相较于传统的抗体检测,适配体更易于大量快速合成,且可和多种检测技术相结合,在蛋白检测方面具有巨大的潜力.水孔蛋白作为生物体内水分跨膜运输的主要途径,了解其表达量的变化在植物水代谢研究中有着重要意义.利用传统的混合列分法构建...相较于传统的抗体检测,适配体更易于大量快速合成,且可和多种检测技术相结合,在蛋白检测方面具有巨大的潜力.水孔蛋白作为生物体内水分跨膜运输的主要途径,了解其表达量的变化在植物水代谢研究中有着重要意义.利用传统的混合列分法构建了8个C端恒定半胱氨酸残基的类肽适配体文库,结合表面等离激元共振成像技术,筛选得到能特异性结合高等植物水孔蛋白PIP2的类肽适配体PPA7,其亲和力 K D高达2.52×10 -9 mol/L.利用PPA7检测了石竹玻璃化和正常植株的水孔蛋白表达量,结果表明,石竹玻璃化植株的水孔蛋白表达量显著高于正常植株.研究提供了一种新的植物蛋白定量检测策略,也为进一步明确水孔蛋白在组培苗玻璃化发生中的作用奠定了基础.展开更多
As a member of the inwardly rectifying channel (Kir) family, Kir2.1 allows to influx the cell more easily than to efflux, a biophysical phenomenon named inward rectification. The function of Kir2.1 is to set the resti...As a member of the inwardly rectifying channel (Kir) family, Kir2.1 allows to influx the cell more easily than to efflux, a biophysical phenomenon named inward rectification. The function of Kir2.1 is to set the resting membrane potential and modulate membrane excitability. It has been reported that residue E224 plays a key role in regulating inward rectification. The mutant Kir2.1 (E224G) displays weaker inward rectification than the WT channel. Gating of Kir2.1 depends on the membrane lipid, PIP<sub>2</sub>, such that the channel gates are closed in the absence of PIP<sub>2</sub>. Here we perform electrophysiological and computational approaches, and demonstrate that E224 also plays an important role in the PIP<sub>2</sub>-dependent activation of Kir2.1 in addition to its influence on inward rectification. The E224G mutant takes 4.5 times longer to be activated by PIP<sub>2</sub>. To probe the mechanism by which E224G slows the channel opening kinetics, we perform targeted molecular dynamics simulations and find that the mutant weakens the interactions between CD-loop and C-linker (H221-R189) and the adjacent G-loops (R312-E303) which are thought to stabilize the open state of the channel in our previous work. These data provide new insights into the regulation of Kir2.1 channel activity and suggest that a common mechanism may be involved in the distinct biophysical processes, such as inward rectification and PIP<sub>2</sub>-induced gating.展开更多
Plasma membrane intrinsic proteins(PIPs),a subclass of aquaporins,play an important role in plant immunity by acting as H2O2 transporters.Their homeostasis is mostly maintained by C-terminal serine phosphorylation.How...Plasma membrane intrinsic proteins(PIPs),a subclass of aquaporins,play an important role in plant immunity by acting as H2O2 transporters.Their homeostasis is mostly maintained by C-terminal serine phosphorylation.However,the kinases that phosphorylate PIPs and manipulate their turnover are largely unknown.Here,we found that Arabidopsis thaliana PIP2;7 positively regulates plant immunity by transporting H_(2)O_(2).Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE 28(CPK28)directly interacts with and phosphorylates PIP2;7 at Ser273/276 to induce its degradation.During pathogen infection,CPK28 dissociates from PIP2;7 and destabilizes,leading to PIP2;7 accumulation.As a countermeasure,oomycete pathogens produce conserved kinase effectors that stably bind to and mediate the phosphorylation of PIP2;7 to induce its degradation.Our study identifies PIP2;7 as a novel substrate of CPK28 and shows that its protein stability is negatively regulated by CPK28.Such phosphorylation could be mimicked by Phytophthora kinase effectors to promote infection.Accordingly,we developed a strategy to combat oomycete infection using a phosphorylation-resistant PIP2;7S273/276A mutant.The strategy only allows accumulation of PIP2;7^(S273/276A) during infection to limit potential side effects on normal plant growth.展开更多
经qRT-PCR,采用ΔCt值分析法、geNorm和NormFinder软件分析法综合比较Act2、TUB-α、TUB-β、18S rRNA、GAPDH、EF-1a、RNA POL II、APRT、TLF9个基因的稳定值,筛选大针茅干旱胁迫下基因表达分析最佳内参基因组合。结果表明,在根中最稳...经qRT-PCR,采用ΔCt值分析法、geNorm和NormFinder软件分析法综合比较Act2、TUB-α、TUB-β、18S rRNA、GAPDH、EF-1a、RNA POL II、APRT、TLF9个基因的稳定值,筛选大针茅干旱胁迫下基因表达分析最佳内参基因组合。结果表明,在根中最稳定的内参基因组合为18S rRNA和EF-1a,在叶中最稳定的内参基因组合为18S rRNA和TLF。在不同干旱胁迫下用大针茅根和叶中PIP2-1和PIP2-2基因表达差异验证最佳内参基因组合的稳定性表明,所筛选的内参基因稳定性较好,可以作为大针茅干旱胁迫基因表达的内参基因。展开更多
文摘相较于传统的抗体检测,适配体更易于大量快速合成,且可和多种检测技术相结合,在蛋白检测方面具有巨大的潜力.水孔蛋白作为生物体内水分跨膜运输的主要途径,了解其表达量的变化在植物水代谢研究中有着重要意义.利用传统的混合列分法构建了8个C端恒定半胱氨酸残基的类肽适配体文库,结合表面等离激元共振成像技术,筛选得到能特异性结合高等植物水孔蛋白PIP2的类肽适配体PPA7,其亲和力 K D高达2.52×10 -9 mol/L.利用PPA7检测了石竹玻璃化和正常植株的水孔蛋白表达量,结果表明,石竹玻璃化植株的水孔蛋白表达量显著高于正常植株.研究提供了一种新的植物蛋白定量检测策略,也为进一步明确水孔蛋白在组培苗玻璃化发生中的作用奠定了基础.
基金Supported by the National Natural Science Foundation for Distinguished Young Scholars of Hebei Province under Grant Nos C2015202340 and C2013202244the Foundation for Outstanding Talents of Hebei Province under Grant No C201400305+3 种基金the National Natural Science Foundation of China under Grant Nos 11247010,11175055,11475053,11347017,31400711 and 11647121the NIH R01 under Grant No HL059949-18the Foundation for the Science and Technology Program of Higher Education Institutions of Hebei Province under Grant No QN2016113the Scientific Innovation Fund for Excellent Young Scientists of Hebei University of Technology under Grant No 2015010
文摘As a member of the inwardly rectifying channel (Kir) family, Kir2.1 allows to influx the cell more easily than to efflux, a biophysical phenomenon named inward rectification. The function of Kir2.1 is to set the resting membrane potential and modulate membrane excitability. It has been reported that residue E224 plays a key role in regulating inward rectification. The mutant Kir2.1 (E224G) displays weaker inward rectification than the WT channel. Gating of Kir2.1 depends on the membrane lipid, PIP<sub>2</sub>, such that the channel gates are closed in the absence of PIP<sub>2</sub>. Here we perform electrophysiological and computational approaches, and demonstrate that E224 also plays an important role in the PIP<sub>2</sub>-dependent activation of Kir2.1 in addition to its influence on inward rectification. The E224G mutant takes 4.5 times longer to be activated by PIP<sub>2</sub>. To probe the mechanism by which E224G slows the channel opening kinetics, we perform targeted molecular dynamics simulations and find that the mutant weakens the interactions between CD-loop and C-linker (H221-R189) and the adjacent G-loops (R312-E303) which are thought to stabilize the open state of the channel in our previous work. These data provide new insights into the regulation of Kir2.1 channel activity and suggest that a common mechanism may be involved in the distinct biophysical processes, such as inward rectification and PIP<sub>2</sub>-induced gating.
基金supported by the National Natural Science Foundation of China(32230089,32070139,32372493,32402315,and 32270208)the China Agricultural Research System(CARS-21)+1 种基金the China Postdoctoral Science Foundation(2022M721655)the Postdoctoral Innovation Talent Support Program(BX20220153).
文摘Plasma membrane intrinsic proteins(PIPs),a subclass of aquaporins,play an important role in plant immunity by acting as H2O2 transporters.Their homeostasis is mostly maintained by C-terminal serine phosphorylation.However,the kinases that phosphorylate PIPs and manipulate their turnover are largely unknown.Here,we found that Arabidopsis thaliana PIP2;7 positively regulates plant immunity by transporting H_(2)O_(2).Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE 28(CPK28)directly interacts with and phosphorylates PIP2;7 at Ser273/276 to induce its degradation.During pathogen infection,CPK28 dissociates from PIP2;7 and destabilizes,leading to PIP2;7 accumulation.As a countermeasure,oomycete pathogens produce conserved kinase effectors that stably bind to and mediate the phosphorylation of PIP2;7 to induce its degradation.Our study identifies PIP2;7 as a novel substrate of CPK28 and shows that its protein stability is negatively regulated by CPK28.Such phosphorylation could be mimicked by Phytophthora kinase effectors to promote infection.Accordingly,we developed a strategy to combat oomycete infection using a phosphorylation-resistant PIP2;7S273/276A mutant.The strategy only allows accumulation of PIP2;7^(S273/276A) during infection to limit potential side effects on normal plant growth.
