK signal networks function in response to abiotic stresses[J].Frontiers in Plant Science, 2018, 9:269.
[32]	KIVAK K J, SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J].Methods, 2001, 25 (4):402-408.
[33]	LINA L, TAKATOSHI K, ANA-BELEN F B, et al.The Arabidopsis nitrate transporter NRT2.5 plays a role in nitrate acquisition and remobilization in nitrogen-starved plants [J].The Plant Journal, 2014, 80:230-241.
[34]	LITTLE D Y, RAO H, OLIVA S, et al.The putative high-affinity nitrate transporter NRT2.1 represses lateral root initiation in response to nutritional cues[J].Proceedings of the National Academy of Science of United States of America, 2005, 102:13693-13698.
[35]	孔敏, 杨学东, 侯喜林, 等.白菜NRT2基因的克隆及表达模式分析[J].园艺学报, 2011, 38 (12):2309-2316.
[36]	ORSEL M, KRAPP A, DANIEL V.F.Analysis of the NRT2 nitrate transporter family in Arabidopsis:Structure and gene expression[J].Plant Physiology, 2002, 129 (2):886-896.
[37]	MAYA K, GUILHEM D, WAFAA R, et al.The NRT2.5 and NRT2.6 genes are involved in growth promotion of Arabidopsis by the plant growth-promoting rhizobacterium (PGPR) strain Phyllobacterium brassicacearum STM196 [J].New Phytologist, 2013, 198:514-524.
[38]	ZHOU J J, FERNÁNDEZ E, GALVÁN A, et al.A high affinity nitrate transport system from Chlamydomonas requires two gene products [J].FEBS Letter, 2000, 466 (2/3):225-227.
[39]	TONG Y P, ZHOU J J, LI Z S, et al.A two-component high-affinity nitrate uptake system in barley[J].The Plant Journal, 2005, 41 (3):442-450.
[40]	KOTUR Z, GLASS A D M.A 150 kDa plasma membrane complex of AtNRT2.5 and AtNAR2.1 is the major contributor to constitutive high-affinity nitrate influx in Arabidopsis thaliana[J].Plant, Cell and Environment, 2015, 38:1490-1502.

Message Board

Cloning and Expression Analysis of MeNRT2.5 Gene in Cassava

doi: 10.15886/j.cnki.rdswxb.2019.02.003

Abstract

References

Proportional views

通讯作者: 陈斌, bchen63@163.com

Proportional views

Related

Cloning and Expression Analysis of MeNRT2.5 Gene in Cassava

doi: 10.15886/j.cnki.rdswxb.2019.02.003

Institute of Tropical Agriculture and Forestry, Hainan University/Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources, Haikou, Hainan 570228, China

HTML

Catalog

Message Board

Cloning and Expression Analysis of MeNRT2.5 Gene in Cassava

doi: 10.15886/j.cnki.rdswxb.2019.02.003

Abstract

References

Proportional views

通讯作者: 陈斌, bchen63@163.com

Proportional views

Related

Cloning and Expression Analysis of MeNRT2.5 Gene in Cassava

doi: 10.15886/j.cnki.rdswxb.2019.02.003

Institute of Tropical Agriculture and Forestry, Hainan University/Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources, Haikou, Hainan 570228, China

HTML

Catalog

Export File

Citation

Format

Content