丙酮醛降解酶基因的克隆与高效表达

由于丙酮醛降解酶(methylglyoxal degradation enzyme,MD)是生物体内代谢丙酮醛的一种关键酶,实现MD的高效表达对调控丙酮醛的代谢途径具有重要意义。所以根据Schizosaccharomyces pombe_Gene DB中MD的基因序列(SPAC22E12.03C)设计引物,以粟酒裂殖酵母(Schizosaccharomyces pombe)c DNA文库为模板通过PCR扩增技术得到目标基因,目的片段全长576 bp。将SPAC22E12.03C连接到p ET-28a(+)上,得到重组质粒p ET-28a(+)-SPAC22E12.03C,并在Escherichia coli BL2l(DE3)中实现表达。同时,对其目的蛋白的表达条件进行了优化,获得最佳表达条件:诱导温度37℃,诱导起始菌体D600 nm为0.5~0.6,诱导剂IPTG浓度为0.8 mmol/L。这为研究粟酒裂殖酵母体内丙酮醛的降解途径打下了坚实基础。     

非生物胁迫下植物体内丙酮醛代谢的研究进展

由于植物固着生长,其无法通过移动来逃避逆境,故非生物胁迫（如极端温度、盐胁迫、干旱或光胁迫等）会伴随着植物的整个生长发育过程,严重胁迫植物的分布、生长、品质和产量,甚至生存。植物只能通过改变自身形态结构以及生理生化反应来适应环境,或者通过释放化学物质来影响周边其他植物的生长发育,以改变微环境,使环境向着更适合自己生长的方向发展。丙酮醛（methylglyoxal,MG）又称之为甲基乙二醛,作为植物体内正常的生理代谢产物可由多条途径产生,其最主要的来源是糖酵解途径,如糖酵解中间体二羟丙酮磷酸和甘油醛3-磷酸去除磷酸基。而植物体内MG的分解主要靠乙二醛酶系统,包括乙二醛酶I、乙二醛酶II以及还原型谷胱甘肽,MG经乙二醛酶降解后形成D-乳酸。在正常生长条件下,植物体内的MG含量维持在较低水平,而当植物遭受非生物胁迫时,其含量会迅速升高;植物体内的MG含量过高会破坏植物细胞的增殖和生存,控制细胞的氧化还原状态以及其他许多方面的新陈代谢过程,最终导致生物大分子蛋白质、DNA、RNA、脂质和生物膜的破坏。因此,MG现在被认为是植物非生物胁迫耐受性的潜在生化标志物,并受到科学界的广泛关注。该文结合最新的研究进展,对非生物胁迫下植物体内丙酮醛合成及降解机制予以综述。     

乙二醛酶1在脑胶质瘤组织中的表达

目的 探讨乙二醛酶1(GLO1)在人脑胶质瘤组织中表达的情况.方法 western blot检测GLO1在29例不同类型的脑胶质瘤与20例脑外伤挫伤脑组织(对照组)中表达的差异.结果 western blot证实GLO1在23例非少突细胞胶质瘤组中的表达比对照组普遍下降(P＜0.01),GLO1在胶质瘤中的表达水平跟胶质瘤分类分级、患者的年龄、性别等无关.结论 GLO1在非少突细胞胶质瘤组织中的低表达可能预示着胶质瘤在肿瘤代谢方面存在新机制.     

外源脯氨酸对番茄体内残留百菌清降解的调控作用

为探索外源脯氨酸(Pro)对番茄残留农药降解代谢的调控作用,以番茄浙杂205为供试植物,以杀菌剂百菌清为供试农药,研究外源Pro对番茄体内喷施百菌清后的农药残留量、活性氧含量、抗氧化酶和解毒酶的影响。研究表明,外源3 mmol·L^-1的Pro预处理显著降低了百菌清处理7 d后番茄体内的农药残留量。与对照相比,外源Pro预处理可显著提高百菌清处理后番茄过氧化物酶(POD)、超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)、单脱氢抗坏血酸还原酶(MDAR)的活性,进一步提高农药处理后12~96 h的GSH/GSSG比值,及谷胱甘肽还原酶(GR)、谷胱甘肽S-转移酶(GST)活性和非蛋白结合态巯基化合物(NPT)含量。因此,外源喷施Pro可提高SOD、MDAR、CAT、POD等抗氧化酶活性,促进GSH的再生,提高GST等解毒酶活性,从而加速番茄体内百菌清的降解代谢,为促进蔬菜残留农药的降解提供一条新途径。     

丛枝真菌对Pb胁迫下烟草的解毒效应

为了探讨丛枝真菌解除Pb胁迫下烟草氧化胁迫毒性的效应,采用盆栽试验,以烟草(Nicotiana tabacum L.)品种中烟100为材料,研究Pb胁迫下接种丛枝真菌对烟草根、叶Pb含量及叶片活性氧、丙二醛、丙酮醛含量及清除活性氧的抗氧化酶活性、酮醛转位酶活性和还原型谷胱甘肽含量的影响。结果表明,接种丛枝真菌后与同质量分数Pb胁迫相比,烟草根中Pb含量显著增加,叶片中Pb含量减少;叶片中过氧化氢含量和超氧阴离子产生速率、丙二醛含量均显著下降,超氧化物歧化酶、过氧化氢酶、抗坏血酸过氧化物酶、过氧化物酶活性相应降低;酮醛转位酶I的活性升高,还原型谷胱甘肽含量增加。接种丛枝真菌可减少烟草叶片中的氧化胁迫,降低叶片Pb含量,提高烟叶质量。     

四种真菌基因组编码的细胞壁降解酶生物信息学分析

为了比较病原性真菌和非病原性真菌基因组编码的植物细胞壁降解酶,用BLAST(Basic Local Alignment Search tool)结合人工二次筛选的方法确定了四种真菌基因组编码的多种植物细胞壁降解酶,并用SignalP来判断细胞壁降解酶能否被分泌到细胞外.结果表明病原性真菌和非病原性真菌基因组编码的植物细胞壁降解酶在种类和数量上存在着明显的差异,其中E3(endoglucanase Ⅱ)仅由病原性真菌基因组编码,而且能够被分泌到细胞外,可能与病原性真菌对植物的致病性有着密切的关系.     

植物病原真菌产漆酶菌株的筛选

 【目的】从常见植物病原真菌中筛选具有漆酶活性的病原菌,对相对酶活力较高的菌株进行漆酶致病力测定,为深入研究漆酶在植物病原真菌致病过程中所发挥的作用及病菌的扩展机理奠定基础。【方法】以2,2’-连氮-双[3-乙基苯并噻唑-6-磺酸](ABTS)为底物,通过平板显色反应筛选得到产漆酶的病原菌,同时利用苯胺蓝(Azure-B)脱色法确定目标菌株降解木质素作用,采用分光光度计法在420 nm下测定漆酶活性,最后利用组织病理学分析漆酶对病原菌致病力的影响。【结果】从20种重要植物病原真菌中筛选得到10种产漆酶的病原菌,且均具有降解木质素的作用,对其中6种颜色变化不同的病原菌进行漆酶活力测定,发现玉米大斑病菌的胞内漆酶活力最高,为18.984 U?mL-1,小孢拟盘多毛孢的胞外漆酶活力最高,为0.919 U?mL-1。致病力测定表明,在玉米大斑病菌的致病过程中,漆酶可以氧化玉米叶片并能够促进病原菌在寄主组织中的扩散。【结论】在产漆酶的植物病原真菌中,漆酶大多以胞内酶形式存在,并具有降解木质素的作用,漆酶可以促进玉米大斑病菌在寄主组织中扩展。     

乙醇酸氧化酶基因超表达载体的构建与水稻的遗传转化

通过RT-PCR从水稻cDNA中扩增得到乙醇酸氧化酶基因(GLO)的完整序列。构建组成型超表达载体并转化水稻,经初步PCR和Southern杂交检测,确认已得到超表达转基因植株。酶活检测表明,其GLO活性最高可提高约70%。GLO基因超表达植株的获得为进一步研究GLO以及光呼吸的生理功能奠定了基础。     

纤维素高效降解真菌-细菌复合系的筛选及其分解特征

真菌-真菌或细菌-细菌复合系可提高纤维素的降解,但人们对真菌与细菌组成复合系的降解情况知之甚少.本文以真菌(LF1,LF3)和细菌(DS6,DS9)各2株组合形成11个复合系,通过比较单一菌种及各复合系在常温20℃下的羧甲基纤维素(CMC)酶活和滤纸糖酶(FPA)酶活来分析分解特征,进而确定高效降解复合系.结果表明,CMC酶活和FPA酶活随时间的变化规律基本一致,单一菌种中LF3最大CMC酶活(Mc)和最大FPA酶活(Mf)为最高,分别为1.99 U和1.18 U;除LF3与细菌组成的复合系之外,其余复合系的最大酶活均明显高于其所含菌种单一培养时的,复合系L13(含LF1和LF3)的CMC和FPA酶活均于168h达到最大,且在所有复合系中其Mc和Mf最高,分别达到了3.95 U和2.23 U;复合系D6L13(含DS6、LF1和LF3)的Mc和Mf仅次干L13,分别达到了3.54 U和1.82U,出现时间分别为168h和144h.     

链状亚历山大藻赤潮衰亡的生理调控

研究了链状亚历山大藻在对数生长期、衰亡期、高氮、低氮条件下,藻细胞中可溶性蛋白含量、超氧化物歧化酶(SOD)活性、丙二醛(MDA)、过氧化氢(H₂O₂)和还原型谷胱甘肽(GSH)含量、光合速率和呼吸速率、DNA降解、端粒酶活性的变化。结果表明:在衰亡期、高氮、低氮条件下链状亚历山大藻细胞中可溶性蛋白、GSH含量、光合速率和呼吸速率下降;SOD活性(低氮条件除外)、H₂O₂、MDA含量上升;端粒酶活性和DNA Ladder随着藻细胞生长而变化,并在衰亡时期,出现了明显的DNA Ladder。研究结果显示链状亚历山大藻衰亡过程的反应表现为:蛋白质合成受阻或降解,产生大量氧化中间产物(MDA,H₂O₂等),抗氧化系统被激活,GSH等非酶抗氧化物质被大量消耗,SOD等酶抗氧化物被激活;另外表现为光合速率和呼吸速率下降;同时活性氧自由基(Reactive Oxygen Species, ROS)的积累诱发了细胞凋亡,核酸内切酶被激活,选择性降解染色质DNA。推测低氮、高氮条件均可以加快藻细胞的衰亡的生理过程,链状亚历山大藻的赤潮衰亡是一种有序的死亡过程。     

Coordinate induction of antioxidant defense and glyoxalase system by exogenous proline and glycinebetaine is correlated with salt tolerance in mung bean

The purpose of this study was to assess the synergistic effects of exogenously applied proline and glycinebetaine (betaine) in antioxidant defense and methylglyoxal (MG) detoxification system in mung bean seedlings subjected to salt stress (200 mmol·L⁻¹ NaCl, 48 h). Seven-day-old mung bean seedlings were exposed to salt stress after pre-treatment with proline or betaine. Salt stress caused a sharp increase in reduced glutathione (GSH) and oxidized glutathione (GSSG) content in leaves, while the GSH/GSSG ratio and ascorbate (AsA) content decreased significantly. The glutathione reductase (GR), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glyoxalase II (Gly II) activities were increased in response to salt stress, while the monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT) and glyoxalase I (Gly I) activities sharply decreased with an associated increase in hydrogen peroxide (H₂O₂) and lipid peroxidation level (MDA). Proline or betaine pre-treatment had little influence on nonenzymatic and enzymatic components as compared to those of the untreated control. However, proline or betaine pre-treated salt-stressed seedlings showed an increase in AsA, GSH content, GSH/GSSG ratio and maintained higher activities of APX, DHAR, GR, GST, GPX, CAT, Gly I and Gly II involved in ROS and MG detoxification system as compared to those of the untreated control and mostly also salt-stressed plants with a simultaneous decrease in GSSG content, H₂O₂ and MDA level. These results together with our previous results suggest that coordinate induction of antioxidant defense and glyoxalase system by proline and betaine rendered the plants tolerant to salinity-induced oxidative stress in a synergistic fashion.     

蒙古沙冬青根围土壤AM真菌PCR-DGGE分析

分子学方法在研究AM真菌多样性中有着重要作用。为了寻找研究蒙古沙冬青根围土壤AM真菌群落的合适引物,选取3对常用引物进行试验,最终选取2对不同引物AM1/NS31和AMV4.5NF/AMDGR,利用DGGE技术研究内蒙古阿拉善和乌拉特后旗2个样地蒙古沙冬青根围土壤AM真菌多样性及其与土壤因子的相关性,阐明极端干旱环境下AM真菌空间分布及土壤因子的生态作用。结果表明:(1)AM1/NS31引物能够更有效扩增样本AM真菌,表现出丰富的DGGE条带数目。(2)2个样地AM真菌群落结构在种类组成上有较高相似性,不同样地和土层AM真菌群落差异性主要体现在丰富度和优势度上。同一样地,0~20cm土层AM真菌丰富度高于20~40cm土层;同一土层,阿拉善样地AM真菌丰富度更高。不同样本AM真菌优势度有较大差异,Rhizophagus intraradices在所有样本中均有较高优势度。(3)土壤速效P、速效N、速效K和有机质对于AM真菌分布有显著影响。     

Comparative analysis of protein kinases and associated domains between Ascomycota and Basidiomycota

Protein kinases play an important role in every aspect of cellular life. In this study, we systemically identified protein kinases from the predicted proteomes of 59 representative fungi from Ascomycota and Basidiomycota. Comparative analysis revealed that fungi from Ascomycota and Basidiomycota differed in the number and variety of protein kinases. Some groups of protein kinases, such as calmodulin/calcium regulated kinases (CMGC) and those with the highest group percentages are the most prevalent protein kinases among all fungal species tested. In contrast, the STE group (homologs of the yeast STE7, STE11 and STE20 genes), was more abundant in Basidiomycetes than in Ascomycetes. Importantly, the distribution of some protein kinase families appeared to be subphylum-specific. The tyrosine kinase-like (TKL) group had a higher protein kinase density in Agaricomycotina fungi. In addition, the distribution of accessory domains, which could have functional implications, demonstrated that usage bias varied between the two phyla. Principal component analysis revealed a divergence between the main functional domains and associated domains in fungi. This study provides novel insights into the variety and expansion of fungal protein kinases between Ascomycota and Basidiomycota.     

The Metabolic Responses of Aspergillus flavus to N-Acetylcysteine, Ascorbate, and H2O2

Aflatoxin, the secondary metabolite of Aspergillus flavus and A. parasiticus, is the most toxic product in nature. In this study, N-acetylcysteine （NAC）, ascorbate, and H2O2 were used to ascertain their effects on fungal metabolic response of A. flavus. The results demonstrated that NAC did not affect fungal growth, but inhibited the aflatoxin Bt production, with the concomitant sporulation reduction. NAC increased the ratio of reduced glutathione （GSH） and oxidized glutathione （GSSG）, but decreased the activity of glutathione reductase （GR）. Ascorbate had similar effect on fungal growth, sporulation, and GR activity, but GSH/GSSG and total glutathione （tGSH, including GSH and GSSG） were significantly increased. H2O2 at high concentration （5 mM） inhibited fungal growth, but the aflatoxin production was increased. At the same time, it reduced GR activity and enhanced tGSH. Though reductive agents had different effects on GSH metabolism, reductive conditions inhibited aflatoxin production and sporulation without any effect on fungal growth. The results in this report confirmed that the relationship between oxidative stress and aflatoxin production is theoretically important in controlling aflatoxin contamination.     

Variations on conserved signaling pathways in biocontrol and development:G protein and MAPK genes of Trichoderma. atroviride and T. virens

Filamentous fungi employ conserved eukaryotic signaling pathway to detect and respond to environmental signals, including the presence of the host. Genetic experiment in which a particular signaling protein is lost, or its activity enhanced, have defined some of the function of heterotrimeric G proteins and MAP kinases in development and virulence. A hallmark of these studies is that orthologs in different species may have different functions. Antagonistic fungal-fungal interactions form …     

玉木耳漆酶基因Aclac的克隆及原核表达

【目的】对玉木耳漆酶基因Aclac进行克隆及原核表达分析,为深入研究漆酶基因在玉木耳子实体发育中的生物学功能提供理论依据。【方法】克隆Aclac基因的cDNA和DNA全长序列,对其进行生物信息学分析,并将目的基因连接至pET-28a质粒上以构建原核表达载体pET28a-Aclac,将其转化大肠杆菌BL21（DE3）中诱导表达。【结果】克隆获得的Aclac基因cDNA序列长度为1734 bp,编码577个氨基酸残基;Aclac基因DNA序列长度为2521 bp,含14个内含子。AcLAC蛋白理论分子量为64.75 kD,理论等电点为5.70,不稳定指数为34.01,无跨膜区域,存在1个信号肽,在4个铜离子结合区高度保守,且含有Cupredoxin超家族蛋白保守功能域。AcLAC蛋白与真菌的漆酶蛋白聚为一支,其中,AcLAC蛋白与木耳属真菌的漆酶蛋白亲缘关系最近。AcLAC融合蛋白在大肠杆菌BL21（DE3）表达宿主中成功表达,分子量为67 kD。【结论】克隆获得的Aclac基因属于Cupredoxin超家族基因,可在原核表达系统中异源表达,推测其与其他真菌漆酶基因在生物学功能上具有一致性,丰富了真菌漆酶资源。     

苍耳提取液与植物内生真菌抗菌活性研究

[目的]为研制高效、低毒、低残留的新型杀菌剂提供理论依据。[方法]采用生长速率法和抑菌圈法,分别以水稻纹枯病菌等6种植物病原真菌和大肠杆菌等3种病原细菌为供试菌种,对苍耳各器官(根、茎、叶、果)提取液、植物内生真菌MG4-23的发酵液及菌丝体提取液进行抑菌活性测定。[结果]苍耳各器官提取液、植物内生真菌发酵液及菌丝体提取液均有抑菌作用。苍耳的丙酮提取液抑菌活性要好于乙醇提取液。苍耳中抑菌活性最高的器官是叶,对6种供试病原真菌的抑制率均在47.8%以上,对3种病原细菌的抑制直径均在0.5 cm以上。植物内生真菌发酵液的抑菌活性也很明显,对6种供试病原真菌的抑制率均在68.1%以上,对3种病原细菌也具有抑制性。苍耳根和植物内生真菌菌丝体提取液的抑菌活性也较明显。[结论]苍耳和植物内生真菌MG4-23均具有较大的开发利用价值。     

薄荷中内生真菌的筛选和初步研究

[目的]从薄荷中筛选出能产生活性物质的内生真菌。[方法]采用组织分离法从薄荷中分离出3株不同的内生真菌P-1、P-2、P-3,对其进行不同pH值梯度培养,运用滤纸片法和对峙培养法,研究它们的抗菌活性。[结果]3株内生真菌在pH值6.5~7.5出现生长最高峰,在pH值4.0~8.5都可以生长,酸碱适应性很强,其中P-3的适应性最强。P-1和P-2对枯草芽孢杆菌、大肠杆菌、金黄色葡萄球菌都有较强的抑制能力,而P-3只对大肠杆菌有很微弱的抑制能力。3株内生真菌对苹果锈病病原菌、苹果白粉病病原菌、苹果落叶病病原菌都有抑制作用,其中P-1的抑制能力较强。[结论]该研究获得了具有抗菌活性的内生真菌。     

在美国使用无纺布菌条防治光肩星天牛的前景

An invasive long-horned beetle,Anoplophora glabripennis,was first reported in the northeastern and midwestern United States and eastern Canada between 1996 and 2004 and has been given the common name Asian longhorned beetle (ALB). This beetle has also been found in several countries in Europe.ALB is difficult to control because larvae are found within the wood of living trees and the long-lived adults often occur high in tree canopies.This species is native to China and Korea and,because it has been a major tree killer in China,government agencies in the U.S.and Canada are working to eradicate ALB from North America.Our laboratory has been developing a microbial control approach targeting ALB adults,based on the Japanese product Biolisa Kamikiri which is used to control cerambycids in orchards.Entomopathogenic fungi are grown within non-woven fiber bands (= fungal bands) and placed around tree trunks and branches where ALB adults become inoculated when walking across bands. We have conducted bioassays with Beauveria brongniartii,Beauveria bassiana and Metarhizium anisopliae against ALB larvae and adults to identify effective isolates and now focus our efforts on M.anisopliae F52 (ARSEF 7711). Caged field trials conducted in China to compare fungal sprays with fungal bands (2000, 2001) demonstrated decreased ALB longevity and fitness for both application methods but longer activity of fungi in cages treated with fungal bands compared with sprays.Uncaged field trials (2001, 2002) yielded faster ALB adult mortality in fungal-treated plots and decreased fitness. Studies in New York City testing the longevity of activity of fungal bands in the field have documented that bands retain＞1×107 conidia·cm-2 (the threshold for activity of Biolisa Kamikiri) for over 3 months.In contrast, studies with unformulated conidia sprayed onto tree trunks in New York documented conidial survival of only a few days.Sublethal effects of exposure of adult female ALB to fungal bands have been investigated further in the laboratory.After either newly eclosed or reproductively active females are exposed to fungal bands,few viable larvae are produced before death of the females.When females are exposed to fungal bands and then caged with males,males become infected.