茶树<i>NPF5</i>基因克隆及其在不同氮素处理下的表达

doi:10.7668/hbnxb.20193422

摘要/Abstract

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2 096,1 440 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1 440 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NO₃^-浓度升高,响应时间更快,且在2 mmol/L NO₃^-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。

关键词: 茶树, 氮转运蛋白, CsNPF5, 氮素响应, 表达分析

Abstract:

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 2 096,1 440 bp,respectively.Sequence analysis suggested that the gene contained three exons and two introns,with an open reading frame(ORF)of 1 440 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 NO₃^- concentration;the peak expression of CsNPF5 in tea leaves appeared after 24 h at 2 mmol/L NO₃^- treatment,which indicated that the gene expression was induced by nitrogen.

Key words: Camellia sinensis, Nitrogen transporter, CsNPF5, Nitrogen response, Expression analysis

唐君, 代祥, 李贵飞, 穆兵, 王枫, 杨亦扬. 茶树NPF5基因克隆及其在不同氮素处理下的表达[J]. 华北农学报, 2023, 38(2): 93-98. doi: 10.7668/hbnxb.20193422.

TANG Jun, DAI Xiang, LI Guifei, MU Bing, WANG Feng, YANG Yiyang. Cloning of NPF5 Gene of Tea Plants and Its Expression under Different Concentration Nitrogen Treatments[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(2): 93-98. doi: 10.7668/hbnxb.20193422.

http://www.hbnxb.net/CN/Y2023/V38/I2/93

图/表 6

表1 PCR扩增的引物序列

Tab.1 Primer sequences of PCR amplification

引物名 Names of primers	引物序列(5'-3') Sequences of primers(5'-3')	用途 Uses
T1	ATGACATTATTGACACTATCAGC	PCR扩增
T2	TCAAGGTGTTATATGAGTGGATG	PCR扩增
T3	ACAGTGTTGCCAGCAAG	定量PCR扩增
T4	CATAGACTGGGACCCAGA	定量PCR扩增
T5	TTGGCATCGTTGAGGGTCT	定量PCR扩增
T6	CAGTGGGAACACGGAAAGC	定量PCR扩增

图1 CsNPF5基因PCR扩增 M.2000 bp DNA Ladder;1.以cDNA为模板目的基因PCR扩增产物;2.以gDNA为模板目的基因PCR扩增产物。

Fig.1 PCR amplification of CsNPF5 gene M.2 000 bp DNA Ladder;1.The PCR amplification product,which was obtained by using cDNA as a template to amplify the target gene;2.The PCR amplification product,which was obtained by using gDNA as a template to amplify the target gene.

图2 CsNPF5基因序列特征阴影部分.指示PTR2结构域在CsNPF5蛋白上的分布;红色的ATG.起始密码子;*.终止密码子。

Fig.2 Sequence characteristics of CsNPF5 gene Shaded area.Indicates the distribution of the PTR2 domain in CsNPF5 protein;Red ATG.Initiation codon;*.Termination codon.

图3 CsNPF5基因结构

Fig.3 Gene structure of CsNPF5

图4 不同植物中CsNPF5同源蛋白的聚类分析节点自展值小于50%不显示。

Fig.4 Phylogenetic analysis of CsNPF5 orthologous proteins from different plants The bootstrap values of nodes less than 50% is not displayed.

图5 不同氮素处理下CsNPF5的相对表达模式不同小写字母表示不同氮素处理下CsNPF5表达差异达显著水平(P<0.05)。

Fig.5 Expression patterns of CsNPF5 gene under different nitrogen treatments Different lowercase letters indicate significant differences (P<0.05)of CsNPF5 expression under different nitrogen treatments.

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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

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