A 13-kilodalton maize mitochondrial protein in E. coli confers sensitivity to Bipolaris maydis toxin

The Texas male-sterile cytoplasm (cms-T) of maize carries the cytoplasmically inherited trait of male sterility. Mitochondria isolated from cms-T maize are specifically sensitive to a toxin (BmT-toxin) produced by the fungal pathogen Bipolaris maydis, race T, and the carbamate insecticide methomyl. A mitochondrial gene unique to cms-T maize, which produces a 13-kilodalton polypeptide associated with cytoplasmic male sterility, was expressed in Escherichia coli. After addition of BmT-toxin or methomyl, inhibition of whole cell respiration and swelling of spheroplasts were observed in Escherichia coli cultures producing the novel mitochondrial protein; these effects are similar to those observed with isolated cms-T mitochondria. The amino-terminal region of the 13-kilodalton polypeptide appears to be essential for proper interaction with the BmT-toxin and methomyl. These results implicate the 13-kilodalton polypeptide in conferring toxin sensitivity to mitochondria of cms-T maize.     

小白菜线粒体DNA提取体系的建立

为了建立小白菜线粒体DNA(mtDNA)提取体系,以小白菜黄化苗为材料,研究了不同提取方法、材料选择等因素对线粒体DNA提取的影响。结果表明,经冰浴研磨小白菜黄化苗,通过简易的密度梯度离心技术快速抽提大量高纯度的线粒体,蛋白酶K裂解线粒体及有机溶剂抽提去除蛋白质,可获得纯度较高的mtDNA,能用于RAPD分析。     

Respiratory control: loss in mitochondria from diseased plants

Mitochondria from healthy oat seedlings oxidized succinate with good respiratory control and high ratios of adenosine diphosphate to oxygen. After treatment with victorin, the pathotoxin responsible for symptoms of Victoria blight of oats, susceptible seedlings yielded mitochondria with little respiratory control and lower ratios of adenosine diphosphate to oxygen. No such effects were obtained with victorin-treated resistant seedlings or when victorin was added directly to mitochondria from healthy susceptible or resistant plants. These data indicate that victorin-induced disease results in a reduction in efficiency of the energy-generating system of isolated mitochondria.     

Growth Inhibitor in Immature Soybean Seeds and 2,4-D-Sprayed Soybean Seedlings

A naturally occurring inhibitor of seed germination has been isolated by ion-exchange chromatography from soybean seeds and seedlings. The inhibitor was present in large amounts in immature seeds and in seedlings sprayed with 2,4-D. The inhibitor acted as an "uncoupler" when applied to soybean root tips or mitochondria.     

3种常用杀虫剂胁迫对小麦防御相关酶活性的影响

为明确常用杀虫剂对小麦防御性的影响,考察了吡虫啉、灭多威和氧乐果处理对小麦苗3种防御相关酶活性的影响。分别使用杀虫剂的田间推荐浓度、高于和低于其推荐浓度1倍的质量浓度3个处理,考察了吡虫啉、灭多威和氧乐果营养液内吸处理小麦苗后5d内,小麦苗中苯丙氨酸解氨酶(PAL)、谷胱甘肽硫转移酶(GSTs)和脂氧合酶(LOX)活性的变化。结果表明:17.5和35.0mg/L吡虫啉处理小麦苗后6h时,小麦苗中PAL均受到抑制,活性显著降低,处理后12h时,PAL活性与对照相比显著上升,分别上升了38.9%和40.0%;100、200和400mg/L灭多威处理小麦苗后12h时,小麦苗中PAL活性显著升高。250、500和1 000mg/L氧乐果处理小麦苗后24h内,小麦苗中PAL活性呈现出先升高再降低的动态过程。而500和1 000mg/L氧乐果处理小麦苗后48h内,小麦苗中GSTs活性呈现出先降低再升高的动态过程。在3种杀虫药剂的胁迫下,小麦苗PAL、GSTs及LOX活性均发生了随着时间而变化的波动,随着处理后时间的继续延长,最后小麦苗3种防御酶的活性又恢复至不受影响的正常状态。一定浓度的吡虫啉、灭多威和氧乐果处理小麦苗,会对小麦苗中PAL、GSTs和LOX的活性及其相关防御性产生一定影响。3种杀虫剂对小麦苗中PAL、GSTs和LOX活性的影响,不仅与杀虫药剂本身的性质有关,还具有一定的剂量效应与时间效应。     

Method for Isolating Mitochondrial DNA from Etiolated Tissue of Cabbage

Isolation of high-quality mitochondrial DNA（mtDNA） is an important premise for researching molecular mechanisms in cytoplasmic male sterility of cabbage（Brassica oleracea L.var.capitata）. An efficient protocol for separation and purification of mitochondria and extraction of mitochondrial DNA（mtDNA） from etiolated tissues of cabbage was developed. We took a method combined mannitol density gradient with differential centrifugation, selected appropriate rotational speed, extended DNase I treating time and changed mitochondria cracking condition. The results showed that the extracted mitochondria in this protocol had complete structure, appeared to ellipsoid and had not been contaminated with other impurities under the Jannus Green B staining. The isolated mitochondrial DNA had high purity and yield through detecting the optical density, nuclear specific primer PCR and agarose gel electrophoresis. The results indicated that mitochondrial DNA extracted by this protocol had high quality and enabled to be used in futher genetic studies.     

6种酚酸类物质对平邑甜茶幼苗根系线粒体及抗氧化酶活性的影响

 【目的】研究对羟基苯甲酸、间苯三酚、丁香酸、苯甲酸、咖啡酸和阿魏酸等6种酚酸类物质对平邑甜茶幼苗根系线粒体和抗氧化酶活性的影响。【方法】育苗基质栽培平邑甜茶幼苗,分别用50 mL浓度为0.8、4.0、20.0 mmol•L-1的6种酚酸类物质处理,测定平邑甜茶幼苗鲜重、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和过氧化物酶（POD）的活性、线粒体膜通透性转运孔（MPTP）开放程度、膜流动性、细胞色素Cyt c/a比值的变化。【结果】0.8、4.0、20.0 mmol•L-1的6种酚酸类物质均抑制了平邑甜茶植株生长,降低了SOD、POD、CAT活性;使线粒体MPTP开放程度增大,线粒体膜流动性降低,细胞色素Cyt c/a比值下降,使线粒体受到不同程度的损伤。与其它处理比,20.0 mmol•L-1苯甲酸处理的抑制效果最显著。【结论】0.8、4.0、20.0 mmol•L-1的6种酚酸类物质都抑制了平邑甜茶抗氧化酶活性和线粒体功能,且浓度越高抑制作用越强;苯甲酸比另外5种酚酸类物质具有更强的抑制作用。     

Ca2+对低氧胁迫下黄瓜幼苗光合效率和细胞超微结构的影响

为阐释Ca2 对低氧胁迫下黄瓜幼苗光合效率和细胞超微结构的影响,以中农8号黄瓜品种为试材,在水培条件下,研究在低氧胁迫下Ca2 与黄瓜幼苗光合效率和叶绿体超微结构的关系.结果表明:低氧胁迫下,气孔限制是导致缺钙植株光合速率(Pn)下降的主要原因,非气孔限制是导致单纯低氧胁迫和低氧高钙处理Pn下降的主要原因;低氧胁迫下,光补偿点提高,光饱和点和最大光合速率降低,高钙可以降低其变化的幅度,缺钙则增大其变化的幅度;低氧胁迫下表观量子效率变化不大,羧化效率有增加的趋势;低氧胁迫下叶绿体和线粒体受到伤害,高钙可以修复或缓解低氧胁迫对叶绿体和线粒体的伤害.总之,高钙对缓解低氧胁迫对黄瓜幼苗光合效率和细胞超微结构的影响均有良好的效应,可有效缓解低氧胁迫造成的伤害.     

Mitochondria and the autophagy-inflammation-cell death axis in organismal aging

Alterations of mitochondrial functions are linked to multiple degenerative or acute diseases. As mitochondria age in our cells, they become progressively inefficient and potentially toxic, and acute damage can trigger the permeabilization of mitochondrial membranes to initiate apoptosis or necrosis. Moreover, mitochondria have an important role in pro-inflammatory signaling. Autophagic turnover of cellular constituents, be it general or specific for mitochondria (mitophagy), eliminates dysfunctional or damaged mitochondria, thus counteracting degeneration, dampening inflammation, and preventing unwarranted cell loss. Decreased expression of genes that regulate autophagy or mitophagy can cause degenerative diseases in which deficient quality control results in inflammation and the death of cell populations. Thus, a combination of mitochondrial dysfunction and insufficient autophagy may contribute to multiple aging-associated pathologies.     

BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis

BAX and BAK are "multidomain" proapoptotic proteins that initiate mitochondrial dysfunction but also localize to the endoplasmic reticulum (ER). Mouse embryonic fibroblasts deficient for BAX and BAK (DKO cells) were found to have a reduced resting concentration of calcium in the ER ([Ca2+]er) that results in decreased uptake of Ca2+ by mitochondria after Ca2+ release from the ER. Expression of SERCA (sarcoplasmic-endoplasmic reticulum Ca2+ adenosine triphosphatase) corrected [Ca2+]er and mitochondrial Ca2+ uptake in DKO cells, restoring apoptotic death in response to agents that release Ca2+ from intracellular stores (such as arachidonic acid, C2-ceramide, and oxidative stress). In contrast, targeting of BAX to mitochondria selectively restored apoptosis to "BH3-only" signals. A third set of stimuli, including many intrinsic signals, required both ER-released Ca2+ and the presence of mitochondrial BAX or BAK to fully restore apoptosis. Thus, BAX and BAK operate in both the ER and mitochondria as an essential gateway for selected apoptotic signals.     

转无毒基因马铃薯抗晚疫病的研究

晚疫病菌(Phytophthora infestans)引起的马铃薯晚疫病是危害全球马铃薯生产的严重病害.通过基因工程方法把外源基因导入植物体内以增强抗病性被证明是一条行之有效的途径.应用植物基因工程技术将具有能激活植物自身防御系统的无毒基因与适合于植物背景、非专一性的病原物诱导启动子组合成嵌合基因构建到植物表达载体中.通过农杆菌或基因枪的介导转化植物,可筛选出高效广谱的抗真菌和细菌病害的转基因植株.本研究从病原细菌Pseudomonas syringae PV.tomato中获得的无毒基因avrD(0*93kb)和从病原真菌Phytophthora parasitica中获得的无毒基因Elicitin(0.294kb)分别与非专一性病原物诱导启动子Pill和BG组成含2个嵌合基因(Pill-avrD,BG-Elicitin)的植物表达载体pYH144和pYHEt.通过农杆菌LBA4404介导转化马铃薯,其中用pYH144载体转化2个品种(克新1号,2号),用pYHEt载体转化3个品种(Desiree,克新2号,4号),通过组织培养分别获得潮霉素(HygromycinB)标记的转基因马铃薯试管苗.将转基因试管苗扩繁,应用马铃薯脱毒微型种薯生产技术获得转无毒基因微型薯,在温室(15～25℃和湿度高)条件下,观察转无毒基因马铃薯植株中对晚疫病菌自然感染的抗性.1998年和1999年(每年的3-5月)的温室试验初步表明用avrD和elicitin基因分别转化的转无毒基因马铃薯植株都具有较明显的对晚疫病菌侵染的抗性,大部分转基因植株不表现或表现轻微的感病症状,对照植株(未转化)则表现明显的感病症状.转基因植株生长正常,且在感染后期(恢复期)生长良好.对照植株在恢复期生长弱和缓慢.在获得较多数量的转无毒基因马铃薯微型种薯的时候,将进行人工接种晚疫病菌和田间种植试验,从中筛选出抗真菌病和细菌病的转基因马铃薯株系.     

mtDNA A3243G 点突变小鼠模型的建立及其致病机制探讨

    利用显微注射线粒体技术建立转人线粒体小鼠模型,研究外源突变mtDNA在不同组织的分布及遗传规律,探讨mtDNA A3243G点突变对线粒体功能的影响.从健康成人及2型糖尿病患者(携带mtDNA 3243A-G突变)血液标本中分离有活性的线粒体,将其显微注射至小鼠受精卵,胚胎移植,产出仔鼠后利用分子生物学方法检测人mtDNA及mtDNA A3243G点突变.获得嵌合体小鼠后,对其空腹血糖和全血乳酸进行测定,并使用荧光法和比色法分析A3243G点突变小鼠重要脏器组织细胞活性氧生成量(ROS)、线粒体复合酶Ⅰ和Ⅳ活力及线粒体ATP合成活力的变化.研究结果显示:在1只雌性(转健康人线粒体)和2只雄性小鼠(转患者线粒体)中检测到人mtDNA,其中2只雄性小鼠携带mtDNA 3243A-G突变;将嵌和体雌鼠与野生型C57BL/6J 雄鼠交配后,在1只后代仔鼠中检测到人mtDNA;人mtDNA仅在嵌合小鼠的部分组织中表达.在含有mtDNA A3243G突变的组织中发现,线粒体复合酶Ⅰ、Ⅳ活力降低,ATP合成速率下降,ROS水平升高,说明A3243G点突变能损伤线粒体正常功能从而导致疾病的发生.综上所述,本研究利用显微注射法成功建立了嵌和小鼠,引入了致病性的点突变,为线粒体疾病的研究提供了良好的思路.     

Hypoxic coordinate regulation of mitochondrial enzymes in mammalian cells

The effect of hypoxic exposure on various mitochondrial enzymes and on cell mitochondrial genomic content was studied in two types of mammalian cells. Hypoxia depressed the activity of six enzymes to the same degree. The kinetics of depression and of recovery during reexposure to normoxia were statistically similar for three marker enzymes. Despite the global and symmetrical decrease in enzyme activities, mitochondrial DNA remained constant. This suggests either symmetrical loss of mitochondrial enzymes from all mitochondria or complete loss of enzymes from a subpopulation of mitochondria with retention of an intact mitochondrial genome.     

Mitochondrial autonomy: incorporation of monosaccharides into glycoprotein by isolated mitochondria

Isolated intact mitochondria selectively incorporate monosaccharides from nucleotide diphosphate monosaccharides into protein. Fucose, mannose, glucose, and galactose were incorporated by the mitochondria into glycoprotein; xylose was not. Structural integrity of the mitochondria was not necessary for the incorporation of monosaccharide into glycoprotein; mitochondria broken by homogenization also incorporated monosaccharide. The monosaccharides incorporated into glycoprotein were localized in the inner mitochondrial membranes, the same membranes which contain the protein into which leucine is incorporated by the isolated mitochondria.     

Role of the ABC transporter Mdl1 in peptide export from mitochondria

ATP-binding cassette (ABC) adenosine triphosphatases actively transport a wide variety of compounds across biological membranes. Here, the ABC protein Mdl1 was identified as an intracellular peptide transporter localized in the inner membrane of yeast mitochondria. Mdl1 was required for mitochondrial export of peptides with molecular masses of approximately 2100 to 600 daltons generated by proteolysis of inner-membrane proteins by the m-AAA protease in the mitochondrial matrix. Proteolysis by the i-AAA protease in the intermembrane space led to the release of similar-sized peptides independent of Mdl1. Thus, two pathways of peptide efflux from mitochondria exist that may allow communication between mitochondria and their cellular environment.     

酶抑制剂与杀虫剂混配对黄曲条跳甲抗药性的抑制作用

研究了磷酸三苯酯(TPP)、胡椒基丁醚(PB)和顺丁烯二酸二乙酯(DEM)3种酶抑制剂对黄曲条跳甲Phyllotretastriolata的杀虫剂敏感性的影响.结果表明:TPP对甲胺磷、敌敌畏、灭多威、氰戊菊酯、氯氰菊酯、氟虫腈和阿维菌素均有显著增效作用;除对敌敌畏的增效作用低于PB外,TPP对其余6种杀虫剂的增效作用均高于PB和DEM;PB对甲胺磷、敌敌畏、灭多威、氰戊菊酯和氯氰菊酯,DEM对甲胺磷、敌敌畏和氯氰菊酯的增效比均大于2.0,但PB对甲胺磷、灭多威以及DEM对甲胺磷、敌敌畏的增效作用不显著;PB对氟虫腈和阿维菌素以及DEM对灭多威、氰戊菊酯、氟虫腈和阿维菌素的增效作用较低或不明显;辛硫磷、毒死蜱和氟虫腈对黄曲条跳甲的毒力远大于甲胺磷、敌敌畏、灭多威、氰戊菊酯和氯氰菊酯.分别采用辛硫磷、毒死蜱和氟虫腈与氰戊菊酯或氯氰菊酯混配后的增效作用显著.故采用酶抑制剂与杀虫剂混配可有效抑制黄曲条跳甲对一些杀虫剂的抗性.     

渗透胁迫对玉米幼苗根尖线粒体ATPase活性与结合态多胺含量的影响

[目的]探讨线粒体膜上结合态多胺与渗透胁迫的关系。[方法]以抗旱性不同的玉米(Zea maysL.)品种农大108(抗旱性较强)和掖单13(抗旱性较弱)幼苗为材料,研究在聚乙二醇(PEG)-6000渗透胁迫下,幼苗根线粒体ATPase水解活性与膜上非共价结合态亚精胺(Spd)含量的关系。[结果]渗透胁迫条件下,抗性强的农大108的ATPase活性下降的幅度明显小于抗性弱的品种掖单13,而农大108的线粒体膜上非共价结合亚精胺的上升幅度明显大于掖单13。外源Spd处理,明显促进了掖单13在胁迫条件下非共价结合Spd含量的增加,同时抑制其在胁迫条件下ATPase活性的降低,从而提高了掖单13幼苗的抗性;Spd生物合成的专一性抑制剂——甲基乙二醛-双(鸟嘌呤腙)(MGBG)处理,显著抑制了农大108在胁迫条件下膜上非共价结合Spd的增加,同时促进其在胁迫条件下ATPase活性的降低,从而明显降低了农大108幼苗的抗性。[结论]线粒体膜上的非共价结合的Spd可能通过维持ATPase活性的稳定来提高玉米幼苗的抗渗透胁迫能力。     

基于线粒体能量代谢途径的金针菇采后纳米包装保鲜机制

【目的】从线粒体水平上揭示不同包装对金针菇能量代谢的影响,为进一步揭示纳米包装保鲜机制提供新思路。【方法】以金针菇为材料,对比不连续密度梯度离心法（DDGCM）、酵母线粒体提取试剂盒方法（YMEKM）和改进普利莱法（IPKM）3种提取方法对金针菇子实体与菌丝体中线粒体功能活性的影响,确定最佳提取方法。通过测定不同包装及不同贮藏期金针菇线粒体中ATP代谢系统物质含量（ATP、ADP、AMP及能荷）和线粒体主要复合体活性,揭示不同包装金针菇采后能量代谢规律。【结果】通过标志性污染物酶和线粒体呼吸速率指标测定,3种方法以IPKM提取的线粒体内乙醇脱氢酶活性及胞液内细胞色素C氧化酶活性最低,呼吸速率最高,证明IPKM所提的线粒体结构完整性最好;IPKM提取的线粒体超氧化物歧化酶活性最高,达到17.82 U·mg^-1 pro,比DDGCM-1和YMEKM分别高7.31%和25.59%。结合透射电镜和健那绿染色结果可知,IPKM提取的线粒体结构相对完整,活性线粒体数量最多。在金针菇冷藏期间,纳米包装金针菇的ATP水平、能荷含量以及线粒体主要复合体活性都显著高于普通PE包装。纳米包装材料通过维持高水平的ATP含量,延缓能荷值的下降,促进了线粒体的氧化磷酸化过程。纳米包装组在第21天的ATP含量仍然比普通PE包装组高113.83 μg·g^-1FW,从而避免金针菇线粒体复合体I、Ⅲ活性降低,减缓线粒体复合体Ⅳ的活性降低。普通PE包装金针菇的线粒体复合体Ⅳ活性在第9天达到第一个峰值,而纳米包装金针菇线粒体复合体IV活性在第15天达到第一个峰值（5.14 U·mg^-1 pro）,显著高于普通PE包装（1.12 U·mg^-1 pro）,相差达到78.23%。证明纳米包装能有效控制线粒体呼吸链中的电子传递不受阻碍,保证细胞能量供应,从而更好地保持金针菇的能量状态。【结论】改进普利莱法对线粒体损伤最小,并维持线粒体活性,适合金针菇采后线粒体能量代谢研究。同时,纳米包装通过维持较高水平的ATP含量与线粒体复合体I、Ⅲ活性并延缓能荷与线粒体复合体Ⅳ活性的下降,进而保持金针菇的能量状态并延缓衰老,延长贮藏期。     

ER tubules mark sites of mitochondrial division

Mitochondrial structure and distribution are regulated by division and fusion events. Mitochondrial division is regulated by Dnm1/Drp1, a dynamin-related protein that forms helices around mitochondria to mediate fission. Little is known about what determines sites of mitochondrial fission within the mitochondrial network. The endoplasmic reticulum (ER) and mitochondria exhibit tightly coupled dynamics and have extensive contacts. We tested whether ER plays a role in mitochondrial division. We found that mitochondrial division occurred at positions where ER tubules contacted mitochondria and mediated constriction before Drp1 recruitment. Thus, ER tubules may play an active role in defining the position of mitochondrial division sites.     

钱塘江三角鲂线粒体基因组测序及其结构特征分析

为了研究钱塘江流域三角鲂(Megalobrama terminalis Richardson)线粒体基因组结构特征及鲌亚科鱼类的系统进化关系,通过PCR扩增、测序、软件拼接获得了钱塘江三角鲂线粒体基因组全序列,GenBank登录号为MN725725。结果表明,钱塘江三角鲂线粒体基因组序列全长为16 621 bp,碱基组成分别为A(31.23%)、G(16.17%)、C(27.87%)和T(24.73%);共有13个蛋白编码基因,22个tRNA基因,2个rRNA基因,NAD6、tRNA-Gln、tRNA-Ala、tRNA-Asn、tRNA-Cys、tRNA-Tyr、tRNA-Ser^(UCN)、tRNA-Glu和tRNA-Pro等基因编码在L链上,其余基因均在H链上编码。钱塘江三角鲂线粒体基因组全序列与蛋白编码基因的A+T含量分别为55.97%和55.86%,具有明显的AT偏好性。线粒体基因中存在2个散在重复序列,分别位于线粒体控制区中终止结合序列区的前端和控制区3'的末端。在22个tRNA基因中,除了tRNA-Ser^(AGY)外,均具有典型的三叶草二级结构。基于BLAST比较,钱塘江三角鲂与黑龙江流域的三角鲂一致性为99.76%,与珠江流域三角鲂一致性为99.87%。基于15种隶属于7属的鲌亚科鱼类线粒体基因组全序列构建的系统进化树,鲂属与鳊属亲缘关系比与鲌属的亲缘关系上较近,与属、半属和细鳊属亲缘关系上较远。