茶树NPF5基因克隆及其在不同氮素处理下的表达  

Cloning of NPF5 Gene of Tea Plants and Its Expression under Different Concentration Nitrogen Treatments

作　　者：唐君 代祥 李贵飞 穆兵 王枫[2] 杨亦扬[1] TANG Jun;DAI Xiang;LI Guifei;MU Bing;WANG Feng;YANG Yiyang(Institute of Leisure Agriculture,Jiangsu Academy of Agricultural Sciences,Jiangsu Key Laboratory for Horticulture Crop Genetic Improvement,Nanjing 210014,China;College of Horticulture,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]江苏省农业科学院休闲农业研究所,江苏省高效园艺作物遗传改良重点实验室,江苏南京210014 [2]南京农业大学园艺学院,江苏南京210095

出　　处：《华北农学报》2023年第2期93-98,共6页Acta Agriculturae Boreali-Sinica

基　　金：江苏省种业振兴揭榜挂帅项目(JBGS(2021)085);江苏省重点研发计划项目(BE2019379)。

摘　　要：NPF转运蛋白家族在植物转运硝态氮过程中起重要作用。为明确茶树中NPF基因的序列特征,用PCR扩增的方法在茶树中分别扩增出一个茶树氮转蛋白基因CsNPF5的DNA和cDNA全长,为2096,1440 bp。序列分析指出,该基因含有3个外显子和2个内含子,开放阅读框(ORF)为1440 bp。序列分析结果显示,该基因编码479个氨基酸,相对分子质量约为53.12 ku,等电点为7.13,其编码蛋白的N端含有一个PTR2 Pfam结构域;亚细胞定位分析指出该基因编码蛋白定位于细胞质内;CsNPF5蛋白与中华猕猴桃NPF蛋白具有较高的同源性,相似度为79.54%;聚类分析指出,CsNPF5及其同源蛋白分别聚类成3个进化分支,且与来自中华猕猴桃NPF18同源蛋白聚到同一进化支上。同时qRT-PCR分析结果表明,在0~48 h氮素处理下CsNPF5在叶中的表达存在不同程度的上调表达,且基因上调表达丰度随NO_(3)^(-)浓度升高,响应时间更快,且在2 mmol/L NO_(3)^(-)处理24 h后,CsNPF5在茶树叶片中表达量出现峰值,这表明该基因表达受氮素诱导。The NPF transporter family plays an important role in plants transporting nitrate nitrogen.To reveal the sequence characteristic of NPF genes,the DNA and cDNA sequences of the nitrogen transporter CsNPF5 gene of tea plants were amplified by using PCR amplification,with full-lengths of 2096,1440 bp,respectively.Sequence analysis suggested that the gene contained three exons and two introns,with an open reading frame(ORF)of 1440 bp,encoding 479 amino acids,having a relative molecular mass of 53.12 ku,an isoelectric point of 7.13 and a PTR2 Pfam domain at the N-terminal end of the encoded protein;subcellular localization analysis indicated that the encoded protein was localized in the cytoplasm;sequence alignment result showed that the CsNPF5 protein had high homology with the Chinese kiwifruit NPF protein,with a similarity of 79.54%;cluster analysis indicated that CsNPF5 and its homologous proteins were classified into three evolutionary branches,and CsNPF5 clustered into the same branch with the homologous protein NPF18 from Chinese kiwifruit.Meanwhile,the qRT-PCR analysis showed that the expression of CsNPF5 was differentially up-regulated in leaves under 0-48 h of nitrogen treatment;up-regulated expression abundance of gene responded faster with increasing NO_(3)^(-) concentration;the peak expression of CsNPF5 in tea leaves appeared after 24 h at 2 mmol/L NO_(3)^(-) treatment,which indicated that the gene expression was induced by nitrogen.

关 键 词：茶树 氮转运蛋白 CsNPF5 氮素响应 表达分析 

分 类 号：S571.03[农业科学—作物学] Q78[生物学—分子生物学]