基于TMT质谱分析技术筛选水稻根尖响应汞胁迫差异表达蛋白

稻米是人群汞暴露的最主要途径之一,为了从蛋白质水平上揭示水稻(Oryza sativa L.)适应重金属汞胁迫的分子机制,本研究以拔节期金优431水稻品种为材料,对比分析对照组和50、150、300 mg·kg~(-1)氯化汞胁迫组,采用同位素相对标记与绝对定量TMT(Tandem Mass Tag)技术,结合定量蛋白质组平行反应监测(parallel reaction monitoring,PRM)技术,鉴定水稻根尖部位响应汞胁迫差异表达蛋白,对所获得差异蛋白进行生物信息学分析,从而筛选出汞胁迫响应显著的潜在靶标蛋白。同时,选择20个差异表达蛋白通过平行反应监测试验进行了蛋白验证。结果表明,在变化倍数≥1.3、P0.05条件下,共鉴定5253种定量蛋白,包含364种差异蛋白,其中258种表达上调,106种表达下调。基因本体分子功能提示,这些差异性蛋白主要参与催化活性、绑定转运活性和抗氧化活性。京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路显著性富集于代谢途径、次生代谢产物生物合成、苯丙烷类生物合成等,错误发现率(false discovery rate,FDR)0.05。成功鉴定并验证到的差异蛋白分别涉及抗氧化还原蛋白、金属螯合肽合成蛋白、金属硫蛋白相关蛋白等。差异表达蛋白中,与抗氧化、重金属胁迫防御响应和信号通路等相关的蛋白总体上调,而与代谢和能量产生与转运相关蛋白表达量总体下调。     

磷高效水稻根系对低磷胁迫响应的差异蛋白分析

以IR71331为材料,通过水培试验,采用双向电泳分离不同磷浓度下（低磷浓度为0.5 mg·L^-1,对照为10 mg·L^-1）水稻生长3 d和6 d根系差异蛋白.结果表明：与对照相比,低磷胁迫下共有29个蛋白,其中3 d时间点有17个蛋白上调、11个下调、1个新增,6 d时间点有8个上调、19个下调、1个抑制表达、1个无明显变化 .经鉴定,其中的10个差异表达蛋白可归为信号转导相关蛋白、基因表达相关蛋白、代谢相关蛋白离子转运相关蛋白4个功能类群.信号转导相关蛋白分别为富含甘氨酸RNA结合蛋白和类似参与磷酸盐饥饿反应调控子;基因表达相关蛋白分别为推定的mRNA前体剪接因子SF2和推定的AAA蛋白酶家族FtsH;代谢相关蛋白分别为腺苷酸琥珀酸裂解酶、丝氨酸蛋白酶抑制剂(serpin)、S-腺苷蛋氨酸合成酶（SAM）和类似MYB类转录因子;离子转运相关蛋白分别为阳离子转运ATP酶和肌浆网膜蛋白.这些蛋白分别参与了信号识别、信号调控、mRNA 的剪接、信号传递、蛋白质降解、细胞体内离子转运和平衡等生理过程.其中serpin、SAM和MYB类转录因子是水稻响应低磷胁迫的关键蛋白.水稻根系对低磷胁迫存在着一个复杂的抗逆信号应答和代谢调控网络,其作用机理可以通过差异表达的蛋白质得以体现.     

水稻叶片对镉胁迫响应的蛋白质差异表达

为揭示水稻镉抗性的分子机理,以抗镉水稻品种P1312777和镉敏感水稻品种IR24为材料,在镉离子浓度为0(对照)、50和100 μmol·L-1条件下水培处理7 d,应用蛋白质组学方法分析了2种水稻叶片对镉胁迫响应的蛋白质差异表达.结果表明:镉胁迫下水稻PI312777叶片中共检测到差异表达蛋白质点31个,通过MALDI-TOF/MS分析,鉴定了其中的24个蛋白质(包括20个不同蛋白质,4个重复检出蛋白质);IR24叶片中共检测到差异表达蛋白质点19个,其中15个蛋白质得到鉴定.PI312777叶片鉴定出的20个蛋白质覆盖了IR24叶片鉴定的15个蛋白质,前者有4个与光合作用相关,11个与细胞防御代谢相关,3个与其他代谢相关,2个为功能未知蛋白.与对照相比,不同浓度镉胁迫下,抗镉水稻PI312777叶片中热激蛋白、谷胱甘肽还原酶、蛋白酶体α亚基6型、果糖1,6-二磷酸醛缩酶、硫氧还蛋白和DNA重组修复蛋白均上调表达;镉敏感水稻IR24叶片中热激蛋白、谷胱甘肽还原酶、蛋白酶体α亚基6型的表达无显著差异,果糖1,6-二磷酸醛缩酶和硫氧还蛋白则下调表达.此外,DNA重组修复蛋白仅在镉胁迫的PI312777叶片中表达.水稻PI312777比IR24具有更强的镉抗性与这些差异表达的蛋白质密切相关.     

嗜碱Bacillus sp. N16-5不同碳源条件下比较蛋白质组分析

为了解碳水化合物对嗜碱微生物代谢途径的影响, 用蛋白质组学方法比较分析了不同碳源条件下培养的嗜碱菌的胞浆蛋白质变化, 试图找到差异表达的蛋白. 分离自内蒙古乌杜淖尔碱湖的嗜碱Bacillus sp. N16-5, 在含有5种不同碳源(葡萄糖、甘露糖、半乳糖、阿拉伯糖和木糖)的培养基中培养. 比较蛋白质组学分析鉴定了61个差异表达蛋白, 它们主要参与碳水化合物代谢、氨基酸转运和代谢、能量的产生和贮存. 结果表明, 不同碳水化合物条件下参与中央代谢途径酶的丰度发生了很大的变化, 尤其是碳代谢调控蛋白A(CcpA)均被上调. 同时发现, 在CcpA参与调控的碳代谢抑制现象中戊糖表现出比己糖更强的效应. 上述结果为进一步理解嗜碱微生物碳水化合物代谢奠定了基础.     

水稻穗发育功能基因对穗期氮肥的响应

为探讨氮肥对水稻(Oryza sativa)穗发育的调控作用, 使用高通量测序技术检测氮肥处理前后水稻叶片和幼穗组织中转录组的变化, 并从中筛选到大量差异表达基因。这些基因的功能涉及转录调控、激素代谢和信号转导、物质代谢和转运、胁迫响应、信号转导(受体)和蛋白质降解等。同时对目前克隆得到的穗发育相关基因进行分析, 发现在氮素穗肥的作用下, 部分重要功能基因的表达量发生了明显变化, 其中一些基因还参与调控水稻株高、抽穗期、分蘖和结实率等性状。对这些差异表达基因的功能研究有助于揭示氮素穗肥调控水稻每穗颖花数的分子机制。     

长春花生物碱合成途径相关基因表达丰度与MeJA处理后基因表达差异分析

长春花(Catharanthus roseus)中含有丰富的次生代谢产物,可产生130多种萜类吲哚生物碱,包括目前临床应用最广的天然植物抗癌药物长春碱(vinblastine)和长春新碱(vincristine)。长春花萜类吲哚生物碱(terpenoid indole alkaloids,TIAs)合成途径包括在内韧皮部相关薄壁细胞、上皮细胞、异细胞和乳管细胞等多种组织细胞内进行的20多步酶促反应。本研究选取TIAs途径上的CPR、LAMT、SLS、STR、SGD、TDC、16-OMT、T16H、D4H等9个酶基因,基于长春花转录组数据库,分析其在各组织器官和MeJA处理下的无菌苗、毛状根和悬浮细胞中的数字表达谱,找出其中可能对MeJA信号响应的基因。本研究发现,MeJA处理后,长春花无菌苗中以上9个基因表达量均发生上调;毛状根中除了TDC,另外8个基因表达丰度均为上调;悬浮细胞中除了D4H,另外8个基因表达丰度均上调。对整个转录组数据进行分析,发现经过MeJA处理后,仅有673条Unigene能在无菌苗、毛状根和悬浮细胞中均表现出差异表达,说明无菌苗、毛状根和悬浮细胞的MeJA应答机制存在较大不同。对这些能在3种体系中均发生差异表达的基因进行功能分类,主要包括初生代谢相关蛋白、次生代谢相关蛋白、植物激素相关蛋白、矿质离子结合蛋白、转运蛋白、信号转导相关蛋白、细胞壁合成与降解相关蛋白、转录因子、植物逆境响应蛋白9个方面。本研究可为通过外源因子调控长春花生物碱的生物合成提供参考。     

燕麦盐胁迫响应基因的差异表达与生理响应的关系

以耐盐燕麦品种VAO-9为材料,通过Illumina测序与数字基因表达谱技术对300mmol/L NaCl处理前后的叶片cDNA文库进行RNA-Seq与DGE分析,同时测定0(CK)、100、200、250、300mmol/L NaCl胁迫下VAO-9幼苗叶片的相对电导率、丙二醛含量和脯氨酸含量,探讨燕麦盐胁迫响应基因的差异表达与生理响应的关系。结果表明:(1)RNA-Seq分析得到Unigenes 65 801条,其基因表达呈现高度的不均一性和冗余性;若差异基因表达谱鉴定分析以log2Ratio≥2且FDR值≤0.001为选择标准,则发现上调和下调表达基因在胁迫0.5h时分别有306和64个,在胁迫3h时分别有639和290个,胁迫24h时分别有1 488和882个。(2)KEGG代谢分析显示,有23 652条Unigenes比对到KEGG中的128条代谢途径,包括与逆境胁迫相关的植物激素信号转导途径、ABC转运蛋白途径、肌醇磷酸代谢途径、渗透调节途径等。(3)在300mmol/L NaCl处理下燕麦叶片的相对电导率、丙二醛含量、脯氨酸含量等生理指标的变化与相关差异表达基因的变化趋势基本一致,说明基因差异表达量与生理反应密切相关。研究认为,在相同的栽培及胁迫处理条件下,可根据植物盐响应生理指标的变化判断耐盐基因的表达情况。     

基于iTRAQ技术筛选水稻根尖甲基汞胁迫响应差异蛋白及其生物信息学分析

为了解水稻(Oryza sativa L.)对甲基汞胁迫响应的分子机制,采用两优302为实验材料,利用同位素相对标记与绝对定量技术(i TRAQ),联合液相色谱-串联质谱技术(LCMS/MS),筛选水稻甲基汞胁迫下的根尖蛋白组差异表达蛋白,并结合生物信息学对差异蛋白进行分析。结果表明,对照组与处理组共定量了3508个蛋白质,在差异倍数(fold change)≥1.20或≤0.83,且P<0.05条件下,筛选出88个差异蛋白,其中32个蛋白表达上调,56个蛋白表达下调,其中15个差异蛋白(包括类萌发素蛋白和脂氧合酶等12个已知蛋白和3个未知蛋白)具有与金属离子结合相关属性。基因本体(gene ontology,GO)分子功能提示,差异性蛋白主要涉及催化活性、结合和转运活性等生物学过程,京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路分析显示,差异蛋白显著富集于内质网蛋白加工、氧化磷酸化、淀粉与蔗糖代谢和苯丙素生物合成等代谢通路。研究结果为今后调控水稻中MeHg的吸收提供依据。     

甜荞柠檬酸转运蛋白基因FeFRD3的克隆及表达分析

该研究采用同源克隆策略,从甜荞中克隆到1个柠檬酸转运蛋白基因FeFRD3(GenBank登录号为MG462907)。FeFRD3基因含一个1 554bp开放阅读框,编码517个氨基酸,预测蛋白分子量为55.83kD,等电点为8.48。生物信息学分析显示,FeFRD3蛋白含有8个跨膜区,定位于质膜和液泡膜上。蛋白序列分析结果表明,FeFRD3与拟南芥、大豆和水稻的FRD3同源蛋白有较高的序列一致性。系统进化树分析表明,FeFRD3属于具有将铁由根向地上部位长距离转运功能的柠檬酸转运蛋白,且与拟南芥AtFRD3亲缘关系最近。qRT-PCR分析结果表明,FeFRD3基因在甜荞根、茎、叶和种子中均有表达,但在根中的表达量最高,在种子中的表达量最低;缺铁胁迫没有影响FeFRD3基因在根中的表达,但高铁胁迫明显诱导了该基因在根中的表达。研究结果为进一步深入研究FeFRD3基因在甜荞铁长距离转运中的功能奠定了基础。     

水胁迫下沙漠小球藻蛋白质组学分析

通过研究小球藻(Chlorella sp.)在干旱胁迫下的蛋白质组变化,从蛋白质表达水平解释小球藻对干旱胁迫的响应机理。以20%的PEG 6000胁迫处理0、6、12、18、24和30 d,提取小球藻总蛋白,利用双向电泳和质谱鉴定技术分析差异蛋白。共鉴定29个差异蛋白,按功能可分为7类:光合作用、能量合成和转化、物质代谢、抗氧化、转运和细胞结构、抗逆和功能未知蛋白质。这些蛋白的表达变化影响着沙漠小球藻的油脂积累,对干旱胁迫应答起到了至关重要的作用,并直接或间接地参与了沙漠小球藻细胞内的光合作用和油脂合成。     

Genetic, proteomic and metabolic analysis of the regulation of energy storage in rice seedlings in response to drought

We used proteomic analysis to determine the response of rice plant seedlings to drought-induced stress. The expression of 71 protein spots was significantly altered, and 60 spots were successfully identified. The greatest down-regulated protein functional category was translation. Up-regulated proteins were mainly related to protein folding and assembly. Additionally, many proteins involved in metabolism (e.g. carbohydrate metabolism) also showed differences in expression. cDNA microarray and GC-MS analysis showed 4756 differentially expressed mRNAs and 37 differentially expressed metabolites. Once these data were integrated with the proteomic analysis, we were able to elucidate the metabolic pathways affected by drought-induced stress. These results suggest that increased energy consumption from storage substances occurred during drought. In addition, increased expression of the enzymes involved in anabolic pathways corresponded with an increase in the content of six amino acids. We speculated that energy conversion from carbohydrates and/or fatty acids to amino acids was increased. Analysis of basic metabolism networks allowed us to understand how rice plants adjust to drought conditions.     

OsNRAMP5, a major player for constitutive iron and manganese uptake in rice

Manganese (Mn) and iron (Fe) are essential mineral micronutrients for plants and their deficiency and or toxicity represents a serious agricultural problem. In rice the information about genes involved in Mn uptake from soil is scarce. Recently, we showed that OsNRAMP5 is a plasma membrane protein involved in Mn and Fe transport. The concentration of Mn in roots, shoots and xylem sap of OsNRAMP5 RNAi (OsNRAMP5i) plants was significantly reduced compared with WT plants. The expression of OsNRAMP5 is not controlled by Fe deficiency in root and was also observed in pistil, ovary, lemma and palea. These data show that rice would utilize OsNRAMP5 for constitutive Fe and Mn uptake, while OsNRAMP5 would also play a role in Fe and Mn transport during flowering and seed development.     

Proteome analysis of cultivar-specific deregulations of Oryza sativa indica and O. sativa japonica cellular suspensions undergoing rice yellow mottle virus infection

We have used two-dimensional gel electrophoresis with mass spectrometry analysis to study the temporal patterns of protein expression during RYMV (Rice yellow mottle virus) infection in rice cells of two cultivars: IR64, Oryza sativa indica, susceptible, and Azucena, O. sativa japonica, partially resistant to RYMV. Proteomic analysis of nonstressed and RYMV inoculated cells showed statistically significant changes in the relative levels of 40 IR64 proteins and 24 Azucena proteins. Protein identification using mass spectrometry was attempted for all the differentially regulated proteins. This global analysis detected 32 hypothetical "new" proteins. Nineteen differentially regulated proteins were identified for IR64 cultivar, while 13 were identified for Azucena cultivar, including proteins in three functional categories: metabolism, stress-related proteins, and translation. These data revealed that a number of proteins regulated by abiotic stress response pathway were activated by RYMV in both cultivars (such as salt-induced protein, heat shock proteins (HSPs), superoxide dismutase (SOD), and others have functions consistent with the susceptibility or partially resistance trait (such as dehydrin, proteins involved in glycolysis pathway).     

Effect of iron plaque outside roots on nutrient uptake by rice (Oryza sativa L.). Zinc uptake by Fe-deficient rice

This solution culture study examined the effect of the deposition of iron plaque on zinc uptake by Fe-deficient rice plants. Different amounts of iron plaque were induced by adding Fe(OH)₃ at 0, 10, 20, 30, and 50 mg Fe/L in the nutrient solution. After 24 h of growth, the amount of iron plaque was correlated positively with the Fe(OH)₃ addition to the nutrient solution. Increasing iron plaque up to 12.1 g/kg root dry weight increased zinc concentration in shoots by 42% compared to that at 0.16 g/kg root dry weight. Increasing the amount of iron plaque further decreased zinc concentration. When the amounts of iron plaque reached 24.9 g/kg root dry weight, zinc concentration in shoots was lower than that in shoots without iron plaque, implying that the plaque became a barrier for zinc uptake. While rice plants were pre-cultured in –Fe and +Fe nutrient solution in order to produce the Fe-deficient and Fe-sufficient plants and then Fe(OH)₃ was added at 20, 30, and 50 mg Fe/L in nutrient solution, zinc concentrations in shoots of Fe-deficient plants were 54, 48, and 43 mg/kg, respectively, in contrast to 32, 35, and 40 mg/kg zinc in shoots of Fe-sufficient rice plants. Furthermore, Fe(OH)₃ addition at 20 mg Fe/L and increasing zinc concentration from 0.065 to 0.65 mg Zn/L in nutrient solution increased zinc uptake more in Fe-deficient plants than in Fe-sufficient plant. The results suggested that root exudates of Fe-deficient plants, especially phytosiderophores, could enhance zinc uptake by rice plants with iron plaque up to a particular amount of Fe.     

水稻依赖抗坏血酸H2O2清除系统在抗铁毒中的作用

根据营养液培养试验从水稻Ａｚｕｃｅｎａ×ＩＲ６４ 发展的一双单倍体（ＤＨ） 群体中筛选出抗铁毒与敏感品系。在铁毒害处理后,各品系的抗坏血酸过氧化物酶（ＡＰ） 、脱氢抗坏血酸还原酶（ＤＲ） 、谷胱甘肽还原酶（ＧＲ） 的活性均有明显提高;水稻受铁毒后的生物量递减量与ＡＰ、ＤＲ、ＧＲ 活性呈负相关。说明抗坏血酸过氧化物酶Ｈ２Ｏ２ 清除系统在水稻抗铁毒中起着十分重要的作用。     

Mitochondrial GPX1 silencing triggers differential photosynthesis impairment in response to salinity in rice plants

The physiological role of plant mitochondrial glutathione peroxidases is scarcely known. This study attempted to elucidate the role of a rice mitochondrial isoform(GPX1) in photosynthesis under normal growth and salinity conditions. GPX1 knockdown rice lines(GPX1s) were tested in absence and presence of 100 mM NaCl for 6 d.Growth reduction of GPX1 s line under non-stressful conditions, compared with non-transformed(NT) plants occurred in parallel to increased H_2O_2 and decreased GSH contents. These changes occurred concurrently with photosynthesis impairment, particularly in Calvin cycle's reactions, since photochemical efficiency did not change.Thus, GPX1 silencing and downstream molecular/metabolic changes modulated photosynthesis differentially. In contrast, salinity induced reduction in both phases of photosynthesis, which were more impaired in silenced plants.These changes were associated with root morphology alterations but not shoot growth. Both studied lines displayed increased GPX activity but H_2O_2 content did not change in response to salinity. Transformed plants exhibited lower photorespiration, water use efficiency and root growth, indicating that GPX1 could be important to salt tolerance. Growth reduction of GPX1 s line might be related to photosynthesis impairment, which in turn could have involved a cross talk mechanism between mitochondria and chloroplast originated from redox changes due to GPX1 deficiency.     

Effect of iron plaque outside roots on nutrient uptake by rice (Oryza sativa L.): Phosphorus uptake

Under anaerobic conditions, ferric hydroxide deposits on the surface of rice roots have been shown to affect the uptake of some nutrients. In the present experiment, different amount of this iron plaque were induced on the roots of rice (Oryza sativa L. cv. TZ88-145) by supplying different Fe(OH)₃ concentrations in nutrient solutions, and the effect of the iron plaque on phosphorus uptake was investigated. Results showed that 1) iron plaque adsorbed phosphorus from the growth medium, and that the amount of phosphorus adsorbed by the plaque was correlated with the amount of plaque; 2) the phosphorus concentration in the shoot increased by up to 72% after 72 h at concentration of Fe(OH)₃ in the nutrient solution from 0 to 30 mg Fe/L, corresponding with amounts of iron plaque from 0.2 to 24.5 mg g^-1 (root d. wt); 3) the phosphorus concentration in the shoots of rice with iron plaque was higher than that without iron plaque though the concentration in the shoot decreased when Fe(OH)₃ was added at 50 mg Fe/L producing 28.3 mg g^-1 (root d. wt) of plaque; and 4) the phosphorus concentrations in Fe-deficient and Fe-sufficient rice plants with iron plaque were the same, although phytosiderophores were released from the Fe-deficient roots. The phytosiderophores evidently did not mobilise phosphorus adsorbed on plaque. The results suggest that iron plaque on rice plant roots might be considered a phosphorus reservoir. This revised version was published online in June 2006 with corrections to the Cover Date.     

低钾胁迫对水稻(Oryza sativa L.)化感潜力变化的影响

研究以国际公认的化感水稻P1312777和非化感水稻Lemont为供体,稗草（Echinochloa cru-galli L.）为受体,采用稻／稗共培体系,研究低钾胁迫对水稻化感潜力变化的影响及其机制。受体稗草的形态指标分析结果表明,低钾胁迫促使化感水稻P1312777对共培稗草的根长、株高和干重的抑制率均升高,增幅远大于非化感水稻Lemont。受体稗草生理生化指标分析结果表明,低钾胁迫下化感与非化感水稻对受体稗草保护酶系（SOD、POD、CAT）及根系活力的抑制作用增强,但化感水稻P1312777比非化感水稻Lemont的抑制程度大,且达极显著差异。实时荧光定量PCR分析结果表明,低钾胁迫下,化感水稻P1312777根部与叶部中酚类代谢的关键酶——苯丙氨酸解氨酶、肉桂酸-4-羟化酶、羟化酶、O-甲基转移酶的基因均上调表达,而非化感水稻根部相应酶均下调表达,叶部除苯丙氨酸解氨酶上调,其余酶也下调表达。而萜类代谢途径关键酶——HMG—CoA还原酶、角鲨烯合酶、单萜烯环化酶、倍半萜烯环化酶、二萜烯环化酶的基因,在两种水稻根部中呈现出相同或相似的表达方式（上调或下调）,即HMG—CoA还原酶上调表达,角鲨烯合酶、单萜烯环化酶、倍半萜烯环化酶、二萜烯环化酶下调表达;而在水稻叶部,非化感水稻Lmont相应酶基因表达方式仍然不变,化感水稻P1312777除了角鲨烯合酶下调表达,其余4个酶均上调表达。水稻根系分泌物中酚类物质的HPLC分析结果表明,低钾胁迫下,化感水稻P1312777根系分泌物中,所检出的酚酸类物质总量是正常营养条件下的2．30倍,而非化感水稻Lemont则是正常营养条件下的0．91倍。综合分析认为低钾胁迫下,化感水稻P1312777抑草能力增强主要是由于酚类代谢途径关键酶基因表达上调,导致酚类代谢途径旺盛,分泌出更多的酚类物质,进而破坏受体稗草保护酶系统,抑制了稗草的正常生长。     

Paraquat toxicity is reduced by metal chelators in rice leaves

The possible mediatory role of transition metals in paraquat (PQ) toxicity in rice leaves was investigated. Metal chelators (2,2'-bipyridine, 8-hydroxylquinoline and 1,10-phenanthroline) reduced PQ toxicity in rice leaves. The reduction of PQ toxicity by 1,10-phenanthroline (PA) is closely associated with the decrease in lipid peroxidation and increase in activities of enzymes detoxifying active oxygen species. Our results support the notion that iron or copper plays a major role in PQ toxicity in detached rice leaves. Reduction of PQ toxicity by PA in detached rice leaves is most likely mediated through chelation of iron or copper and an increase in superoxide dismutase and glutathione reductase activities.