Effects of heterologously overexpressing Manihot esculenta Crantz MeNRT2.6 gene in Arabidopsis thaliana on nitrogen utilization efficiency

ZHAI Min; CAO Yingjie; LIN Chenyu; ZHAO Pingjuan; LI Wenbin; YU Xiaoling; LIAO Wenbin; RUAN Mengbin; ZOU Liangping; LI Zhuang

doi:10.7671/j.issn.1001-411X.202410014

Journal of South China Agricultural University > 2025 > Uncorrected proof > DOI: 10.7671/j.issn.1001-411X.202410014

ZHAI Min, CAO Yingjie, LIN Chenyu, et al. Effects of heterologously overexpressing Manihot esculenta Crantz MeNRT2.6 gene in Arabidopsis thaliana on nitrogen utilization efficiency[J]. Journal of South China Agricultural University, 2025, 46(0): 1-12. DOI: 10.7671/j.issn.1001-411X.202410014

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Effects of heterologously overexpressing Manihot esculenta Crantz MeNRT2.6 gene in Arabidopsis thaliana on nitrogen utilization efficiency

ZHAI Min^{1, 2, 3,},
CAO Yingjie^{2, 4},
LIN Chenyu^{2, 3, 4},
ZHAO Pingjuan^{2, 3},
LI Wenbin^{2, 3},
YU Xiaoling^{2, 3},
LIAO Wenbin^{2, 3},
RUAN Mengbin^{2, 3},
ZOU Liangping^{2, 3, ,},
LI Zhuang^1, ,

1.
College of Agriculture, Sichuan Agricultural University, Chengdu 611130, China
2.
Institute of Tropical Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
3.
Sanya Research Institute/Institute of Tropical Agricultural Resources, Chinese Academy of Tropical Agricultural Sciences, Sanya 572024, China
4.
School of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China

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Abstract

Abstract

Objective
The high-affinity nitrate transporter family 2 (NRT2), when upregulated under low nitrogen conditions, enhances plants’ efficient nitrogen uptake and plays a crucial role in improving plant tolerance to low nitrogen stress. This study focused on further functional characterization of the MeNRT2.6 gene in cassava (Manihot esculenta Crantz), by examining the effects of heterologous expression of cassava MeNRT2.6 in Arabidopsis thaliana on nitrogen use efficiency, to provide both theoretical foundation and technical support for targeted molecular breeding aimed at improving nitrogen utilization efficiency.
Method
The MeNRT2.6 gene, which was identified through transcriptome screening as being highly expressed in cassava roots, stems and leaves under low nitrogen conditions, was selected as the research subject. The MeNRT2.6 gene fragment was obtained by PCR amplification using cDNA from cassava cultivar C3 as template. Bioinformatics analysis and tissue expression profiling were performed, and the tissue expression pattern and subcellular localization of MeNRT2.6 were determined combining a protoplast transient expression system. The transgenic A. thaliana lines heterologously expressing MeNRT2.6 were generated via the floral dip method. The SPAD values and the activities of four key nitrogen metabolism enzymes, including nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (GOGAT), under low and high nitrate conditions were measured.
Result
Phylogenetic analysis showed that MeNRT2.6 was most closely related to PtNRT2.7 from Populus trichocarpa, with 74.5% amino acid sequence similarity. Its promoter sequence contains stress- and hormone-responsive cis-elements. MeNRT2.6 protein was localized to the cell membrane. Under 0.5 mmol/L nitrate nitrogen treatment, MeNRT2.6 expression was induced in cassava roots, stems and leaves depending on the treatment method. At 39.5 mmol/L nitrate nitrogen treatment, MeNRT2.6 expression was specifically induced in leaves regardless of treatment method. Furthermore, A. thaliana plants heterologously expressing MeNRT2.6 showed increased root length, plant height and fresh weight under 0.5 mmol/L nitrate nitrogen treatment, along with enhanced activities of key nitrogen metabolism enzymes GOGAT, NR.
Conclusion
The MeNRT2.6 promoter contains low nitrogen-responsive elements. Heterologous expression of MeNRT2.6 in A. thaliana improved nitrogen use efficiency, providing valuable insights for developing nitrogen-efficient cassava germplasms.
- MeNRT2.6 gene,
- Heterologous expression,
- Nitrate nitrogen stress,
- Nitrogen utilization,
- Evaluation indicator

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References (45)

References

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Effects of heterologously overexpressing Manihot esculenta Crantz MeNRT2.6 gene in Arabidopsis thaliana on nitrogen utilization efficiency

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