茶树硝态氮转运蛋白NRT1.1基因的克隆及表达分析

doi:10.13305/j.cnki.jts.2016.05.009

茶叶科学 ›› 2016, Vol. 36 ›› Issue (5): 505-512.doi: 10.13305/j.cnki.jts.2016.05.009

茶树硝态氮转运蛋白NRT1.1基因的克隆及表达分析

杨亦扬^1,2, 胡雲飞¹, 万青¹, 李荣林¹, 王枫³, 阮建云^2,*

1. 江苏省农业科学院园艺研究所江苏省高效园艺作物遗传改良重点实验室,江苏南京 210014;
2. 中国农业科学院茶叶研究所农业部茶树生物学与资源利用重点实验室,浙江杭州 310008;
3. 南京农业大学园艺学院作物遗传与种质创新国家重点实验室,江苏南京 210095

收稿日期:2016-04-25 出版日期:2016-10-15 发布日期:2019-08-26
通讯作者: *,jruan@mail.tricaas.com
作者简介:杨亦扬,女,博士,副研究员,主要从事茶树生理与营养研究。
基金资助:
国家自然科学基金（31400587）、江苏省自然科学基金（BK20160590）、茶树生物学与资源利用国家重点实验室开放基金（SKLTOF20150114）、江苏省农业科技自主创新资金（CX(16)1003）

Cloning and Expression Analysis of Nitrate Transporter NRT1.1 Gene in Tea Plant (Camellia sinensis (L.))

YANG Yiyang^1,2, HU Yunfei¹, WAN Qing¹, LI Ronglin¹, WANG Feng³, RUAN Jianyun^2,*

1. Institute of Horticulture, Jiangsu Academy of Agricultural Science, Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Nanjing 210014, China;
2. Tea Research of Institute, Chinese of Academy of Agricultural Science, Key Laboratory of Tea Biology and Resources Utilization, Ministry of Agriculture, Hangzhou 310008, China;
3. College of Horticulture, Nanjing Agricultural University, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing 210095, China

Received:2016-04-25 Online:2016-10-15 Published:2019-08-26

摘要/Abstract

摘要： 以茶树（Camellia sinensis (L.)）品种龙井43为试材,采用PCR结合RACE技术,克隆得到硝态氮转运蛋白基因（NRT1.1）的cDNA全长序列,基因序列全长1 880 bp,其中开放阅读框（ORF）1 788 bp,编码595个氨基酸,预测蛋白质分子量为65.9 kD,理论等电点为8.99,命名为CsNRT1.1。序列分析表明,CsNRT1.1与葡萄NRT1.1氨基酸序列的相似性最高。通过生物信息学分析,对CsNRT1.1的氨基酸理化性质、亲/疏水性、跨膜区域及亚细胞定位进行了预测。实时定量PCR表达分析表明,茶树根和叶片中CsNRT1.1在1 mol·L^-1 NO₃^-处理5 min内均受到抑制,叶部CsNRT1.1表达量0.5 h后即达到最大值,24 h内各个时间点均高于根部,根中表达量CsNRT1.1始终低于对照。本研究结果为研究茶树对NO₃^-的吸收、转运和调控机理提供了分子生物学基础。

关键词: 茶树, 硝态氮, NRT1.1基因, 实时定量PCR

Abstract: A full length cDNA sequence of Nitrate transport gene (NRT1.1) was obtained from tea plant (Camellia sinensis (L.)) cultivar ‘Longjing 43’ by polymerase chain reaction (PCR) and rapid amplification of cDNA ends PCR (RACE-PCR). The length of nucleotide sequence of this gene was 1 880 bp, containing a complete open reading frame (1 788 bp) to encode 595 amino acids. The putative protein had an isoelectric point of 8.99 and a calculated molecular weight of 65.9 kD. CsNRT1.1 was highly homologous to the gene NRT1.1 in Vitis vinifera by sequence alignment. Several parameters of these sequences, including sequences composition, physicochemical property, topological structure of transmembrane regions, hydrophobicity or hydrophilicity, subcellular localization were predicted by bioinformatics tools. Quantitative real-time PCR analysis showed that the expression of CsNRT1.1 in roots and leaves were inhibited after incubation in 1 mol·L^-1 NO₃^- for 5 min. The expressions of CsNRT1.1 in roots were always lower than that of CK within 24 h. Its expressions in leaves were higher than those in roots with its peak

Key words: Camellia sinensis, nitrate, NRT1.1, RT-PCR

中图分类号:

S571.1
Q51

杨亦扬, 胡雲飞, 万青, 李荣林, 王枫, 阮建云. 茶树硝态氮转运蛋白NRT1.1基因的克隆及表达分析[J]. 茶叶科学, 2016, 36(5): 505-512. doi: 10.13305/j.cnki.jts.2016.05.009.

YANG Yiyang, HU Yunfei, WAN Qing, LI Ronglin, WANG Feng, RUAN Jianyun. Cloning and Expression Analysis of Nitrate Transporter NRT1.1 Gene in Tea Plant (Camellia sinensis (L.))[J]. Journal of Tea Science, 2016, 36(5): 505-512. doi: 10.13305/j.cnki.jts.2016.05.009.

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茶树硝态氮转运蛋白NRT1.1基因的克隆及表达分析

Cloning and Expression Analysis of Nitrate Transporter NRT1.1 Gene in Tea Plant (Camellia sinensis (L.))

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