运用PCR和RT-PCR技术从萝卜中克隆出RsIRT1基因,其cDNA包含一个长度为1 038 bp的开放阅读框,编码345个氨基酸,蛋白质相对分子质量为36.37×103。TMHMM Server v2.0分析RsIRT1蛋白有8个跨膜结构域,推测为一种膜蛋白;蛋白序列同源分...运用PCR和RT-PCR技术从萝卜中克隆出RsIRT1基因,其cDNA包含一个长度为1 038 bp的开放阅读框,编码345个氨基酸,蛋白质相对分子质量为36.37×103。TMHMM Server v2.0分析RsIRT1蛋白有8个跨膜结构域,推测为一种膜蛋白;蛋白序列同源分析表明,萝卜RsIRT1(AFJ05595)与拟南芥AtIRT1(XP-002869968)、遏蓝菜TcIRT1-P(CAL25151)的遗传距离最近。半定量RT-PCR表达分析表明,正常铁营养时RsIRT1基因在萝卜花瓣、花蕾、叶片和根中均不表达,缺铁胁迫及缺铁加镉(100 mg·L-1)胁迫下表达,且缺铁加镉胁迫下,叶片和根中RsIRT1的表达量均高于缺铁胁迫。研究结果表明RsIRT1基因受外源缺铁和镉胁迫所诱导,RsIRT1参与金属铁和镉的吸收和转运过程。展开更多
Iron(Fe)is an essential micronutrient for all organisms.Fe availability in the soil is usually much lower than that required for plant growth,and Fe deficiencies seriously restrict crop growth and yield.Calcium(Ca2+)i...Iron(Fe)is an essential micronutrient for all organisms.Fe availability in the soil is usually much lower than that required for plant growth,and Fe deficiencies seriously restrict crop growth and yield.Calcium(Ca2+)is a second messenger in all eukaryotes;however,it remains largely unknown how Ca2+regulates Fe deficiency.In this study,mutations in CPK21 and CPK23,which are two highly homologous calcium-dependent protein kinases,conferredimpaired growth and rootdevelopment under Fe-deficient conditions,whereas constitutively active CPK21 and CPK23 enhanced plant tolerance to Fe-deficient conditions.Furthermore,we found that CPK21 and CPK23 interacted with and phosphorylated the Fe transporter IRONREGULATED TRANSPORTER1(IRT1)at the Ser149 residue.Biochemical analyses and complementation of Fe transport in yeast and plants indicated that IRT1 Ser149 is critical for IRT1 transport activity.Taken together,these findings suggest that the CPK21/23-IRT1 signaling pathway is critical for Fe homeostasis in plants and provides targets for improving Fe-deficient environments and breeding crops resistant to Fe-deficient conditions.展开更多
Epitope tags are widely used for detecting,modifying,or purifying proteins of interest,but their range of application is often limited.Recently,the rationally designed ALFA tag and its ALFA nanobody have expanded the ...Epitope tags are widely used for detecting,modifying,or purifying proteins of interest,but their range of application is often limited.Recently,the rationally designed ALFA tag and its ALFA nanobody have expanded the repertoire of epitope tags and emerged as a highly versatile system characterized in various animal models,outperforming existing tags.Here,we evaluated the ALFA tag/ALFA nanobody technology in plants and demonstrated its application for in planta protein detection across multiple compartments and cellular structures,protein-protein interaction studies,protein immunoprecipitation,inducedproximity approaches,and super-resolution microscopy.Most importantly,we highlight the potential of the ALFA tagging technology for proteins that are difficult to tag due to topological or functional constraints.We provide proof of concept for the ALFA tag technology in the detection and functional analysis of the Arabidopsis IRT1 Fe transporter.Overall,this versatile and validated toolbox of ALFA tag and ALFA nanobody applications will serve as a valuable resource for functional studies in plants.展开更多
基金supported by the National Natural Science Foundation of China(32222008,32100215,31900236)Northwest A&F University(Z111021604)+1 种基金the open funds of China Postdoctoral Science Foundation(2018M643740)Natural Science Basic Research Plan in Shaanxi Province of China(2019JQ-150).
文摘Iron(Fe)is an essential micronutrient for all organisms.Fe availability in the soil is usually much lower than that required for plant growth,and Fe deficiencies seriously restrict crop growth and yield.Calcium(Ca2+)is a second messenger in all eukaryotes;however,it remains largely unknown how Ca2+regulates Fe deficiency.In this study,mutations in CPK21 and CPK23,which are two highly homologous calcium-dependent protein kinases,conferredimpaired growth and rootdevelopment under Fe-deficient conditions,whereas constitutively active CPK21 and CPK23 enhanced plant tolerance to Fe-deficient conditions.Furthermore,we found that CPK21 and CPK23 interacted with and phosphorylated the Fe transporter IRONREGULATED TRANSPORTER1(IRT1)at the Ser149 residue.Biochemical analyses and complementation of Fe transport in yeast and plants indicated that IRT1 Ser149 is critical for IRT1 transport activity.Taken together,these findings suggest that the CPK21/23-IRT1 signaling pathway is critical for Fe homeostasis in plants and provides targets for improving Fe-deficient environments and breeding crops resistant to Fe-deficient conditions.
基金supported by research grants from the French National Research Agency(grant nos.ANR-21-CE20-0046 to G.V.and 22-PESV0002 to J.N.)the French Laboratory of Excellence(project“TULIP”grant nos.ANR-10-LABX-41 and ANR-11-IDEX-0002-02 to G.V.).
文摘Epitope tags are widely used for detecting,modifying,or purifying proteins of interest,but their range of application is often limited.Recently,the rationally designed ALFA tag and its ALFA nanobody have expanded the repertoire of epitope tags and emerged as a highly versatile system characterized in various animal models,outperforming existing tags.Here,we evaluated the ALFA tag/ALFA nanobody technology in plants and demonstrated its application for in planta protein detection across multiple compartments and cellular structures,protein-protein interaction studies,protein immunoprecipitation,inducedproximity approaches,and super-resolution microscopy.Most importantly,we highlight the potential of the ALFA tagging technology for proteins that are difficult to tag due to topological or functional constraints.We provide proof of concept for the ALFA tag technology in the detection and functional analysis of the Arabidopsis IRT1 Fe transporter.Overall,this versatile and validated toolbox of ALFA tag and ALFA nanobody applications will serve as a valuable resource for functional studies in plants.
