Function Analyses of Rice Nitrate Transporter Gene OsNPF7.9 in Nitrogen Accumulation and Transport

doi:10.16819/j.1001-7216.2017.7031 457

Abstract

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

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

CLC Number:

Q786
S511.01

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.

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.

Figures/Tables 9

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

Fig. 1. Phylogenetic of NPF in plants.

Fig. 2. Transmembrane topology of OsNPF7.9.

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

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

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

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

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

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

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