茶树硝态氮转运蛋白 NRT1.1基因的克隆及表达分析

摘要

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 at 0.5 h. Our results provides favorably help to reveal NO3- uptake and utilization in tea plants.%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1880 bp，其中开放阅读框（ORF）1788 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。