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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (8): 2131-2142.doi: 10.3724/SP.J.1006.2024.44002

• RESEARCH NOTES • Previous Articles     Next Articles

Identification of nitrate transporter protein 1/peptide transporter protein family 6.4 gene (ScNPF6.4) and functional analysis of its regulation of tillering in sugarcane

LI Xu-Juan1,2(), LI Chun-Jia1,2, TIAN Chun-Yan1,2, KONG Chun-Yan1,2, XU Chao-Hua1,2, LIU Xin-Long1,2,*()   

  1. 1National Key Laboratory for Biological Breeding of Tropical Crops, Kunming 650205, Yunnan, China
    2Sugarcane Research Institute, Yunnan Academy of Agricultural Sciences / Yunnan Key Laboratory of Sugarcane Genetic Improvement, Kaiyuan 661699, Yunnan, China
  • Received:2024-01-02 Accepted:2024-04-01 Online:2024-08-12 Published:2024-04-24
  • Contact: * E-mail: lxlgood868@163.com
  • Supported by:
    Yunnan Seed Laboratory(202205AR070001-09);National Natural Science Foundation of China(31760412);National Key Research and Development Program of China(2022YFD2301102-20);Yunnan Science and Technology Talent and Platform Program(202205AM070001)

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Abstract:

Sugarcane is an important sugar and energy crop, and tillering is one of the important traits affecting its yield. Nitrate transporter 1 (NRT1) / peptide transporter (PTR) family (NPF) protein plays an important role in plant vegetative growth and development, mining and exploitation of NPF genes in sugarcane could lay an important foundation for genetic regulation of tillering in sugarcane. In this study, the full-length cDNA sequence of the NPF6.4 gene (ScNPF6.4) was obtained from sugarcane variety ROC22 by combining pre-transcriptomic data with RT-PCR. Subsequently, the sequence structure, physicochemical properties and phylogeny were analyzed. The tissue-specific expression of the gene in sugarcane at seedlings stage, the expression in axillary bud germination, and the expression pattern in responsive to hormone treatments were analyzed. Finally, the gene was genetically transformed into rice to verify the over-expression for functional verification. The results showed that the cDNA of ScNPF6.4 contained an open reading frame of 1806 bp encoding 601 amino acids, which belonged to the major facilitator superfamily (MFS) protein with a molecular weight of 63.9 kD and a theoretical isoelectric point of 9.23, and included 12 transmembrane helical regions. The protein was hydrophobic in nature and had no signal peptide, whereas it belonged to a class of stable non-secretory proteins. In addition, phylogenetic analysis illustrated that it belonged to the NPF family and 6.4 subfamily of proteins. Likewise, subcellular localization indicated that the protein was localized in the endoplasmic reticulum. The tissue-specific analysis revealed that the relative expression level of ScNPF6.4 was the highest in roots and lower in leaf and stem bases of sugarcane seedling. Moreover, ScNPF6.4 was up-regulated at the axillary bud sprouting stage of different sugarcane varieties, and appropriate concentrations of exogenous plant hormones such as 6-BA, ABA, GA3, IBA, ethylene, and SLs could induce the up-regulated its expression in sugarcane at seedling stage, while ectopic over-expression of ScNPF6.4 increased the tillering number and induced early heading in rice. Thus, it could be inferred that ScNPF6.4 could regulate the germination of sugarcane tiller buds, and its expression was up-regulated by exogenous phytohormones, whereas the over-expression of this gene could increase the tillering numbers and induce early heading in rice. This study provides an important genetic resource for the breeding of sugarcane to improve tillering ability, which is beneficial for the early and rapid growth and high cane yield formation.

Key words: sugarcane, ScNPF6.4, tillering, exogenous plant hormone, functional analysis

Table 1

Primers used in this study"

引物名称Primer name 引物序列Primer sequence (5°-3°) 引物作用 Primer function
NPF-54F GTCGTCCTCGCTCCCAGTC 基因克隆
Gene cloning
NPF-2143R CGCCGTATGCCTCCTATGTT
NPF-138F ATGGTTTCCGCTGGGG
NPF-1943R CTACACGTCCATTCCTTCG
Q-ScNPF-208F TCCAACTCCAAGTCCGCCAAC 表达分析(甘蔗)
Relative expression analysis (in sugarcane)
Q-ScNPF-358R CAAGCTCACGCCGGTGGC
GAPDH-F CACGGC CAC TGGAAGCA 内参基因(甘蔗)
Reference gene (in sugarcane)
GAPDH-R TCC TCAGGG TTC CTG ATG CC
Q-ScNPF6.4-F CGCGAACGCCCGTGGTCCGC 表达分析(水稻)
Relative expression analysis (in rice)
Q-ScNPF6.4-R CGGACACGTTCGCCTTGAGACCCCCC
OsActin TGCTATGTACGTCGCCATCCA 内参基因(水稻)
Reference gene (in rice)
OsActin AATGAGTAACCACGCTCCGTC

Fig. S1

Electrophoretic results of PCR amplification products of ScNPF6.4 coding region"

Fig. S2

Gene structure of ScNPF6.4 The yellow bar and the black line represent exon and intron, respectively."

Fig. 1

Nucleotide sequence and derived amino acid sequence of ScNPF6.4"

Fig. 2

Phylogenetic tree of NRT1 protein of sugarcane and NPF proteins of other plants ScNPF6.4 was marked in red. The NPFs used in the phylogenetic tree are from S. spontaneum L. (SsNPF), Zea mays (ZmNPF), Sorghum bicolor (SbNPF), Setaria italica (SiNPF), Oryza sativa (OsNPF), Hordeum vulgare (HvNPF), Nicotiana attenuata (NaNPF), Solanum lycopersicum (SlNPF), Helianthus annuus (HaNPF), Glycine max (GmNPF), and Arabidopsis thaliana (AtNPF)."

Fig. S3

Secondary structure prediction of ScNPF6.4 Red e is the extended strand, green t is the beta turn, blue h is the alpha helix, and orange c is the random coil."

Fig. S4

Predicted transmembrane structure of ScNPF6.4"

Fig. 3

Subcellular localization of ScNPF6.4 in rice protoplast GFP: green fluorescent light channel; Bright: bright field; Merged: overlapping. Bar: 10 μm."

Fig. 4

Relative expression patterns of ScNPF6.4 genes A: the relative expression level of ScNPF6.4 in different tissues of ROC22; B: the relative expression level of ScNPF6.4 at different developmental stages of axillary buds of Huanan 56-21, Guitang 17 and ROC22; C: the relative expression level of ScNPF6.4 under different exogenous plant hormones treatments. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as an internal reference gene. The error bar represents the mean ± standard deviation (n = 3) of the repeats. Different lowercase letters above the bars indicate significant difference between treatments, as determined by the Duncan’s new multiple range test (P < 0.05). bud 0: dormant bud; bud 1: budding bud; bud 2: enlarged bud; bud 3: elongation bud; bud 4: sharp leaf bud. 6-BA: 6-benzylaminopurine (400 mg L-1); ABA: abscisic acid (4 mg L-1); GA3: gibberellin A3 (200 mg L-1); IBA: indole-3-butytric acid (400 mg L-1); Ethephon: 100 mg L-1; SLs: strigolactones (20 mg L-1)."

Fig. 5

Spatiotemporal expression and phenotypic analysis of ScNPF6.4 overexpression transgenic rice Rice activator protein gene (OsActin) is used as the internal reference gene. The error bar represents the mean ± standard deviation (n = 3) of the repeats. Different lowercase letters above the bars indicate a significant difference between treatments, as determined by the Duncan’s new multiple range test at P < 0.05."

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