玉米ZmSOD基因克隆及其在干旱胁迫下的表达

为探讨超氧化物歧化酶(superoxide dismutase,SOD)基因在玉米抗逆反应中的作用,研究选用2个抗旱性有明显差别的玉米品种为试验材料,采用RT-PCR、实时荧光定量PCR(qRT-PCR)及二维凝胶电泳(2-DE)耦联的基质辅助激光解吸电离/飞行时间质谱(MALDI-TOF)法,对ZmSOD基因的序列结构及其在干旱胁迫下的表达特性进行分析。结果表明：(1)从干旱敏感品种‘登海605’(DH605)和抗旱品种‘蠡玉35’(LY35)玉米叶片中分别成功克隆出ZmSOD1和ZmSOD2基因。DH605中ZmSOD1开放阅读框(ORF)全长456 bp,编码151个氨基酸,编码的蛋白等电点为5.76,分子量为15.05 kD。LY35中ZmSOD2ORF全长459 bp,编码152个氨基酸,编码的蛋白等电点为5.65,分子量为15.11 kD;ZmSOD1和ZmSOD2是亲水性稳定蛋白,都含有Cu/Zn-SOD结构域,蛋白序列N端无信号肽及无跨膜结构域。同源性和系统进化分析表明,玉米ZmSOD和谷子Cu/Zn-SOD2的亲缘关系最近,相似性高达96.05%。(2)在干旱条件下,ZmSOD1在DH605中转录水平明显降低,LY35中ZmSOD2转录水平显著升高;2-DE耦联的质谱分析显示：ZmSOD1在DH605中表达量明显降低,LY35中ZmSOD2表达量无显著性变化,却检测到Mn-SOD蛋白表达量显著增加。(3)相关分析显示,干旱条件下,DH605叶片中ZmSOD1转录水平和其蛋白丰度及SOD酶活性呈极显著性正相关,LY35叶片中ZmSOD2转录水平与SOD酶活性呈显著性正相关。研究结果为进一步探索SOD基因在调节玉米抗性以及逆境胁迫应答过程中的作用机制提供了基础。     

Expression of Mitochondrial Cytochrome C Oxidase Chaperone Gene (COX20) Improves Tolerance to Weak Acid and Oxidative Stress during Yeast Fermentation

Introduction

Saccharomyces cerevisiae is the micro-organism of choice for the conversion of fermentable sugars released by the pre-treatment of lignocellulosic material into bioethanol. Pre-treatment of lignocellulosic material releases acetic acid and previous work identified a cytochrome oxidase chaperone gene (COX20) which was significantly up-regulated in yeast cells in the presence of acetic acid.

Results

A Δcox20 strain was sensitive to the presence of acetic acid compared with the background strain. Overexpressing COX20 using a tetracycline-regulatable expression vector system in a Δcox20 strain, resulted in tolerance to the presence of acetic acid and tolerance could be ablated with addition of tetracycline. Assays also revealed that overexpression improved tolerance to the presence of hydrogen peroxide-induced oxidative stress.

Conclusion

This is a study which has utilised tetracycline-regulated protein expression in a fermentation system, which was characterised by improved (or enhanced) tolerance to acetic acid and oxidative stress.     

Gene Expression Analysis of Cold and Freeze Stress in Baker's Yeast

We used mRNA differential display to assess yeast gene expression under cold or freeze shock stress conditions. We found both up- and down-regulation of genes, although repression was more common. We identified and sequenced several cold-induced genes exhibiting the largest differences. We confirmed, by Northern blotting, the specificity of the response for TPI1, which encodes triose-phosphate isomerase; ERG10, the gene for acetoacetyl coenzyme A thiolase; and IMH1, which encodes a protein implicated in protein transport. These genes also were induced under other stress conditions, suggesting that this cold response is mediated by a general stress mechanism. We determined the physiological significance of the cold-induced expression change of these genes in two baker's yeast strains with different sensitivities to freeze stress. The mRNA level of TPI1 and ERG10 genes was higher in freeze-stressed than in control samples of the tolerant strain. In contrast, both genes were repressed in frozen cells of the sensitive strain. Next, we examined the effects of ERG10 overexpression on cold and freeze-thaw tolerance. Growth of wild-type cells at 10°C was not affected by high ERG10 expression. However, YEpERG10 transformant cells exhibited increased freezing tolerance. Consistent with this, cells of an erg10 mutant strain showed a clear phenotype of cold and freeze sensitivity. These results give support to the idea that a cause-and-effect relationship between differentially expressed genes and cryoresistance exists in Saccharomyces cerevisiae and open up the possibility of design strategies to improve the freeze tolerance of baker's yeast.     

Root Morphology and Gene Expression Analysis in Response to Drought Stress in Maize (Zea mays)

Water-deficit stress tolerance is a complex trait, and water deficit results in various physiological and chemical changes in maize (Zea mays L.) and exacerbates pre-harvest aflatoxin contamination. The objective of this study was to characterize the variations in morphology, physiology, and gene expression in two contrasting inbred lines, Lo964 and Lo1016, in order to understand the differences in response to water-deficit stress. The results revealed that Lo964 was less sensitive to water-deficit stress, and had a strong lateral root system and a higher root/shoot ratio in comparison to Lo1016. In response to water-deficit stress by comparing stressed versus well-watered conditions, abscisic acid syntheses were increased in leaves, roots, and kernels of both Lo964 and Lo1016, but by different magnitudes. Indole-3-acetic acid (IAA) was undetectable in the leaves and roots of either genotype regardless of treatments, but increases of 58% and 8% in IAA concentration were observed in 20 DAP kernels, in response to water-deficit stress, respectively. The expression of the MIPS was up-regulated 7-fold in leaf tissues of Lo964 compared to Lo1016 at watered conditions, but decreased significantly to similar levels in both genotypes at water-deficit conditions. ZmPR10 and ZmFer1 expressions tended to up-regulate although ZmPR10 was expressed higher in root tissue while ZmFer1 was expressed higher in leaf tissue. Further study is needed to confirm if Lo964 has reduced aflatoxin contamination associated with the drought tolerance in the field in order to utilize the resistant trait in breeding.     

水稻(Oryza sativa L.)苹果酸酶(OsNADP-ME3)基因在逆境下的表达特性研究

水稻苹果酸酶（NADP-ME）是多基因家族,由3个胞质型NADP-ME和1个质体型NADP-ME构成。本研究针对水稻胞质型成员（命名为NADP-ME3）（NM₀01061367）进行初步的功能解析。克隆获得的NADP-ME3基因的cDNA序列全长为2240bp,其中5’非翻译区为151bp,3’非翻译区为376bp,开放读码框（ORF）长1713bp,编码570个氨基酸。为研究NADP-ME3在逆境胁迫下的表达量变化,Northern blot检测结果显示,在NaCl、NaHCO3和PEG胁迫条件下,NADP-ME3随胁迫处理时间的不同表达量呈现不同程度的变化,推断NADP-ME3可能与非生物胁迫有应答关系,将NADP-ME3转入拟南芥中并通过观察转基因拟南芥在非生物胁迫下表型变化,发现NADP-ME3能够在一定程度上提高植物对非生物胁迫的耐受性。     

氧化应激诱导的细胞衰老过程中细胞生长相关基因的表达

应激诱导的细胞早衰与复制性细胞衰老有相似的细胞表型,但其机制不尽相同.分析二者的衰老相关基因表达特点对了解应激因素诱导细胞衰老的机制有重要意义. 本文对过氧化氢诱导的HeLa细胞早衰过程中的关键衰老相关基因及其转录后调控因子的表达做了分析.结果发现,在复制性衰老过程中明显降低的cyclin A、cyclin B1、c-fos及HuR,在温和过氧化氢诱导的细胞早衰过程中并无明显改变;在氧化应激诱导的细胞早衰过程中,p21与p16表达升高,AUF1则降低,与复制性衰老过程一致;p21 mRNA半衰期在复制性衰老过程中无明显变化,但在氧化应激诱导的细胞早衰过程中则显著延长.上述结果提示,尽管氧化应激诱导的细胞早衰与复制性衰老存在相似基因表达变化,调控机制则不尽相同.     

玉米ZmPHO2基因家族克隆及其低磷胁迫下的表达

PHO2(编码一个泛素结合酶E2)作为磷高亲和转运体PHT1的负调控子,在维持植物体内磷的动态平衡中发挥重要作用。该研究以拟南芥和水稻中的PHO2为基础,从玉米自交系B73基因组中鉴定出9个ZmPHO2基因家族成员,在系统进化关系上将其分为3类。在玉米自交系178中克隆了上述9个基因的CDS全长序列,保守结构域分析发现,ZmPHO2蛋白质序列中均有1个由约130个氨基酸组成的泛素结合酶E2催化结构域(UBCc),其中包含1个重要的保守氨基酸(半胱氨酸)。实时荧光定量结果表明,低磷胁迫处理后,所有ZmPHO2基因均有表达,并呈现不同的表达模式,主要表现为叶与根之间的组织差异和玉米自交系178与9782之间的基因型差异,而在同一组织多数基因间的表达差异不明显。其中,ZmPHO2;H2在自交系9782的根中持续下调表达,但在叶中持续上调表达,表明ZmPHO2;H2可能参与调控磷素在叶与根之间的运输,以维持地上部分和地下部分磷的平衡。     

一个水稻多逆境响应基因OsMsr3克隆与表达分析

为深入探讨水稻对逆境的反应机理并寻找新的植物耐逆基因,采用Affymetrix水稻表达芯片(含51279个转录本)分析了培矮64S全基因组在不同逆境(高温、干旱、低温)胁迫下、不同生育时期叶片和穗中的表达谱,从中筛选出一个受多种逆境诱导表达的基因OsMsr3(Oryza sativa L.multiple stresses responsive gene3,GenBank登陆号为 FJ383169).定量实时PCR分析结果进一步证实了此基因在逆境条件下的诱导表达模式.用 RT-PCR方法扩增获得了包含其完整开放阅读框的cDNA克隆,序列分析表明,其ORF大小为480 bp,编码一个具有160个氨基酸残基的低分子量热激蛋白,推测分子量约为18.0 kD;pI约为6.8.对其编码的蛋白质进行分析,发现其羧基端存在一个HSP20的蛋白保守结构域,与其他植物中的低分子量热激蛋白的相似性介于33.7%～97.5%.对其可能的启动子序列分析,发现6类与逆境反应有关的顺式作用元件.推测该基因在逆境反应中起着重要的作用,进一步的研究正在进行中.     

Oxidative Stress Induced Age Dependent Meibomian Gland Dysfunction in Cu,Zn-Superoxide Dismutase-1 (Sod1) Knockout Mice

Purpose

The purpose of our study was to investigate alterations in the meibomian gland (MG) in Cu, Zn-Superoxide Dismutase-1 knockout (Sod1 ^−/−) mouse.

Methods

Tear function tests [Break up time (BUT) and cotton thread] and ocular vital staining test were performed on Sod1 ^−/− male mice (n = 24) aged 10 and 50 weeks, and age and sex matched wild–type (+/+) mice (n = 25). Tear and serum samples were collected at sacrifice for inflammatory cytokine assays. MG specimens underwent Hematoxylin and Eosin staining, Mallory staining for fibrosis, Oil Red O lipid staining, TUNEL staining, immunohistochemistry stainings for 4HNE, 8-OHdG and CD45. Transmission electron microscopic examination (TEM) was also performed.

Results

Corneal vital staining scores in the Sod1 ^−/− mice were significantly higher compared with the wild type mice throughout the follow-up. Tear and serum IL-6 and TNF-α levels also showed significant elevations in the 10 to 50 week Sod1 ^−/− mice. Oil Red O staining showed an accumulation of large lipid droplets in the Sod1 ^−/− mice at 50 weeks. Immunohistochemistry revealed both increased TUNEL and oxidative stress marker stainings of the MG acinar epithelium in the Sod1 ^−/− mice compared to the wild type mice. Immunohistochemistry staining for CD45 showed increasing inflammatory cell infiltrates from 10 to 50 weeks in the Sod1 ^−/− mice compared to the wild type mice. TEM revealed prominent mitochondrial changes in 50 week Sod1 ^−/− mice.

Conclusions

Our results suggest that reactive oxygen species might play a vital role in the pathogensis of meibomian gland dysfunction. The Sod1 ^−/− mouse appears to be a promising model for the study of reactive oxygen species associated MG alterations.     

Expression of Yeast NDI1 Rescues a Drosophila Complex I Assembly Defect

Defects in mitochondrial electron transport chain (ETC) function have been implicated in a number of neurodegenerative disorders, cancer, and aging. Mitochondrial complex I (NADH dehydrogenase) is the largest and most complicated enzyme of the ETC with 45 subunits originating from two separate genomes. The biogenesis of complex I is an intricate process that requires multiple steps, subassemblies, and assembly factors. Here, we report the generation and characterization of a Drosophila model of complex I assembly factor deficiency. We show that CG7598 (dCIA30), the Drosophila homolog of human complex I assembly factor Ndufaf1, is necessary for proper complex I assembly. Reduced expression of dCIA30 results in the loss of the complex I holoenzyme band in blue-native polyacrylamide gel electrophoresis and loss of NADH:ubiquinone oxidoreductase activity in isolated mitochondria. The complex I assembly defect, caused by mutation or RNAi of dCIA30, has repercussions both during development and adulthood in Drosophila, including developmental arrest at the pupal stage and reduced stress resistance during adulthood. Expression of the single-subunit yeast alternative NADH dehydrogenase, Ndi1, can partially or wholly rescue phenotypes associated with the complex I assembly defect. Our work shows that CG7598/dCIA30 is a functional homolog of Ndufaf1 and adds to the accumulating evidence that transgenic NDI1 expression is a viable therapy for disorders arising from complex I deficiency.     

刚毛柽柳ThDREB基因在酵母中的表达及抗逆能力分析

DREB蛋白能特异地与DRE（dehydration responsive element）顺式作用元件结合,从而调控下游多个与逆境相关基因的表达,在植物对多种逆境胁迫的应答反应中起重要调节作用。为了研究刚毛柽柳ThDREB基因是否具有抗逆功能,将ThDREB基因插入到酵母表达载体pYES2中构建成重组载体,转入酿酒酵母（Saccharomyces cerevisiae）中获得重组型酵母。分别比较转ThDREB基因酵母和转空载体对照酵母在山梨醇、H₂O₂、CdCl₂、NaCl、Na₂CO₃、MgCl₂、-20℃胁迫处理之后的存活能力。结果显示,ThDREB基因能有效提高转基因酵母的抗干旱、盐、碱、氧化、重金属及低温胁迫的能力,表明ThDREB基因可能参与了柽柳多种抗逆调控过程。     

Gene Expression Profiles Deciphering Rice Phenotypic Variation between Nipponbare (Japonica) and 93-11 (Indica) during Oxidative Stress

Rice is a very important food staple that feeds more than half the world''s population. Two major Asian cultivated rice (Oryza sativa L.) subspecies, japonica and indica, show significant phenotypic variation in their stress responses. However, the molecular mechanisms underlying this phenotypic variation are still largely unknown. A common link among different stresses is that they produce an oxidative burst and result in an increase of reactive oxygen species (ROS). In this study, methyl viologen (MV) as a ROS agent was applied to investigate the rice oxidative stress response. We observed that 93-11 (indica) seedlings exhibited leaf senescence with severe lesions under MV treatment compared to Nipponbare (japonica). Whole-genome microarray experiments were conducted, and 1,062 probe sets were identified with gene expression level polymorphisms between the two rice cultivars in addition to differential expression under MV treatment, which were assigned as Core Intersectional Probesets (CIPs). These CIPs were analyzed by gene ontology (GO) and highlighted with enrichment GO terms related to toxin and oxidative stress responses as well as other responses. These GO term-enriched genes of the CIPs include glutathine S-transferases (GSTs), P450, plant defense genes, and secondary metabolism related genes such as chalcone synthase (CHS). Further insertion/deletion (InDel) and regulatory element analyses for these identified CIPs suggested that there may be some eQTL hotspots related to oxidative stress in the rice genome, such as GST genes encoded on chromosome 10. In addition, we identified a group of marker genes individuating the japonica and indica subspecies. In summary, we developed a new strategy combining biological experiments and data mining to study the possible molecular mechanism of phenotypic variation during oxidative stress between Nipponbare and 93-11. This study will aid in the analysis of the molecular basis of quantitative traits.     

Protective Role of Sirtuin3 (SIRT3) in Oxidative Stress Mediated by Hepatitis B Virus X Protein Expression

Background/Aim

The hepatitis B virus (HBV) infection is accompanied by the induction of oxidative stress, especially mediated by HBV X protein (HBx). Oxidative stress has been implicated in a series of pathological states, such as DNA damage, cell survival and apoptosis. However, the host factor by which cells protect themselves under this oxidative stress is poorly understood.

Methodology/Principal Findings

In this study, we first confirmed that HBV infection significantly induced oxidative stress. Moreover, viral protein HBx plays a major role in the oxidative stress induced by HBV. Importantly, we found that mitochondrial protein SIRT3 overexpression could decrease reactive oxygen species (ROS) induced by HBx while SIRT3 knockdown increased HBx-induced ROS. Importantly, SIRT3 overexpression abolished oxidative damage of HBx-expressing cells as evidenced by γH₂AX and AP sites measurements. In contrast, SIRT3 knockdown promoted HBx-induced oxidative damage. In addition, we also observed that oxidant H₂O₂ markedly promoted HBV replication while the antioxidant N-acetyl-L-cysteine (NAC) inhibited HBV replication. Significantly, SIRT3 overexpression inhibited HBV replication by reducing cellular ROS level.

Conclusions/Significance

Collectively, these data suggest HBx expression induces oxidative stress, which promotes cellular oxidative damage and viral replication during HBV pathogenesis. Mitochondrial protein SIRT3 protected HBx expressing-cells from oxidative damage and inhibited HBV replication possibly by decreased cellular ROS level. These studies shed new light on the physiological significance of SIRT3 on HBx-induced oxidative stress, which can contribute to the liver pathogenesis.     

番茄AT-hook基因家族的鉴定及胁迫条件下的表达分析

AT-hook蛋白家族在植物生长发育、器官构建及逆境胁迫和激素信号应答中发挥重要作用。本研究在番茄基因组范围内,利用生物信息学方法对番茄AT-hook基因家族的成员、分布、结构和功能进行分析。结果表明,番茄AT-hook家族包含32个成员,分为3种类型,其中类型Ⅰ含有13个成员;遗传进化分析表明番茄AT-hook基因成员与拟南芥家族基因具有相似分类。利用实时荧光定量PCR对番茄32个基因开展组织表达分析,结果表明AT-hook基因具有表达差异,主要在根和花中表达较高。氧化胁迫分析结果表明,32个基因受ABA、SA、盐、高温和低温诱导表达,其中部分基因显著上调或下调表达,很可能参与了番茄逆境胁迫条件下的防御应答反应。本研究结果将为番茄AT-hook家族基因的深入研究提供依据,为进一步解析番茄AT-hook基因的功能奠定基础。     

差异显示技术研究NaHCO3 胁迫下星星草基因表达

用差异显示技术研究NaHCO3胁迫下星星草(Puccinellia tenuiflora)基因的表达。经Reverse Northern检测, 获得了7个差异表达的基因片段。其中, 6个为胁迫后诱导表达, 1个为胁迫后抑制表达。序列同源性分析表明, 胁迫诱导表达的6个基因片段中, 1个与钙依赖性蛋白激酶(CDPKs)基因同源性较高,其余5个可能为新序列, 胁迫后抑制表达的基因片段与假定的adaptor蛋白基因同源性较高。本研究为进一步研究星星草的抗盐机理奠定了基础。     

差异显示技术研究NaHCO_3胁迫下星星草基因表达

用差异显示技术研究NaHCO3胁迫下星星草(Puccinelliatenuiflora)基因的表达。经ReverseNorth-ern检测,获得了7个差异表达的基因片段。其中,6个为胁迫后诱导表达,1个为胁迫后抑制表达。序列同源性分析表明,胁迫诱导表达的6个基因片段中,1个与钙依赖性蛋白激酶(CDPKs)基因同源性较高,其余5个可能为新序列,胁迫后抑制表达的基因片段与假定的adaptor蛋白基因同源性较高。本研究为进一步研究星星草的抗盐机理奠定了基础。