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

doi:10.7668/hbnxb.20193422

Abstract

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

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.

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Figures/Tables 6

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

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.

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.

Fig.3 Gene structure of CsNPF5

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

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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