Specific Protein Properties of Setcreasea pupurea Boom under Copper Stress

[Objective] This study was to investigate the expression of the specific protein in Setcreasea purpurea Boom under copper stress, with the aim to clarify the copper tolerance mechanism of S. purpurea. [Method] Methods of water culture, elec- trophoresis and chromatography were used to analyze the molecular weight of the specific protein in the copper hyperaccumulator S. purpurea, as well as its expression time and the minimum copper concentration for the expression. And the specific protein was isolated and purified. [Result] Under copper stress, the minimum concentra- tion of copper to induce the expression of the specific protein from S. purpurea was 50 umol/L, and the expression time of the protein was in the 4th week with the molecular weight of 89.4 kDa. [Conclusion] The results show that the copper tolerance of S. purpurea is closely related with the expression of the specific protein.

Exogenous Nitric Oxide Involved in Subcellular Distribution and Chemical Forms of Cu^(2+) Under Copper Stress in Tomato Seedlings

Nitric oxide（NO）,a bioactive signaling molecule,serves as an antioxidant and anti-stress agent under abiotic stress.A hydroponics experiment was conducted to investigate the effects of sodium nitroprusside（SNP）,a NO donor,on tomato seedlings exposed to 50 μmol L-1CuCl 2.The results show that copper is primarily stored in the soluble cell sap fraction in the roots,especially after treatment with Cu＋SNP treatment,which accounted for 66.2% of the total copper content.The copper concentration gradually decreased from the roots to the leaves.In the leaves,exogenous NO induces the storage of excess copper in the cell walls.Copper stress decreases the proportion of copper integrated with pectates and proteins,but exogenous NO remarkably reverses this trend.The alleviating effect of NO is blocked by hemoglobin.Thus,exogenous NO is likely involved in the regulation of the subcellular copper concentrations and its chemical forms under copper stress.Although exogenous NO inhibited the absorption and transport of excess copper to some extent,the copper accumulation in tomato seedlings significantly increased under copper stress.The use of exogenous NO to enhance copper tolerance in some plants is a promising method for copper remediation.

Hydrogen Sulfide Promotes Wheat Seed Germination and Alleviates Oxidative Damage against Copper Stress

With the enhancement of copper （Cu） stress, the germination percentage of wheat seeds decreased gradually. Pretreatment with sodium hydrosulfide （NariS）, hydrogen sulfide （H2S） donor alleviated the inhibitory effect of Cu stress in a dose- dependent manner; whereas little visible symptom was observed in germinating seeds and radicle tips cultured in NariS solutions. It was verified that H2S or HS- rather than other sulfur-containing components derived from NariS attribute to the potential role in promoting seed germination against Cu stress. Further studies showed that NariS could promote amylase and esterase activities, reduce Cu-induced disturbance of plasma membrane integrity in the radicle tips, and sustain lower levels of malondialdehyde and H202 in germinating seeds. Furthermore, NariS pretreatment increased activities of superoxide dismutase and catalase and decreased that of lipoxygenase, but showed no significant effect on ascorbate peroxidase. Alternatively, NariS prevented uptake of Cu and promoted the accumulation of free amino acids in seeds exposed to Cu. In addition, a rapid accumulation of endogenous H2S in seeds was observed at the early stage of germination, and higher level of H2S in NaHS-pretreated seeds. These data indicated that H2S was involved in the mechanism of germinating seeds＇ responses to Cu stress.

Cu Stress-Induced Transcriptome Alterations in Sorghum and Expression Analysis of the Transcription Factor-Encoding Gene SbWRKY24

Sorghum is not only an important bio-energy crop but also a vital raw material for brewing.Exogenous copper affects the growth and metabolism of crops in specific ways.This study identified 8475 differentially expressed genes(DEGs)by high-throughput transcriptome sequencing in the sorghum cultivar‘Jinnuoliang 2’after 24 h of treatment with 10 mM CuSO4.Using GO analysis,476 genes were functionally annotated,which were mainly related to catabolism and biosynthetic processes.Additionally,90 pathways were annotated by employing the KEGG analysis.Among them,glutathione metabolism and peroxisome were induced,while photosynthesis,photosynthesis-antenna protein,and carbon sequestration of photosynthetic organisms were inhibited.Of the DEGs,399 were identified to encode transcription factors belonging to 49 families.This study also identified a WRKY transcription factor-encoding gene SbWRKY24 from the transcriptome data.For studying its function,the relative expression levels of SbWRKY24 in roots and leaves post-treatment with different growth hormones and exposure to a variety of abiotic stresses were detected by RT-qPCR.SbWRKY24 showed treatment-and tis-sue-specific expression patterns,indicating its unique role in stress tolerance.This study lays a theoretical basis for the functional exploration of SbWRKY24,elucidating the mechanism of copper resistance,and elaborating on the stress responses in sorghum.It also guides the exploration of the molecular mechanism of copper ions inducing intracellular signal transduction pathways.

Copper Ions Stimulate the Proliferation of Hepatic Stellate Cells via Oxygen Stress in vitro

This study examined the effect of copper ions on the proliferation of hepatic stellate cells （HSCs） and the role of oxidative stress in this process in order to gain insight into the mechanism of he- patic fibrosis in Wilson＇s disease. LX-2 cells, a cell line of human HSCs, were cultured in vitro and treated with different agents including copper sulfate, N-acetyl cysteine （NAC） and buthionine sulfoxi- mine （BSO） for different time. The proliferation of LX-2 cells was measured by non-radioactive cell proliferation assay. Real-time PCR and Westem blotting were used to detect the mRNA and protein ex- pression of platelet-derived growth factor receptor 13 subunit （PDGFI3R）, ELISA to determine the level of glutathione （GSH） and oxidized glutathione （GSSG）, dichlorofluorescein assay to measure the level of reactive oxygen species （ROS）, and lipid hydroperoxide assay to quantify the level of lipid peroxide （LPO）. The results showed that copper sulfate over a certain concentration range could promote the pro- liferation of LX-2 cells in a time- and dose-dependent manner. The effect was most manifest when LX-2 cells were treated with copper sulfate at a concentration of 100 ~tmol/L for 24 h. Additionally, copper sulfate could dose-dependently increase the levels of ROS and LPO, and decrease the ratio of GSH/GSSG in LX-2 cells. The copper-induced increase in mRNA and protein expression of PDGF^R was significantly inhibited in LX-2 cells pre-treated with NAC, a precursor of GSH, and this phenome- non could be reversed by the intervention of BSO, an inhibitor of NAC. It was concluded that copper ions may directly stimulate the proliferation of HSCs via oxidative stress. Anti-oxidative stress therapies may help suppress the copper-induced activation and proliferation of HSCs.

Grafting Enhances Copper Tolerance of Cucumber Through Regulating Nutrient Uptake and Antioxidative System

An experiment was carried out to determine plant growth, mineral uptake, lipid peroxidation, antioxidative enzymes, and antioxidant of cucumber plants （Cucumis sativus L. cv. Xintaimici） under copper stress, either ungrafted or grafted onto the rootstock （Cucurbitaficifolia）. Excess Cu inhibited growth, photosynthesis, and pigment synthesis of grafted and ungrafted cucumber seedlings and significantly increased accumulation of Cu in roots besides reducing mineral uptake. Cu concentration in roots of grafted cucumber plants was significantly higher than that of ungrafted plants and obviously lower in leaves. The accumulation of reactive oxygen species （ROS） significantly increased in cucumber leaves under Cu stress and resulted in lipid peroxidation, and the levels of ROS and lipid peroxidation were greatly decreased by grafting. Activities of protective enzymes （superoxide dismutase, SOD; peroxidase, POD; catalase, CAT; ascorbate peroxidase, APX; dehydroascorbate reductase, DHAR; glutathione reductase, GR） and the contents of ascorbate and glutathione in leaves of grafted plants were significantly higher than those of ungrafted plants under Cu stress. Better performance of grafted cucumber plants were attributed to the higher ability of Cu accumulation in their roots, better nutrient status, and the effective scavenging system of ROS.

Grafting Raises the Cu Tolerance of Cucumber Through Protecting Roots Against Oxidative Stress Induced by Cu Stress

A greenhouse experiment was carried out to determine plant growth, reactive oxygen species （ROS） metabolism in roots and functions of plasma membrane （PM） and tonoplast in cucumber seedlings （Cucumis sativus L. ev. Xintaimici） treated with 40 μmol L^-1 CuSO4·5H2O, which were either ungrafted or grafted onto the rootstock （Cucurbitaficifolia）. Cu treatment inhibited growth, induced significant accumulation of H2O2 and led to serious lipid peroxidation in cucumber roots, and the ROS-scavenging enzymes activities in grafted seedlings roots were significantly higher than that of ungrafted plants, thus less accumulation in grafted cucumber roots induced by Cu. As a result, lipid peroxidation in roots decreased. Furthermore, the activities of H^-ATPase, H＋-PPase and Ca^2＋-ATPase in PM and/or tonoplast in grafted cucumber seedlings under Cu stress were obviously higher than that in ungrafted plants, resulting into higher ability in grafted plants to expulse the excess H＋, promote the cytoplasm alkalinization, regulate the intracellular Ca^2＋ concentration and brought the cytoplasma concentration of free Ca2＋ to extremely low level under Cu stress.

Grafting Increases the Copper Tolerance of Cucumber Seedlings by Improvement of Polyamine Contents and Enhancement of Antioxidant Enzymes Activity

The aim of the study is to determine whether grafting could improve antioxidant enzyme activities and polyamine contents in leaves of cucumber plants（Cucumis sativus L.cv.Xintaimici） under copper stress.Grafted（using Cucurbita ficifolia as rootstock） and ungrafted cucumber seedlings were cultured in deep flow technique（DFT） with the Cu2＋ concentration of 40 μmol L-1.The results showed that on the 9th day of copper stress treatment,the contents of malondialdehyde（MDA） and hydrogen peroxide（H2O2）,superoxide radical（） producing rate,and electrolyte leakage percentage were significantly lower in grafted seedlings in comparison to those of the ungrafted seedlings,whereas the activities of antioxidants such as superoxide dismutase（SOD,EC 1.15.1.1）,peroxidase（POD,EC 1.11.1.7）,ascorbate peroxidase（APX,EC 1.11.1.11）,catalase（CAT,EC 1.11.1.6）,glutathione reductase（GR,EC 1.6.4.2）,and monodehydroascorbate reductase（MDHAR,EC 1.6.5.4） of grafted seedlings were also significantly higher than that of ungrafted seedlings,and the contents of free proline and soluble protein of grafted seedlings were significantly higher than that of ungrafted seedlings.Cu2＋ treatment increased the putrescine（Put） level and lowered the spermidine（Spd） and spermine（Spm） levels,thereby reducing the Put/（Spd ＋ Spm） ratio in leaves of grafted and ungrafted seedlings.Grafting markedly reversed these Cu-induced effects for all three PAs and partially restored the Put/（Spd ＋ Spm） ratio in leaves.These results suggest that grafting can enhance the tolerance of cucumber seedlings to Cu2＋ stress by increasing the activities of antioxidants and the levels of endogenous Spd and Spm,decreasing the Put/（Spd ＋ Spm） ratio and the levels of ROS,promoting free proline and soluble protein synthesis in cucumber seedling leaves.

The maize late embryogenesis abundant protein Zm DHN13 positively regulates copper tolerance in transgenic yeast and tobacco

Late embryogenesis abundant (LEA) proteins accumulate in the late stage of plant seed development, and are upregulated in most plants during drought, cold, heat, or salinity stress. LEA proteins can be classified by amino-acid sequence into seven groups. Dehydrins belong to LEA protein group Ⅱ. In previous studies, the maize KS type dehydrin ZmDHN13 increased the tolerance of transgenic tobacco to oxidative stress. In the present study, ZmDHN13 was identified under copper stress conditions, and the protein was then characterized using transgenic yeast and tobacco plants to investigate its functions. ZmDHN13 bound Cu2+. Its overexpression in transgenic tobacco conferred tolerance to copper stress by binding metals and reducing the accumulation of reactive oxygen species (ROS). Three conserved domains displayed a cooperative effect under copper stress conditions.

Gene Expression and Activities of SOD in Cucumber Seedlings Were Related with Concentrations of Mn^(2+),Cu^(2+),or Zn^(2+) Under Low Temperature Stress

Effects of increasing Mn^2＋, Cu^2＋, or Zn^2＋ on SOD expressions were studied in cucumber seedlings under low temperature stress. Both gene expressions and activities of Cu/Zn-SOD and Mn-SOD in cucumber seedling leaves were induced by increasing Mn^2＋, Cu^2＋, or Zn^2＋ under low temperature stress, especially 48 h afterwards. The activities of Cu/Zn-SOD and Mn-SOD at 0 and 48 h after treatment were in accordance with their gene expression levels, which implied that the transcriptional regulation plays key roles in regulating their activities at the early stage of low temperature stress. Gene expressions of Cu/Zn-SOD and Mn-SOD declined at 96 h, but Cu/Zn-SOD and Mn-SOD activities still remain high, which suggested that Cu/Zn-SOD and Mn-SOD activities might be regulated by other factors after transcription at the later stage of low temperature stress. Therefore, we concluded that the increasing Mn^2＋, Cu^2＋, or Zn^2＋ could increase the capacity of scavenging ROS in cucumber seedlings under low temperature stress by inducing gene expressions of Cu/ Zn-SOD and Mn-SOD, elevating activities of Cu/Zn-SOD, Mn-SOD, or regulating other factors after transcription.

Deformation behavior of dispersion-strengthened copper at high temperature

The deformation behavior of dispersion-strengthened copper with different compositions was investigated by hot compression simulation tests on a Gleeble-1500 thermal-mechanical simulator. The microstructure during deformation at high temperature was also studied. The result shows that at the beginning of hot compression simulation, the flowing stress of the dispersion-strengthened copper quickly attains a peak value and the stress shows a greater decrease when the temperature is higher and the strain rate is lower. The dispersion particles lead to an obvious increase in the recrystallization temperature. Under experimental conditions, dynamic recovery is the main softening method. The constitutive equation at high temperature of 1.2%Al2O3-0.4%WC/Cu is obtained.

IMPROVING THE MECHANICAL PROPERTIES OF COPPER ALLOYS BY THERMO-MECHANICAL PROCESSING

Systematic physical simulation of thermo-mechanical processing routes has been ap-plied on a Gleeble 1500 simulator to four copper alloys (mass %) Cu-0.57Co-0.32Si,Cu-0.55Cr-0.065P, Cu-0.22Zr-0.035Si and Cu-1.01Ni-0.43Si aimed at clarifying theinfluences of processing conditions on their final properties, strength and electricalconductivity. Flow curves were determined over wide temperature and strain rateranges. Hardness was used as a measure of the strength level achieved. High hard-ness was obtained as using equal amounts (strains 0.5) of cold deformation beforeand after the precipitation annealing stage. The maximum values achieved for theCu-Co-Si, Cu-Cr-P, Cu-Zr-Si and Cu-Ni-Si alloys were 190, 165, 178 and 193 HV5,respectively. A thermo-mechanical schedule involving the hot deformation-ageing-colddeformation stages showed even better results for the Cu-Zr-Si alloy. Consequently,the processing routes were designed based on simulation test results and wires of 5 and2mm in diameters have been successfully processed in the industrial scale.

Flow Stress Behavior of Cu13Zn Alloy Deformed at Elevated Temperature

The flow stress behavior of Cu13Zn alloy was investigated by compression tests carried out at 650 ℃, 700 ℃, 750 ℃, 850 ℃, and constant strain rates of 0.05 s -1 , 0.1 s -1 , 0.5 s -1 , 1 s -1 , 5 s -1 , respectively. The results show that the flow stress increases with the increase of strain and reaches a steady state stress, and the saturated stress ( σ s) increases with the increase of the strain rate and the decrease of temperature. Flow stress curves of the alloy deformed at elevated temperatures can be simulated effectively by the model proposed by Zhou and Clode, and the flow stress is described as a function of strain, strain rate and temperature. Material constants values are: Q =270.43 kJ/mol, α =0.020 94, A =1.747×10 11 s -1 and n = 3.549 mm 2·N -1 , the deformation mechanisms of the alloy are self diffusion and dynamic recovery.

樟树对铜胁迫的生长和生理响应及铜富集转运特性分析

以前期在铜污染矿区修复中筛选出的耐铜樟树(Cinnamomum camphora(L.)Presl)为材料,通过盆栽模拟实验,以不添加铜为对照(CK),设置50、150、300、600、900、1200 mg/kg 6个处理浓度,胁迫处理60 d后,测定樟树的生物量积累、叶绿素含量、生理生化指标及樟树对铜离子的富集和转移量,分析不同浓度铜处理对耐铜樟树生长、生理生化的影响,以及铜在不同组织的富集转运规律。结果显示,樟树对铜胁迫表现出低浓度(150 mg/kg)促进生长,高浓度抑制生长的趋势。超氧化物歧化酶(Superoxide dismutase,SOD)、过氧化物酶(Peroxidase,POD)、过氧化氢酶(Catalase,CAT)、过氧化氢(Hydrogen peroxide,H_(2)O_(2))、游离脯氨酸和可溶性糖含量等生理指标在900 mg/kg处理时没有显著升高;谷胱甘肽还原酶(Glutathione reductase,GR)随着处理浓度的升高逐渐降低;丙二醛(Malondialdehyde,MDA)、谷胱甘肽(Glutathione,GSH)含量则随着处理浓度的升高逐渐升高。各器官铜含量的大小顺序为根>叶>茎,樟树主要将铜富集在根系,往地上茎和叶的转移率很低,极大地降低了对茎、叶的铜毒害作用,且随着处理浓度的增加,铜由根系向叶片的转移率IF(Leaf/Root)由对照处理的0.078逐降至1200 mg/kg处理时的0.007;铜由根系向茎的转移率IF(Stem/Root)由对照处理的0.06逐降至1200 mg/kg处理时的0.005。综合其生长、生理及铜的富集转运特性,樟树对铜具有较好的耐受性,耐受浓度可达900 mg/kg,是可用于铜污染土壤种植的理想材料。

Effect of strain rate on the compressive deformation behaviors of lotus-type porous copper

Lotus-type porous copper was fabricated by unidirectional solidification, and compressive experiments were subsequently conducted in the strain rate range of 10-3-2400 s-1 with the compressive direction parallel to the pores. A GLEEBLE-1500 thermal-mechanical simulation system and a split Hopkinson pressure bar （SHPB） were used to investigate the effect of strain rate on the compressive deforma-tion behaviors of lotus-type porous copper. The influence mechanism of strain rate was also analyzed by the strain-controlling method and by high-speed photography. The results indicated that the stress-strain curves of lotus-typed porous copper consist of a linear elastic stage, a plateau stage, and a densification stage at various strain rates. At low strain rate （〈1.0 s^-1）, the strain rate had little influence on the stress-strain curves; but when the strain rate exceeded 1.0 s^-1, it was observed to strongly affect the plateau stage, showing obvious strain-rate-hardening characteristics. Strain rate also influenced the densification initial strain. The densification initial strain at high strain rate was less than that at low strain rate. No visible inhomogeneous deformation caused by shockwaves was observed in lotus-type porous copper during high-strain-rate deformation. However, at high strain rate, the bending deformation characteristics of the pore walls obviously differed from those at low strain rate, which was the main mechanism by which the plateau stress exhibited strain-rate sensitivity when the strain rate exceeded a certain value and exhibited less densification initial strain at high strain rate.

肝豆补肾汤通过抑制铁死亡和内质网应激改善肝豆状核变性模型TX小鼠卵巢组织损伤

目的观察肝豆补肾汤对肝豆状核变性(hepatolenticular degeneration,HLD)小鼠卵巢损伤的保护作用,并探究其分子机制。方法以TX小鼠作为HLD模型,将其分为HLD组、青霉胺组和肝豆补肾汤组,另设DL同系小鼠作为正常对照组。测量小鼠体质量、卵巢质量、卵巢系数。采用促性腺激素促排卵法观察小鼠的排卵情况。采用苏木精—伊红(hematoxylin-eosin,HE)染色法观察小鼠卵巢的组织形态,透射电子显微镜下观察卵巢组织的超微结构。采用比色法测定血清铁含量,TBA法检测卵巢组织中丙二醛(malondialdehyde,MDA)含量,微量酶标法检测卵巢组织中还原型谷胱甘肽(glutathione,GSH)及氧化型谷胱甘肽(oxidized glutathione,GSSG)水平。采用Western blot法检测小鼠卵巢组织铁死亡相关标志物前列腺素内过氧化物合酶2(prostaglandin-endoperoxide synthase 2,PTGS2)和谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)水平,及内质网应激通路相关蛋白[葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)、蛋白激酶核糖核酸样内质网激酶(protein kinase RNA-like endoplasmic reticulum kinase,PERK)、磷酸化PERK(phosphorylated PERK,p-PERK)、真核起始因子2α(eukaryotic initiation factor 2 alpha-subunit,eIF2α)、磷酸化eIF2α(phosphorylated eIF2α,p-eIF2α)、活化转录因子4(activating transcription factor 4,ATF4)和C/EBP同源蛋白(C/EBP homologous protein,CHOP)]的表达水平。结果HE染色显示HLD组小鼠卵细胞形态结构受损严重,闭锁卵泡显著增加;透射电子显微镜下HLD组小鼠线粒体皱缩明显,出现内质网肿胀和脱颗粒等内质网应激表现。与正常对照组比较,HLD组小鼠卵巢质量、排卵数均显著降低(P<0.05),血清铁及卵巢组织中MDA、GSSG水平显著升高(P<0.05),卵巢组织中GSH水平、GSH/GSSG显著降低(P<0.05),卵巢组织中PTGS2、GRP78、p-PERK、p-eIF2α、ATF4、CHOP表达水平均显著升高(P<0.05),GPX4表达水平显著降低(P<0.05)。与HLD组比较,肝豆补肾汤组小鼠的卵泡形态、线粒体和内质网结构均显著改善,促排卵后排卵数显著升高(P<0.05),血清铁及卵巢组织中MDA和GSSG水平显著降低(P<0.05),卵巢组织中GSH水平和GSH/GSSG显著升高(P<0.05),卵巢组织中PTGS2、GRP78、p-PERK、p-eIF2α、CHOP表达水平显著降低(P<0.05),卵巢组织中GPX4表达水平显著升高(P<0.05)。结论肝豆补肾汤可减轻TX小鼠铜沉积诱导的卵巢损伤,其机制可能与抑制铁死亡和PERK通路介导的内质网应激有关。

红树植物功能性状对盐和铜胁迫的响应

目前,室内通过人工控制方式开展红树林对环境胁迫的响应研究,忽视了不同红树植物物种的生长习性,且以单一胁迫为主。通过野外生长与人工控制相结合方式,研究5个时期(2018.6—2020.8)盐和铜胁迫对3种红树植物(秋茄、拉关木和木榄)11种功能性状的影响,使用单因素和双因素方差分析研究不同时期不同树种下盐和铜胁迫对叶片功能性状的影响,回答红树植物功能性状对盐和铜单一及复合胁迫响应的科学问题。结果表明,不同时期下,三种红树植物功能性状具有明显差异。随着时间增长,三种植物的碳含量和冠层高度均呈升高趋势,其他功能性状因物种不同而表现出不同趋势;与乡土红树植物(秋茄和木榄)相比,外来入侵物种(拉关木)具有更快的生长速度。不同时期下,拉关木比秋茄和木榄具有更好耐盐性,三个物种的植物生长发育对铜胁迫的响应均不敏感。大部分时期下,单一盐胁迫对拉关木的植物功能性状无显著影响(P<0.05),对秋茄和木榄的碳含量、冠层高度和比叶面积有显著影响(P<0.05);单一铜胁迫对三个物种的绝大多数功能性状无显著影响。另外,盐-铜复合胁迫对秋茄叶片碳、磷、氮磷比及冠层高度有显著影响(P<0.05),对拉关木叶片碳、铜含量、冠层高度、比叶面积有显著影响(P<0.05),仅对木榄SPAD值有显著影响(P<0.05)。在红树林保护修复工作中,研究成果可为红树林退化原因和机理阐释、红树林生长监测评估、生境治理以及树种选择提供一定的理论依据。

基于转录组的海州香薷COPT铜转运蛋白家族鉴定及响应铜胁迫表达分析

为了解海州香薷(Elsholtzia splendens)COPT(copper transporter)铜转运蛋白对铜胁迫的响应规律,运用生物信息学方法对海州香薷转录组数据库中的COPT家族成员进行鉴定与分析,构建植物表达载体探究蛋白的亚细胞定位.结果显示:海州香薷转录组中有11个EsCOPT成员,它们编码的蛋白质由72~159个氨基酸组成,等电点介于4.72~9.92,平均相对分子质量为12.870×10^(3).11个成员均为膜蛋白,系统进化树分析显示EsCOPT蛋白分别属于3个亚组.正常条件下,EsCOPT2和EsCOPT9表达量较高,铜处理后,除EsCOPT5的表达无明显变化外,其他EsCOPTs的表达均呈下降趋势.全长EsCOPT9编码的蛋白具有3个跨膜结构域且N末端较短,共定位实验表明EsCOPT9-GFP定位于液泡膜上.

埃洛石纳米管对铜胁迫下小麦幼苗生长的影响

在水培条件下设置3个铜(Cu)处理[20 mg/L Cu^(2+)(20Cu)、50 mg/L Cu^(2+)(50Cu)和100 mg/L Cu^(2+)(100Cu)],研究外源添加50 mg/L埃洛石纳米管(HNTs)对Cu胁迫下小麦幼苗生长、气孔大小、叶绿体色素含量、根系氧化损伤、植株内Cu^(2+)含量的影响,以期为HNTs缓解重金属污染对作物生长的影响提供理论依据。结果表明,外源添加HNTs显著提高了Cu胁迫下小麦幼苗的株高、根长,20Cu+HNTs、50Cu+HNTs、100Cu+HNTs处理株高分别提高了13.10%、26.45%、28.30%,根长分别提高了62.87%、56.68%、92.34%;外源添加HNTs显著提高了Cu胁迫下小麦幼苗干、鲜质量,叶绿素、类胡萝卜素含量,根系活力,降低了根系氧化损伤程度,其中,叶绿素a含量分别提高了34.34%、18.09%、54.76%,叶绿素b含量分别提高了44.83%、25.93%、43.75%,根系活力分别提高了38.16%、339.48%、453.56%;与100Cu处理相比,100Cu+HNTs处理小麦幼苗根系、叶片中Cu^(2+)含量分别降低了19.18%、37.02%。综上,外源添加HNTs能够降低小麦幼苗中Cu^(2+)含量,缓解重金属Cu对小麦幼苗的毒性,促进其生长。

滇水金凤耐铜相关基因CAX3和CCS的克隆及表达分析

植物CAX(Ca2+/H+反向转运蛋白)和CCS(铜锌超氧化物歧化酶铜伴侣蛋白)分别是重要的液泡膜转运蛋白和铜伴侣蛋白,通过不同机制在缓解重金属胁迫中发挥着重要作用。本课题组前期研究发现滇水金凤(Impatiens uliginosa)对铜胁迫具有很好的耐受性,并通过转录组测序筛选出耐铜相关基因。在此基础上,本研究以野生滇水金凤为材料,通过RT-PCR技术克隆获得耐铜相关基因CAX3和CCS,分别命名为IuCAX3和IuCCS,其cDNA序列长度分别为1320 bp和975 bp,分别编码439个和324个氨基酸。生物信息学分析结果表明,IuCAX3为不稳定蛋白,具有caca2超家族的保守结构域,定位在质膜、液泡上;IuCCS为稳定蛋白,具有PLN02957超家族的保守结构域,定位在叶绿体上。系统进化分析结果显示,IuCAX3和IuCCS均与喜马拉雅凤仙花的相应基因聚为一支,相似性分别为98.18%和89.81%。通过设置不同的铜处理浓度(0、5、10、15、20、25 mg·L^(-1))研究了IuCAX3和IuCCS基因在滇水金凤叶片中的表达情况,qRT-PCR分析结果表明,铜处理能诱导两个基因大幅上调表达,且铜浓度为15 mg·L^(-1)时的上调幅度最大,并均在处理12 d达到最高水平;随处理时间延长,IuCAX3的表达量先升高后降低,IuCCS的表达量在铜浓度不高于15 mg·L^(-1)时呈先升后降再升的趋势,但当铜浓度高于15 mg·L^(-1)后则呈先升后降的趋势,而且铜处理下IuCCS的表达水平明显高于IuCAX3,因此推测IuCAX3基因主要在铜胁迫前期起作用,而IuCCS基因在铜胁迫后期仍发挥重要作用。本研究结果可为深入研究滇水金凤耐铜分子机制提供一定的参考依据。