华南农业大学学报2025，Vol.46Issue(4)：459-470,12.DOI:10.7671/j.issn.1001-411X.202410014
拟南芥异源过表达木薯MeNRT2.6基因对氮素利用效率的影响
Effects of heterologously overexpressing Manihot esculenta Crantz MeNRT2.6 gene in Arabidopsis thaliana on nitrogen utilization efficiency
摘要
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 functional characterization of the MeNRT2.6 gene in cassava(Manihot esculenta Crantz),by examining the effects of heterologous overexpression 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 overexpressing 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.Under 39.5 mmol/L nitrate nitrogen treatment,MeNRT2.6 expression was specifically induced in leaves regardless of treatment method.Furthermore,A.thaliana plants heterologously overexpressing 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 and NR.[Conclusion]The MeNRT2.6 promoter contains low nitrogen-responsive elements.Heterologous overexpression of MeNRT2.6 in A.thaliana improved nitrogen use efficiency,providing valuable insights for developing nitrogen-efficient cassava germplasms.关键词
木薯/MeNRT2.6基因/异源过表达/硝态氮胁迫/氮素利用/评价指标Key words
Manihot esculenta Crantz/MeNRT2.6 gene/Heterologous overexpression/Nitrate nitrogen stress/Nitrogen utilization/Evaluation indicator分类
农业科技引用本文复制引用
翟敏,李壮,曹颖杰,林晨俞,赵平娟,李文彬,于晓玲,廖文彬,阮孟斌,邹良平..拟南芥异源过表达木薯MeNRT2.6基因对氮素利用效率的影响[J].华南农业大学学报,2025,46(4):459-470,12.基金项目
海南省科技专项(ZDYF2023XDNY179) （ZDYF2023XDNY179）
海南省自然科学基金(322MS146) （322MS146）
中国热带农业科学院热带生物技术研究所基本科研业务费(1630052022026) （1630052022026）
