水稻硝酸盐转运蛋白基因OsNPF7.9在氮素积累和转运中的 功能研究

doi:10.16819/j.1001-7216.2017.7031 457

摘要/Abstract

摘要：

目的植物NPF家族成员具有转运硝酸盐、小肽等的功能。对水稻OsNPF7.9基因的功能研究能够为水稻氮素高效利用的分子机制提供理论基础。方法利用不同的生物软件对OsNPF7.9蛋白的生物学信息进行了预测分析;用OsNPF7.9基因启动子融合GUS报告基因的转基因水稻进行OsNPF7.9的组织定位观察;利用半定量RT-PCR和pOsNPF7.9::GUS转基因水稻分析OsNPF7.9受氮素调控特征;构建了OsNPF7.9的超表达水稻株系,并进行生理指标测定。结果 OsNPF7.9是细胞质膜蛋白,有12个跨膜结构域,在第6~7个结构域之间有一个大的亲水环。组织定位结果显示OsNPF7.9在根、叶片、根茎结合处和花中都有表达。RT-PCR和pOsNPF7.9::GUS转基因水稻的GUS染色结果都表示,OsNPF7.9的表达不受氮素形态和浓度的影响。OsNPF7.9基因超表达后,不仅显著增加水稻的地上部硝酸盐含量以及根系向地上部的硝酸盐转运,而且增加了地上部的总氮浓度、总氮积累以及根系向地上部的总氮转运。结论 OsNPF7.9参与硝酸盐从根系向地上部的转运,并且OsNPF7.9超表达可以提高水稻氮素积累和转运能力。

关键词: 水稻, 氮素, OsNPF7.9, 硝酸盐转运蛋白

Abstract:

【Objective】 NPF family in plants can transport nitrate and peptide, etc. Functional analyses of OsNPF7.9 can lay a theoretical basis for studying molecular mechanism of high nitrogen use-efficiency.【Method】 Bioinformatics of OsNPF7.9 was predicted with different biological softwares. The OsNPF7.9 promoter-GUS transgenic rice was obtained for analyzing the spatial expression pattern of OsNPF7.9. And the expression patterns of OsNPF7.9 under different N supply levels were observed using semi-quantitative RT-PCR and pOsNPF7.9::GUS transgenic rice. OsNPF7.9 overexpressing rice was obtained, and their different physiological indexes were measured. 【Result】 OsNPF7.9 is localized on plasma membrane with 12 transmembrane domains and a big hydrophilic loop between the 6th and the 7th transmembrane domain. Tissue expression pattern showed that OsNPF7.9 was expressed in roots, leaves, root-shoot junction and flowers. Both RT-PCR and GUS staining of pOsNPF7.9::GUS transgenic rice indicated that the expression of OsNPF7.9 was not affected by N concentration and form. Overexpression of OsNPF7.9 could significantly increase not only nitrate concentration in shoots and roots-shoots nitrate ratio; but also total nitrogen concentration and accumulation in shoots, and roots-shoots total nitrogen ratio. 【Conclusion】OsNPF7.9 participated nitrate transport from roots to shoots, and overexpression of OsNPF7.9 could increase N accumulation and transport from roots to shoots.

Key words: rice, nitrogen, OsNPF7.9, nitrate transporter

中图分类号:

Q786
S511.01

冯慧敏, 陆宏, 王汉卿, 李昕玥. 水稻硝酸盐转运蛋白基因OsNPF7.9在氮素积累和转运中的功能研究[J]. 中国水稻科学, 2017, 31(5): 457-465.

Huimin FENG, Hong LU, Hanqing WANG, Xinyue LI. Function Analyses of Rice Nitrate Transporter Gene OsNPF7.9 in Nitrogen Accumulation and Transport[J]. Chinese Journal OF Rice Science, 2017, 31(5): 457-465.

图/表 9

表1 本研究所用的半定量RT-PCR引物序列

Table 1 Primers for semi-RT PCR in the study.

基因名称 Gene	引物序列 Primer sequence(5′-3′)	退火温度 Annealing temperature/℃		循环数 No. of cycles	产物大小 Product size/ bp
OsActin	F:GGAACTGGTATGGTCAAGGC R: AGTCTCATGGATAACCGCAG		55	32	792
OsNPF7.9	F: GCAACGACGGAGACATGAAG R: GCGAGGCCAAGGACAAAGAT		58	28	500

图1 植物NPF家族进化树分析

Fig. 1. Phylogenetic of NPF in plants.

图2 OsNPF7.9跨膜结构分析

Fig. 2. Transmembrane topology of OsNPF7.9.

图3 OsNPF7.9水稻原生质体亚细胞定位

Fig. 3. Sub-celluar localization of OsNPF7.9 in rice protoplast.

图4 OsNPF7.9启动子融合GUS转基因水稻的组织定位

Fig. 4. Tissue expression of pOsNPF7.9::GUS transgenic rice.

图5 OsNPF7.9在不同形态氮素和不同浓度下的表达

Fig. 5. Expression of OsNPF7.9 supplied with various forms of nitrogen at different N concentrations.

图6 OsNPF7.9超表达水稻的分子鉴定

Fig. 6. Identification of OsNPF7.9-overexpressing rice.

图7 OsNPF7.9 超表达水稻的生长和NO3–含量

Fig. 7. Growth and NO3– concentration in the OsNPF7.9overexpressing rice.

图8 OsNPF7.9超表达水稻的总氮浓度、积累和转运

Fig. 8. Total N concentration, accumulation and transport of OsNPF7.9 overexpression rice.

参考文献 28

[1]	Feng Q, Zhang Y, Hao P, Wang S, Fu G, Huang Y, Li Y, Zhu J, Liu Y, Hu X, Jia P, Zhang Y, Zhao Q, Ying K, Yu S, Tang Y, Weng Q, Zhang L, Lu Y, Mu J, Lu Y, Zhang L S, Yu Z, Fan D, Liu X, Lu T, Li C, Wu Y, Sun T, Lei H, Li T, Hu H, Guan J, Wu M, Zhang R, Zhou B, Chen Z, Chen L, Jin Z, Wang R, Yin H, Cai Z, Ren S, Lv G, Gu W, Zhu G, Tu Y, Jia J, Zhang Y, Chen J, Kang H, Chen X, Shao C, Sun Y, Hu Q, Zhang X, Zhang W, Wang L, Ding C, Sheng H, Gu J, Chen S, Ni L, Zhu F, Chen W, Lan L, Lai Y, Cheng Z, Gu M, Jiang J, Li J, Hong G, Xue Y, Han B.Sequence and analysis of rice chromosome 4.Nature, 2002, 420(6912): 316-320.
[2]	Sasaki T, Matsumoto T, Yamamoto K, Sakata K, Baba T, Katayose Y, Wu J, Niimura Y, Cheng Z, Nagamura Y, Antonio B A, Kanamori H, Hosokawa S, Masukawa M, Arikawa K, Chiden Y, Hayashi M, Okamoto M, Ando T, Aoki H, Arita K, Hamada M, Harada C, Hijishita S, Honda M, Ichikawa Y, Idonuma A, Iijima M, Ikeda M, Ikeno M, Ito S, Ito T, Ito Y, Ito Y, Iwabuchi A, Kamiya K, Karasawa W, Katagiri S, Kikuta A, Kobayashi N, Kono I, Machita K, Maehara T, Mizuno H, Mizubayashi T, Mukai Y, Nagasaki H, Nakashima M, Nakama Y, Nakamichi Y, Nakamura M, Namiki N, Negishi M, Ohta I, Ono N, Saji S, Sakai K, Shibata M, Shimokawa T, Shomura A, Song J, Takazaki Y, Terasawa K, Tsuji K, Waki K, Yamagata H, Yamane H, Yoshiki S, Yoshihara R, Yukawa K, Zhong H, Iwama H, Endo T, Ito H, Hahn J H, Kim H I, Eun M Y, Yano M, Jiang J, Gojobori T.The genome sequence and structure of rice chromosome 1.Nature, 2002, 420(6913): 312-316.
[3]	Yang J L, Li Y Y, Zhang Y J, Zhang S S, Wu Y R, Wu P, Zheng S J.Cell wall polysaccharides are specifically involved in the exclusion of aluminum from the rice root apex.Plant Physiol, 2008, 146(2): 602-611.
[4]	Parker J L, Newstead S.Molecular basis of nitrate uptake by the plant nitrate transporter NRT1.1.Nature, 2014, 507(7490): 68-72.
[5]	Krapp A, David L C, Chardin C, Girin T, Marmagne A, Leprince A S, Chaillou S, Ferrario-Méry S, Meyer C, Daniel-Vedele F.Nitrate transport and signalling in Arabidopsis.J Exp Bot, 2014, 65(3): 789-798.
[6]	Léran S, Varala K, Boyer J C, Chiurazzi M, Crawford N, Daniel-Vedele F, David L, Dickstein R, Fernandez E, Forde B, Gassmann W, Geiger D, Gojon A, Gong J M, Halkier B A, Harris J M, Hedrich R, Limami A M, Rentsch D, Seo M, Tsay Y F, Zhang M, Coruzzi G, Lacombe B.A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants.Trends Plant Sci, 2014, 19(1): 5-9.
[7]	Xu G, Fan X, Miller A J.Plant nitrogen assimilation and use efficiency.Annu Rev Plant Biol, 2012, 63(1): 153-182.
[8]	Wang Y Y, Hsu P K, Tsay Y F.Uptake, allocation and signaling of nitrate.Trends Plant Sci, 2012, 17(8): 458-467.
[9]	Tsay Y F 1, Schroeder J I, Feldmann K A, Crawford N M. The herbicide sensitivity gene CHL1 of Arabidopsis encodes a nitrate-inducible nitrate transporter. F 1, Schroeder J I, Feldmann K A, Crawford N M. The herbicide sensitivity gene CHL1 of Arabidopsis encodes a nitrate-inducible nitrate transporter.Cell, 1993, 72(5): 705-713.
[10]	Liu K H, Huang C Y, Tsay Y F.CHL1 is a Dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake.Plant Cell, 1999, 11(5): 865-874.
[11]	Liu K H, Tsay Y F.Switching between the two action modes of the dual-affinity nitrate transporter CHL1 by phosphorylation. EMBO J, 2003, 22(5): 1005-1013.
[12]	Guo F Q, Wang R, Chen M, Crawford N M.The Arabidopsis dual-affinity nitrate transporter gene AtNRT1.1 (CHL1) is activated and functions in nascent organ development during vegetative and reproductive growth.Plant Cell, 2001, 13(8): 1761-1778.
[13]	Ho C H, Lin S H, Hu H, Tsay Y F.CHL1 functions as a nitrate sensor in plants.Cell, 2009, 138(6): 1184-1194.
[14]	Lin S H, Kuo H F, Canivenc G, Lin C S, Lepetit M, Hsu P K, Tillard P, Lin H L, Wang Y Y, Tsai C B, Gojon A, Tsay Y F.Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport.Plant Cell, 2008, 20(9): 2514-2528.
[15]	Chen C Z, Lv X F, Li J Y, Yi H Y, Gong J M.Arabidopsis NRT1.5 is another essential component in regulation of nitrate reallocation and stress tolerance.Plant Physiol, 2012, 159(4): 1582-1590.
[16]	Li J Y, Fu Y L, Pike S M, Bao J, Tian W, Zhang Y, Chen C Z, Zhang Y, Li H M, Huang J, Li LG, Schroeder J I, Gassmann W, Gong J M.The Arabidopsis nitrate transporter NRT1.8 functions in nitrate removal from the xylem sap and mediates cadmium tolerance.Plant Cell, 2010, 22(5):1633-1646.
[17]	Pike S, Gao F, Kim M J, Kim S H, Schachtman D P, Gassmann W.Members of the NPF3 transporter subfamily encode pathogen-inducible nitrate/nitrite transporters in grapevine and Arabidopsis.Plant Cell Physiol, 2014, 55(1):162-170.
[18]	Tal I, Zhang Y, Jørgensen M E, Pisanty O, Barbosa I C, Zourelidou M, Regnault T, Crocoll C, Olsen C E, Weinstain R, Schwechheimer C, Halkier B A, Nour-Eldin H H, Estelle M, Shani E. The Arabidopsis NPF3 protein is a GA transporter.Nat Commun, 2016, 7: 11486.
[19]	David L C, Berquin P, Kanno Y, Seo M, Daniel-Vedele F, Ferrario-Méry S.N availability modulates the role of NPF3.1, a gibberellin transporter, in GA-mediated phenotypes in Arabidopsis.Planta, 2016, 244(6): 1315-1328.
[20]	Fang Z, Xia K, Yang X, Grotemeyer M S, Meier S, Rentsch D, Xu X, Zhang M.Altered expression of the PTR/NRT1 homologue OsPTR9 affects nitrogen utilization efficiency, growth and grain yield in rice.Plant Biotechnol J, 2013, 11(4): 446-458.
[21]	Xia X, Fan X, Wei J, Feng H, Qu H, Xie D, Miller A J, Xu G.Rice nitrate transporter OsNPF2.4 functions in low-affinity acquisition and long-distance transport.J Exp Bot, 2015, 66(1): 317-331.
[22]	Li Y, Ouyang J, Wang Y Y, Hu R, Xia K, Duan J, Wang Y, Tsay Y F, Zhang M.Disruption of the rice nitrate transporter OsNPF2.2 hinders root-to-shoot nitrate transport and vascular development.Sci Rep, 2015, 5: 9635.
[23]	Hu R, Qiu D, Chen Y, Miller A J, Fan X, Pan X, Zhang M.Knock-Down of a tonoplast localized low-affinity nitrate transporter osnpf7.2 affects rice growth under high nitrate supply.Front Plant Sci, 2016, 7: 1529.
[24]	Tang Z, Fan X, Li Q, Feng H, Miller A J, Shen Q, Xu G.Knockdown of a rice stellar nitrate transporter alters long-distance translocation but not root influx.Plant Physiol, 2012, 160(4): 2052-2063.
[25]	Ai P, Sun S, Zhao J, Fan X, Xin W, Guo Q, Yu L, Shen Q, Wu P, Miller A J, Xu G.Two rice phosphate transporters, OsPht1;2 and OsPht1;6, have different functions and kinetic properties in uptake and translocation.Plant J, 2009, 57(5): 798-809.
[26]	Tsay Y F, Chiu C C, Tsai C B, Ho C H, Hsu P K.Nitrate transporters and peptide transporters.FEBS Lett, 2007, 581(12): 2290-2300.
[27]	Parker J L, Newstead S.Molecular basis of nitrate uptake by the plant nitrate transporter NRT1.1.Nature, 2014, 507(7490): 68-72.
[28]	Fan X, Feng H, Tan Y, Xu Y, Miao Q, Xu G.A putative 6-transmembrane nitrate transporter OsNRT1.1b plays a key role in rice under low nitrogen. J Integr Plant Biol, 2016, 58(6): 590-599.