Proteomics of Arabidopsis redox proteins in response to methyl jasmonate

Protein redox regulation is increasingly recognized as an important switch of protein activity in yeast, bacteria, mammals and plants. In this study, we identified proteins with potential thiol switches involved in jasmonate signaling, which is essential for plant defense. Methyl jasmonate (MeJA) treatment led to enhanced production of hydrogen peroxide in Arabidopsis leaves and roots, indicating in vivo oxidative stress. With monobromobimane (mBBr) labeling to capture oxidized sulfhydryl groups and 2D gel separation, a total of 35 protein spots that displayed significant redox and/or total protein expression changes were isolated. Using LC–MS/MS, the proteins in 33 spots were identified in both control and MeJA-treated samples. By comparative analysis of mBBr and SyproRuby gel images, we were able to determine many proteins that were redox responsive and proteins that displayed abundance changes in response to MeJA. Interestingly, stress and defense proteins constitute a large group that responded to MeJA. In addition, many cysteine residues involved in the disulfide dynamics were mapped based on tandem MS data. Identification of redox proteins and their cysteine residues involved in the redox regulation allows for a deeper understanding of the jasmonate signaling networks.     

The FUSCA genes of Arabidopsis: negative regulators of light responses

More than 200 fusca mutants of Arabidopsis have been isolated and characterised, defining 14 complementation groups. Mutations in at least nine FUSCA genes cause light-dependent phenotypic changes in the absence of light: high levels of anthocyanin accumulation in both the embryo and the seedling, inhibition of hypocotyl elongation, apical hook opening, and unfolding of cotyledons. In double mutants, the fusca phenotype is epistatic to the hy phytochromedeficiency phenotype, indicating that the FUSCA genes act downstream of phytochrome. By contrast, the accumulation of anthocyanin is suppressed by mutations in TT and TTG genes, which affect the biosynthesis of anthocyanin, placing the FUSCA genes upstream of those genes. Regardless of the presence or absence of anthocyanin, fusca mutations limit cell expansion and cause seedling lethality. In somatic sectors, mutant fus1 cells are viable; expressing tissue-specific phenotypes: reduced cell expansion and accumulation of anthocyanin in subepidermal tissue, formation of ectopic trichomes but no reduced cell expansion in epidermal tissue. Our results suggest a model of FUSCA gene action in light-induced signal transduction.     

拟南芥ABA敏感突变体asm1的分离与鉴定

以拟南芥(Arabidopsis thaliana)为研究材料,从T-DNA突变体库中筛选分离得到1株脱落酸(ABA)敏感突变体asm1(ABA sensitive mutant 1,asm1),在含有ABA的培养基中,与野生型相比,asm1突变体的根伸长明显受到抑制,且其种子萌发结果显示asm1对ABA同样表现出敏感特性。在生长发育方面,asm1突变体抽苔时间提前,植株矮化,并且荚果长度明显小于野生型。利用远红外成像系统分析发现,在干旱胁迫下asm1突变体叶面温度高于野生型;失水率分析显示突变体失水率降低以及水分散失减少。遗传学分析表明,asm1是单基因隐性突变且与一个T-DNA插入共分离;通过图位克隆成功获得候选基因ASM1。RT-PCR结果显示,在突变体中ASM1的表达受到抑制,并且能够调控多种ABA信号通路和胁迫应答基因的表达水平。研究结果表明,ASM1可能参与调控ABA信号转导并应答干旱胁迫。     

拟南芥多聚ADP核糖聚合酶Ⅰ的原核表达与活性检测

采用RT-PCR技术获得了拟南芥多聚ADP核糖聚合酶[poly(ADP-ribose)polymerase,PARP]PARP1基因的全长cDNA,转入原核表达载体pET32a并转化宿主菌Origami(DE3),加入终浓度为0.3mmol/L IPTG,在16℃下诱导可获得较多的可溶重组蛋白。纯化TRX-PARP1,在反应液中加入NAD+和断裂DNA,通过SDS-PAGE凝胶电泳和Western blotting分析,TRX-PARP1分子量可随着时间的延长逐渐增大,产生向上的弥散,表明蛋白质连上了ADP核糖分子;与此对比,作为参照的标签蛋白TRX无此现象。实验结果显示原核表达拟南芥PARP1能够催化自身多聚ADP核糖化修饰,为深入研究植物多聚ADP核糖聚合酶的功能奠定了基础。     

Intragenic recombination in the CSR1 locus of Arabidopsis

Four classes of herbicides are known to inhibit plant acetolactate synthase (ALS). In Arabidopsis, ALS is encoded by a single gene, CSR1. The dominant csr1-1 allele encodes an ALS resistant to chlorsulfuron and triazolopyrimidine sulfonamide while the dominant csr1-2 allele encodes an ALS resistant to imazapyr and pyrimidyl-oxy-benzoate. The molecular distance between the point mutations in csr1-1 and csr1-2 is 1369 bp. Here we used multiherbicide resistance as a stringent selection to measure the intragenic recombination frequency between these two point mutations. We found this frequency to be 0.008 ± 0.0028. The recombinant multiherbicide-resistant allele, csr1-4, provides an ideal marker for plant genetic transformation.     

二氧化硫增强拟南芥植株对干旱的适应性

以模式植物拟南芥为材料,研究SO_2对植物干旱适应性的影响。采用分光光度法检测植物干旱生理指标的变化,并用半定量RT-PCR技术分析了拟南芥热激基因和干旱响应基因的转录水平。研究发现:4周龄拟南芥植株暴露于30mg/m3的SO_2后,6—72h间叶面气孔开度显著低于对照并逐渐减小,在暴露48h和72h时,热激转录因子HsfA2和热激基因Hsp17.7、Hsp17.6B、Hsp17.6C转录上调,干旱响应基因DREB2A、DREB2B和RD29A表达增强;在SO_2熏气72h后进行干旱胁迫,干旱期间SO_2预暴露植株的叶片相对含水量高于非熏气干旱处理组,植株萎蔫程度比后者明显减轻,且SO_2预暴露植株的地上组织中可溶性糖和脯氨酸含量升高,超氧化物歧化酶活性提高,丙二醛含量降低。结果表明:SO_2能降低气孔开度、提高抗氧化能力、上调热激基因和干旱响应基因转录,并能促进干旱期间植物细胞内渗透调节物质的合成和积累,促使抗氧化酶活性提高,从而降低干旱胁迫对植株造成的氧化损伤,增强拟南芥对干旱的适应性。植物通过基因转录应答、酶活性改变、渗透调节物质积累等,在适应环境高浓度SO_2的同时,提高了对干旱的适应性。     

CRISPR/Cas9介导靶向敲除拟南芥GGB基因突变体的鉴定

GGB是抗旱负调控基因。为了获得拟南芥ggb突变体材料,构建了以拟南芥U6启动子驱动GGB sgRNA的CRISPR/Cas9基因组编辑载体。将构建好的编辑载体利用农杆菌介导的浸花法转化野生型拟南芥。对转基因后代GGB基因的测序结果分析发现,在靶位点处有缺失4个碱基和增加1个T碱基的2种突变体产生。分别对野生型拟南芥和上述2种ggb突变体进行半定量RT PCR分析结果显示,突变体材料中几乎检测不到GGB基因表达,说明获得了GGB基因敲除突变体。对野生型和ggb突变体叶片失水率、耐旱表型及单株种子量的测定结果表明,与野生型相比,拟南芥GGB基因突变后,叶片失水率显著减少,抗旱性明显增强,而单株种子量却并没有改变。研究表明,GGB是一种理想的作物分子育种的候选靶基因,获得的突变体为今后从农作物中克隆的GGB同源基因进行功能互补验证提供了有用的遗传材料。     

拟南芥bso-1突变体的基因定位及表型分析

通过EMS化学诱变在拟南芥Columbia(Col-0)野生型突变体库中筛选获得1株器官显著增大的突变体,命名为big size organ1(bso-1)。遗传分析表明,bso-1受单个隐性核基因控制。表型观察发现,突变体植株的幼苗、花、果荚及种子与野生型相比都表现出明显的增大。组织切片结果显示,突变体种子的增大主要由胚细胞个体增大导致胚体积增大而实现,因此突变体种子的重量也较野生型有明显增加。利用图位克隆方法将相关基因初步定位在4号染色体上SSLP标记T5L19与F28M11之间58kb区间内,生物信息学分析显示此区间内未见调控植物器官大小发育相关的已知基因的报道。该研究结果为进一步克隆bso-1突变体相关基因及探讨其在控制植物器官发育尤其是种子发育过程中的作用奠定了基础。     

生物钟PRR蛋白促进拟南芥幼苗中花青素的合成

生物钟(circadian clock)是激发植物生理特征节律性表达,并使之维持稳定的保守内源调节机制。PRR(PSEUDO-RESPONSE REGULATOR)蛋白家族是生物钟中央振荡器的重要组成部分,调控植物的种子萌发、下胚轴伸长和开花等多种生命过程。花青素(anthocyanin)是植物次生代谢产物,对植物的繁衍、生长发育和抵抗逆境胁迫具有重要作用。该研究以拟南芥(Arabidopsis thaliana)为对象,探讨生物钟PRR蛋白对花青素生物合成的调控功能和分子机制。结果表明：(1)在PRR基因单突变体及多突变体幼苗中,花青素的积累明显降低,某些花青素合成相关基因的表达也显著降低。(2)相反,在PRR5过表达幼苗中,花青素的积累以及某些花青素合成相关基因的表达则显著升高。(3)蛋白相互作用结果显示,PRR5蛋白能与MYB75、TT8、MYB90及MYB113等花青素调控蛋白相互作用,并形成复合物。(4)遗传学分析结果显示,拟南芥PRR5诱导幼苗中花青素的合成依赖于MYB家族花青素调控蛋白。综上认为,生物钟PRR蛋白可能通过PRR5与MYB75、TT8等相互作用,促进拟南芥幼...     

Gene expression profiling of mouse host response to Listeria monocytogenes infection

The purpose of this study was to evaluate gene expression profiles in the liver and blood for prediction of infection severity from Listeria monocytogenes (LM). Mice were injected with medium broth (control) or a nonlethal or lethal dose of LM and sacrificed 6 h later. Gene expression changes were determined using Affymetrix MGU74Av2 GeneChips and confirmed by real-time polymerase chain reaction analysis. We identified discernable genes whose gene expression profiles can be used in pattern recognition to predict and classify samples in differently treated groups, with >or=90% accuracy in liver samples and 80% accuracy in blood at prediction; however, different genes were predictive in each tissue. Our results suggest that gene expression profiling in response to LM in mice may be able to distinguish samples in groups with varying severity of infection and provide information in finding molecular mechanisms and early biomarkers for subsequent conventional clinical endpoints.     

Analysis of the frequency of inheritance of transposed Ds elements in Arabidopsis after activation by a CaMV 35S promoter fusion to the Ac transposase gene

The Ac/Ds transposon system of maize shows low activity in Arabidopsis. However, fusion of the CaMV 35S promoter to the transposase gene (35S::TPase) increases the abundance of the single Ac mRNA encoded by Ac and increases the frequency of Ds excision. In the experiments reported here it is examined whether this high excision frequency is associated with efficient re-insertion of the transposon. This was measured by using a Ds that carried a hygromycin resistance gene (HPT) and was inserted within a streptomycin resistance gene (SPT). Excision of Ds therefore gives rise to streptomycin resistance, while hygromycin resistance is associated with the presence of a transposed Ds or with retention of the element at its original location. Self-fertilisation of most individuals heterozygous for Ds and 35S::TPase produced many streptomycin-resistant (strep^r) progeny, but in many of these families a small proportion of strep^r seedlings were also resistant to hygromycin (hyg^r). Nevertheless, 70% of families tested did give rise to at least one strep^r, hyg^r seedling, and over 90% of these individuals carried a transposed Ds. In contrast, the Ac promoter fusion to the transposase gene (Ac::TPase) produced fewer strep^rhyg^r progeny, and only 53% of these carried a transposed Ds. However, a higher proportion of the strep^r seedlings were also hyg^r than after activation by 35S::TPase. We also examined the genotype of strep^r, hyg^r seedlings and demonstrated that after activation by 35S::TPase many of these were homozygous for the transposed Ds, while this did not occur after activation by Ac::TPase. From these and other data we conclude that excisions driven by 35S::TPase usually occur prior to floral development, and that although a low proportion of strep^r progeny plants inherit a transposed Ds, those that do can be efficiently selected with an antibiotic resistance gene contained within the element. Our data have important implications for transposon tagging strategies in transgenic plants and these are discussed.     

转CkLEA4基因拟南芥种子萌发期的抗逆性分析

该研究在实验室前期研究的基础上,将受脱水、盐胁迫和ABA诱导的柠条锦鸡儿CkLEA4基因转入野生型拟南芥,并利用实时荧光定量PCR从8株纯合体中筛选出3个表达量不同的株系,比较野生型和转CkLEA4基因过表达拟南芥种子在不同胁迫处理下的萌发率,以探讨CkLEA4基因在植物抵抗逆境胁迫中的功能。结果发现:(1)在不同浓度NaCl、甘露醇及ABA处理下,转CkLEA4基因过表达拟南芥种子的萌发率均高于野生型,随着NaCl、甘露醇及ABA浓度增加,各株系萌发率均降低,但野生型的萌发率下降幅度均高于3个过表达株系,并且在200mmol/L NaCl和400mmol/L甘露醇处理下,过表达株系子叶绿化率均显著高于野生型。(2)在低浓度ABA处理下,CkLEA4过表达植株子叶的绿化率也高于野生型。研究表明,柠条锦鸡儿CkLEA4基因提高了拟南芥种子萌发阶段对盐、ABA及渗透胁迫的耐受性。     

罗汉果色氨酸转氨酶基因SgTAR2的克隆及表达分析

色氨酸转氨酶基因家族,是直接参与植物生长素生物合成途径的关键酶基因。该研究在罗汉果转录组测序的基础上,结合RACE技术克隆罗汉果色氨酸转氨酶基因SgTAR2的全长cDNA序列和DNA序列;并对其进行生物信息学分析和时空表达分析。结果表明:克隆所得SgTAR2的cDNA全长序列2078bp,最长ORF为1332bp,编码443个氨基酸,Gen Bank登录号为KU949381,其编码蛋白具有2个蒜氨酸酶保守结构域和多个5'-PLP结合位点,推测其可能参与催化色氨酸转氨基作用、化学防御作用、生长素生物合成等生物学过程;SgTAR2基因DNA长为4103bp,含有4个内含子和5个外显子,其内含子具有多个高水平转录调控因子和多个与激素、环境等胁迫响应相关的作用元件,暗示SgTAR2基因内含子协同调控罗汉果生长素合成、抗胁迫反应、形态发育等生物学过程。实时荧光定量结果显示,SgTAR2基因在罗汉果各组织器官均有表达,在雌蕊和15d幼果期表达量较高,暗示该基因参与罗汉果果实早期发育。该研究结果表明SgTAR2参与了生长素介导的罗汉果不同生长发育过程,特别对幼果及花的起始发育和器官形态建成等具有重要意义。     

Biotic and abiotic stress down-regulate miR398 expression in Arabidopsis

MicroRNA398 targets two Cu/Zn superoxide dismutases (CSD1 and CSD2) in higher plants. Previous investigations revealed both decreased miR398 expression during high Cu²⁺ or paraquat stress and increased expression under low Cu²⁺ or high sucrose in the growth medium. Here, we show that additional abiotic stresses such as ozone and salinity also affect miR398 levels. Ozone fumigation decreased miR398 levels that were gradually restored to normal levels after relieved from the stress. Furthermore, miR398 levels decreased in Arabidopsis leaves infiltrated with avirulent strains of Pseudomonas syringae pv. tomato, Pst DC3000 (avrRpm1 or avrRpt2) but not the virulent strain Pst DC3000. To our knowledge, miR398 is the first miRNA shown to be down-regulated in response to biotic stress (P. syringae). CSD1, but not CSD2, mRNA levels were negatively correlated with miR398 levels during ozone, salinity and biotic stress, suggesting that CSD2 regulation is not strictly under miR398 control during diverse stresses. Overall, this study further establishes a link between oxidative stress and miR398 in Arabidopsis.     

冷胁迫条件下拟南芥Rubisco相互作用蛋白质的比较分析

1,5-二磷酸核酮糖羧化酶/加氧酶(Rubisco,EC 4.1.1.39)在生物适应环境变化的过程中起到重要的作用.位于叶绿体中与冷胁迫密切相关的非常重要的复合酶——Rubisco,其相互作用的蛋白质至今没有系统的研究.对拟南芥进行4种处理:a.持续在20℃生长(对照);b.4℃4 h冷胁迫;c.4℃24 h冷胁迫;d.4℃24 h冷胁迫后放入20℃恢复24 h.然后利用免疫共沉淀、十二烷基硫酸钠聚丙烯酰胺凝胶电泳及基质辅助激光解析电离飞行时间质谱技术,在冷胁迫条件下研究了拟南芥光合抑制与Rubisco相互作用蛋白质解聚之间的关系.在鉴定出的5个与冷胁迫相关的Rubisco相互作用蛋白质中,AAA-型ATP酶家族蛋白和糖基转移酶对Rubisco活性及植物适应冷胁迫起着重要的作用.研究结果表明,Rubisco复合酶体系的解聚可能是低温胁迫下拟南芥光合速率降低的主要原因.