形成素结合蛋白调节粗糙脉孢菌菌丝极性生长发育

微丝骨架在真菌菌丝极性生长中具有重要的功能,而其动态解聚 聚合特性是其实现功能的前提.形成素作为肌动蛋白结合蛋白,是微丝骨架动态调控因子之一,而形成素结合蛋白对于形成素发挥功能非常关键,但是对其在真菌极性生长发育中的功能还未见报道.本文以丝状真菌粗糙脉孢菌为材料,利用同源重组基因敲除技术,通过电击转化、分生孢子过膜以及PCR鉴定的方法,获得了形成素结合蛋白基因缺失突变菌株（FBPKO）.进一步利用平板生长方法并结合细胞壁染色对突变菌株的表型进行分析. 结果显示, 与野生型相比,在分生孢子接种后24 h内突变菌株FBPKO的菌丝生长明显减慢且分支异常.这些结果表明, 形成素结合蛋白调节着粗糙脉孢菌菌丝早期的极性生长发育.     

粗糙脉孢菌26 S蛋白酶体三个亚基缺失菌株的构建及表型分析

【目的】通过构建26S蛋白酶体的3个亚基RPN4、RPN7、RPN10的缺失突变菌株,研究这些缺失突变体的表型,进而探究这3个亚基在蛋白酶体中的作用。【方法】采用同源重组基因敲除技术、电转化、粗糙脉胞菌杂交、子囊孢子萌发及PCR鉴定等方法分别获得3个调节亚基的基因缺失突变体。利用racetube和平板生长法进行突变体表型检测。【结果】得到rpn4和rpn10的缺失突变纯合体及rpn7缺失突变异核体菌株。【结论】与野生型相比,rpn7KO(ku70RIP背景)突变体的菌丝生长及产生分生孢子的能力显著减弱;rpn4KO突变体在生长初期的菌丝生长缓慢,而后期的产孢能力与野生型无显著差异;rpn10KO突变菌株的表型介于上述两种突变体的表型之间。这些结果表明26S蛋白酶体的这3个亚基对脉胞菌的生长和发育至关重要。     

裂殖酵母Cnb1参与胞质分裂过程

丝/苏氨酸特异性钙调磷酸酶(Calcineurin, CN)是一种在真核生物中广泛存在的蛋白, 是参与转录调控的重要分子。裂殖酵母中的CN是由催化亚基Ppb1和调节亚基Cnb1组成的异源二聚体。文章报道了裂殖酵母中cnb1+的缺失引起细胞生长速度缓慢, 产生多隔膜现象, 胞质分裂受阻滞。胞质分裂过程中, Cnb1与Ppb1组成CN复合物, 与收缩环在分裂平面上共定位, 并与收缩环一起收缩。cnb1Δ菌株的隔膜成熟过程存在缺陷, 微管出现纵穿隔膜的现象。上述结果说明Cnb1可能参与隔膜的成熟过程。此外, 还检测了cnb1D菌株中胞裂蛋白的信号。胞裂蛋白包括Spn1、Spn2、Spn3和Spn4, 它们是引导隔膜降解的重要分子。结果显示, 在cnb1D菌株中, 80%左右的细胞在隔膜处缺失Spn2和Spn3的信号, 20%左右的细胞缺失Spn1和Spn4的信号。由于胞裂蛋白的蛋白表达量在cnb1D中没有降低, 因此胞裂蛋白信号的消失不是转录缺陷引起的, 这暗示Cnb1可能采用了不依赖转录的方式来调控胞裂蛋白环的稳定性。以上结果提示, Cnb1可能通过影响隔膜的成熟及胞裂蛋白环的稳定性参与调节裂殖酵母的胞质分裂过程。     

花粉粒和花粉管中的微丝骨架

自从1972年Franke等第一次报道大君子兰和麝香百合花粉管中存在直经6nmn的肌动蛋白微丝以来,近十几年来这方面的研究工作已迅速展开,积累了丰富的研究资科。借助于荧光探针、免疫荧光标记、透射电镜等技术手段,已先后对三十几种植物的花粉粒和(或)花粉管的微丝做过观察,揭示了花粉萌发和花粉管生长过程中肌动蛋白微丝的三维结构及其在时间和空间上变化的规律,并对微丝在花粉萌发和花粉管生长的生理活动中的重要作用获得了比较一致的认识。     

丝裂原活化蛋白激酶基因CfMKK1调控果生炭疽菌的生长发育和致病力

【目的】由果生炭疽菌引起的炭疽病是油茶的主要病害,造成油茶产量下降。本文研究果生炭疽菌中丝裂原活化蛋白激酶CfMkk1的生物学功能,旨在为解析油茶炭疽病菌的致病机理提供依据。【方法】根据同源重组原理构建CfMKK1基因敲除载体片段,采用PEG介导法将载体导入原生质体中筛选获得突变体菌株DCfmkk1;PCR扩增果生炭疽菌含有启动子的CfMKK1基因回补片段,构建回补载体pYF11::CfMKK1;采用PEG介导法把回补载体转化至突变体的原生质体中,荧光筛选回补菌株ΔCfmkk1-C。测定野生型菌株、突变体菌株DCfmkk1及基因回补菌株ΔCfmkk1-C在营养生长、附着胞形成、胁迫应答和致病力等生物学表型。【结果】与野生型和回补菌株相比,CfMKK1基因敲除突变体ΔCfmkk1菌丝生长速率明显减缓;在含刚果红的PDA培养基上菌丝生长受到明显抑制,无法穿透玻璃纸,丧失了侵染寄主的能力;而且无法形成附着胞。【结论】研究结果表明CfMKK1基因参与调控油茶果生炭疽菌的生长发育、附着胞形成、致病力以及响应外界胁迫过程。     

基于报告基因敲入构建果生刺盘孢细胞核荧光标记菌株

运用基因敲入技术,将GFP、mCherry整合到果生刺盘孢 Colletotrichum fructicola组蛋白histone H1位点,实现融合表达,获得细胞核荧光标记菌株。基于标记菌株可以对分生孢子、营养菌丝、附着胞、侵染菌丝等结构中的细胞核进行实时活体观察。果生刺盘孢存在性亲和的“+”、“-”型菌株分化,GFP“+”型菌株和mCherry“-”型菌株接触形成明显杂交线,杂交线上单子囊内含红绿两种孢子,表明“+”、“-”型菌株间发生杂交;杂交线上子囊壳壁表达mCherry,表明由“-”型菌株发育而来。本研究构建的核荧光标记菌株将是研究果生炭疽菌细胞周期调控和有性繁殖过程的重要材料。     

小麦条锈菌胞间菌丝的超微结构和细胞化学研究

本文应用电镜技术和细胞化学方法,对小麦条锈菌寄主胞间菌丝的超微结构进行了研究。观察发现:小麦条锈菌胞间菌丝有两种类型,即具隔膜菌丝和无隔膜菌丝。在胞间菌丝中,多核现象极为普遍。常规染色和细胞化学染色结果表明:胞间菌丝的细胞壁由四层组成,隔膜由三层构成,细胞壁的内层与隔膜的外层相连,细胞壁和隔膜中含有蛋白质和多糖物质。隔膜的发育可分三个阶段,即隔膜突的形成,隔膜壁的延伸和隔膜孔结构的形成。本研究中还观察到胞间菌丝间的融合现象。本文的研究结果表明:小麦条锈菌胞间菌丝的一些特征显然不同于其它锈菌。     

将盘基网柄菌用于本科细胞生物学综合性实验教学初探——电穿孔转化绿色荧光蛋白

盘基网柄菌(Dictyostelium discoideum)是一个应用广泛的模式生物,非常适合用来研究胞质分裂、细胞运动、吞噬作用、趋化性、趋电性、信号转导以及个体发育过程中的细胞分化。该实验主要介绍电穿孔技术转化绿色荧光蛋白标记肌动蛋白基因质粒(Lifeact-GFP)进入盘基网柄菌活细胞中,抗性筛选(潮霉素B)获得阳性克隆子,最后借助荧光显微镜观察绿色荧光蛋白标记的微丝在盘基网柄菌的分布情况。综合性实验训练可提高学生的学习兴趣和综合运用理论知识的能力,进而可培养学生的科学研究思维。     

天麻抗真菌蛋白对木霉菌丝的作用位点

天麻抗真菌蛋白(Gastrodia Antifungal Protein,GAFP)能强烈抑制腐生真菌菌丝的生长,在天麻限制和防止蜜环菌［Armillariella mellea (Vahl. ex Fr.)Karst.］侵染球茎的防卫机制中起重要作用。本文报告GAFP抗菌机理研究的部分内容——GAFP对木霉菌丝的作用位点。用荧光试剂异硫氰酸荧光素(Fluorescein isothiocyanate,FITC)标记GAFP,试验表明,标记后的GAFP与未标记的GAFP对木霉菌丝生长均有抑制作用。在荧光显微镜下观察GAFP在木霉菌丝上的作用位点,发现被“标记GAFP”作用后的菌丝边缘有荧光,并主要集中在木霉菌丝的顶端和菌丝横隔处,表明GAFP对木霉的作用位点在菌丝的细胞壁上。     

三唑酮对玉米弯孢病菌超微结构和细胞化学的影响

三唑酮(triadimenfon)属于麦角甾醇类生物合成抑制剂(ergosterol biosynthesis inhibitors．EBI),具有较广的抗真菌谱,明确其对玉米弯孢菌发育的影响可为该杀菌剂的田间应用提供理论依据。利用电镜技术和细胞化学技术观察的结果表明,玉米率孢菌经三唑酮处理后,菌丝生长明显受到抑制,表现为菌落生长速度减慢、菌丝分枝增多,且不观则地肿大和缢缩,出现许多瘤状突起,处理菌丝明显畸形。透射电镜观察结果表明,三唑酮可引起菌丝细胞壁不规则增厚,特别是菌丝顶端细胞壁增厚尤为明显:菌丝细胞隔膜发育受阴而表现畸形;菌丝细胞外有大量电子染色深的外渗物质。细胞化学标记定位结果表明,真菌细胞壁主要成分β-1,3-葡聚糖和几丁质的含量在药剂处理后发生很大变化,其标记密度明显低于未处理的对照菌丝,表明病菌细胞壁的结构和功能受到明显的不利影响。论文对弯孢菌受三唑酮影响后胞壁成份变化与其它真菌不同的原因进行了讨论。     

利用荧光蛋白标记西瓜枯萎病菌的过氧化物酶体及细胞核

西瓜枯萎病是一种世界范围的西瓜毁灭性病害,其病原菌为尖孢镰刀菌西瓜专化型(Fusarium oxysporum f.sp.niveum,FON)。研究病原菌生长发育和侵染的机制是解决病害的根本途径。利用荧光蛋白对细胞或细胞器进行标记,是病原菌研究中的重要方法。该研究利用绿色荧光蛋白和红色荧光蛋白对FON的细胞核和过氧化物酶体进行了荧光标记。通过农杆菌介导转化(Agrobacterium tumefaciens-mediated transformation,AtMT),该文将3种不同的荧光定位载体分别导入FON,获得了细胞核红色荧光标记的转化子(潮霉素抗性,含mCherry-H2B融合蛋白),以及过氧化物酶体绿色(潮霉素抗性,含GFP-PTS1融合蛋白)和红色(潮霉素抗性,含DsRED-PTS1融合蛋白)荧光标记的转化子各1种。在标记细胞核的菌株中,菌丝、孢子都可见明亮、圆形的红色荧光点,荧光点与DAPI染色标记的细胞核区域完全重合。在过氧化物酶体标记的菌株中,菌丝、孢子中可见明亮的红色或绿色荧光成小点状分布,符合过氧化物酶体的分布特征,而且在脂类物质诱导的条件下,荧光点的数量明显增加。此外,该文还利用细胞壁荧光染色剂卡氏白对3种荧光蛋白标记菌株进行染色。结果显示,卡氏白染色产生的蓝色荧光与红、绿荧光蛋白的荧光在FON中互不干扰。转化子继代培养和初步分析表明,其表型与野生型无差异,菌株继代后荧光表达稳定、定位明显。该结果为进一步研究FON细胞器动态、生长发育与致病分子机制提供了方法和工具。     

Use of green fluorescent protein to quantify the growth of Colletotrichum during infection of tobacco

To develop a quantitative assay of fungal growth inside plant tissues, strains of Colletotrichum destructivum and Colletotrichum orbiculare were transformed with a modified green fluorescent protein (GFP) gene fused with a glyceraldehyde-3-phosphate dehydrogenase promoter from Aspergillus nidulans. Transformants expressed GFP in culture and had the same growth rate and general appearance as the wild type. GFP was observed in all fungal structures during infection of leaves of Nicotiana benthamiana, except for the melanized appressoria and setae. The timing and appearance of the fungal structures in the host appeared to be identical to that of the wild type. GFP accumulation in inoculated leaves of N. benthamiana was quantified in leaf extracts using a fluorescence microplate reader, and the quantity of fluorescence was strongly correlated with the growth of the fungus as measured by the amount of fungal actin gene expression using Northern blot hybridizations. These results demonstrated that assaying green fluorescence levels from a GFP-transformed fungus is an accurate, fast and easy means of quantifying fungal growth inside host plant cells.     

Analysis of cell division using fluorescently labeled actin and myosin in living PtK2 cells

Actin and the light chains of myosin were labeled with fluorescent dyes and injected into interphase PtK2 cells in order to study the changes in distribution of actin and myosin that occurred when the injected cells subsequently entered mitosis and divided. The first changes occurred when stress fibers in prophase cells began to disassemble. During this process, which began in the center of the cell, individual fibers shortened, and in a few fibers, adjacent bands of fluorescent myosin could be seen to move closer together. In most cells, stress fiber disassembly was complete by metaphase, resulting in a diffuse distribution of the fluorescent proteins throughout the cytoplasm with the greatest concentration present in the mitotic spindle. The first evidence of actin and myosin concentration in a cleavage ring occurred at late anaphase, just before furrowing could be detected. Initially, the intensity of fluorescence and the width of the fluorescent ring increased as the ring constricted. In cells with asymmetrically positioned mitotic spindles, both protein concentration and furrowing were first evident in the cortical regions closest to the equator of the mitotic spindle. As cytokinesis progressed in such asymmetrically dividing cells, fluorescent actin and myosin appeared at the opposite side of the cell just before furrowing activity could be seen there. At the end of cytokinesis, myosin and actin were concentrated beneath the membrane of the midbody and subsequently became organized in two rings at either end of the midbody.     

Plant actin filament and microtubule interactions during anaphase--telophase transition: effects of antagonist drugs

F-actin and microtubule co-distribution and interaction were studied during anaphase-telophase. Rapid and drastic changes in the cytoskeleton during these particular stages were studied in isolated plant endosperm cells of the blood lily. These wall-free cells can be considered as natural dividing protoplasts. As identified previously, an F-actin cytoskeletal network characterized the plant cortex and formed an elastic cage around the spindle, remaining throughout interphase, mitosis and cytokinesis. Actin was specifically labeled by fluorescent phalloidin and/or monoclonal antibodies. Gold-labelled secondary antibodies were used for ultrastructural observations and silver-enhancement was applied for video-enhanced microscopy. Microtubule and microfilament dynamics and interaction were studied using drug antagonists to actin (cytochalasins B, D) and to tubulin (colchicine). This permitted precise correlations to be made between chromosome movement inhibition and alteration in the actin/tubulin cytoskeleton. During anaphase chromosome migration, the cortical actin network was stretched along the microtubular spindle, while it remained homogeneous when anaphase was inhibited by colchicine. Cytochalasins did not inhibit chromosome movement but altered actin distribution. A new population of actin filaments appeared at the equator in late anaphase before the microtubular phragmoplast was formed and contributed to cell plate formation. Our conclusion is that F-actin-microtubule interaction may contribute to the regulatory mechanism of plant cytokinesis.     

Mechanism of the formation of contractile ring in dividing cultured animal cells. II. Cortical movement of microinjected actin filaments

The contractile ring in dividing animal cells is formed primarily through the reorganization of existing actin filaments (Cao, L.-G., and Y.-L. Wang. 1990. J. Cell Biol. 110:1089-1096), but it is not clear whether the process involves a random recruitment of diffusible actin filaments from the cytoplasm, or a directional movement of cortically associated filaments toward the equator. We have studied this question by observing the distribution of actin filaments that have been labeled with fluorescent phalloidin and microinjected into dividing normal rat kidney (NRK) cells. The labeled filaments are present primarily in the cytoplasm during prometaphase and early metaphase, but become associated extensively with the cell cortex 10-15 min before the onset of anaphase. This process is manifested both as an increase in cortical fluorescence intensity and as movements of discrete aggregates of actin filaments toward the cortex. The concentration of actin fluorescence in the equatorial region, accompanied by a decrease of fluorescence in polar regions, is detected 2-3 min after the onset of anaphase. By directly tracing the distribution of aggregates of labeled actin filaments, we are able to detect, during anaphase and telophase, movements of cortical actin filaments toward the equator at an average rate of 1.0 micron/min. Our results, combined with previous observations, suggest that the organization of actin filaments during cytokinesis probably involves an association of cytoplasmic filaments with the cortex, a movement of cortical filaments toward the cleavage furrow, and a dissociation of filaments from the equatorial cortex.     

Actin organization and dynamics in filamentous fungi

Growth and morphogenesis of filamentous fungi is underpinned by dynamic reorganization and polarization of the actin cytoskeleton. Actin has crucial roles in exocytosis, endocytosis, organelle movement and cytokinesis in fungi, and these processes are coupled to the production of distinct higher-order structures (actin patches, cables and rings) that generate forces or serve as tracks for intracellular transport. New approaches for imaging actin in living cells are revealing important similarities and differences in actin architecture and organization within the fungal kingdom, and have yielded key insights into cell polarity, tip growth and long-distance intracellular transport. In this Review, we discuss the contribution that recent live-cell imaging and mutational studies have made to our understanding of the dynamics and regulation of actin in filamentous fungi.     

Insight into Actin Organization and Function in Cytokinesis from Analysis of Fission Yeast Mutants

Actin is a key cytoskeletal protein with multiple roles in cellular processes such as polarized growth, cytokinesis, endocytosis, and cell migration. Actin is present in all eukaryotes as highly dynamic filamentous structures, such as linear cables and branched filaments. Detailed investigation of the molecular role of actin in various processes has been hampered due to the multifunctionality of the protein and the lack of alleles defective in specific processes. The actin cytoskeleton of the fission yeast, Schizosaccharomyces pombe, has been extensively characterized and contains structures analogous to those in other cell types. In this study, primarily with the view to uncover actin function in cytokinesis, we generated a large bank of fission yeast actin mutants that affect the organization of distinct actin structures and/or discrete physiological functions of actin. Our screen identified 17 mutants with specific defects in cytokinesis. Some of these cytokinesis mutants helped in dissecting the function of specific actin structures during ring assembly. Further genetic analysis of some of these actin mutants revealed multiple genetic interactions with mutants previously known to affect the actomyosin ring assembly. We also characterize a mutant allele of actin that is suppressed upon overexpression of Cdc8p-tropomyosin, underscoring the utility of this mutant bank. Another 22 mutant alleles, defective in polarized growth and/or other functions of actin obtained from this screen, are also described in this article. This mutant bank should be a valuable resource to study the physiological and biochemical functions of actin.     

猕猴桃间座壳菌Diaporthe actinidiae的绿色荧光蛋白基因标记及侵染过程

果实软腐病是猕猴桃贮藏期间最严重的真菌病害,猕猴桃间座壳Diaporthe actinidiae是该病检出率最高且致病力最强的病原菌.该病菌从花前期开始侵染,至果实贮藏期才表现症状,侵染至发病周期较长,可借助荧光标记对其侵染过程进行研究.本研究采用PEG介导原生质体转化的技术,运用携带GFP及潮霉素抗性基因的双元载体p...     

In vitro approaches to study actin and microtubule dependent cell processes

Actin and microtubules represent complex polymer systems that play essential roles during many cellular processes including chromosome segregation, cytokinesis and motility. The dynamic nature of actin and microtubules together with their regulation by a myriad of proteins makes their study both fascinating and challenging. Over the past few years there has been an increasing move towards development of in vitro systems to facilitate the elucidation of the molecular basis of actin and microtubule dependent cell processes. This review focuses on some of the recent developments using in vitro assays to dissect the cellular role of the actin and microtubule cytoskeleton.