转PvPGIP2基因小麦的获得与纹枯病抗性鉴定

多聚半乳糖醛酸酶抑制蛋白(PGIP)是一种植物防卫蛋白,可阻止一些病原真菌的侵害。本研究克隆出扁豆PvP-GIP2基因编码序列,构建了受玉米泛素(ubiquitin)启动子控制的PvPGIP2基因表达载体pA25-PvPGIP2;采用基因枪法将pA25-PvPGIP2转化小麦推广品种扬麦18幼胚愈伤组织4000块,获得了203株再生植株。PCR检测出阳性植株65株,转化率为1.625%。对转PvPGIP2基因小麦T1～T2植株,进行外源基因的PCR、RT-PCR、荧光定量RT-PCR(Q-RT-PCR)分析和小麦纹枯病抗性鉴定。结果表明,转入的PvPGIP2能够在转基因小麦中遗传、转录与表达;PvPGIP2基因的表达提高了转基因植株对小麦纹枯病的抗性。     

马铃薯抗菌肽SN1基因的克隆、原核表达及其抑菌活性

马铃薯抗菌肽SN1是一种新型抗菌肽。为明确SN1是否抑制小麦重要土传真菌小麦纹枯菌(禾谷丝核菌)、小麦根腐菌(平脐蠕孢菌)的生长,本文克隆了马铃薯抗菌肽基因SN1的全长编码序列,将该基因编码序列亚克隆到原核表达载体pGEX-4T-1上,构建成GST-SN1融合蛋白表达载体。经异丙基-β-D-硫代半乳糖苷(IPTG)诱导,获得了以包涵体形式表达的GST-SN1重组蛋白。经过裂解、洗涤、溶解、复性等处理,获得了纯化的GST-SN1融合蛋白。体外抑菌试验表明,SN1显著抑制禾谷丝核菌、平脐蠕孢菌菌丝生长,可作为上述植物病害抗性育种的潜在基因。     

荧光蛋白报告基因在植物寄生真菌研究中的应用

综述菌根真菌、植物内生菌和植物病原真菌等植物寄生真菌转荧光蛋白基因研究现状.介绍转化载体的构建、转化方法及特基因的检测方法,以及转荧光蛋白基因技术在植物寄生真菌侵染过程研究中的应用,指出真菌转荧光蛋白基因存在的问题和展望.     

几丁质酶基因与抗真菌蛋白基因、葡聚糖酶基因双价表达载体的构建及农杆菌工程菌株的重组

几丁质酶（Chitinase,Chi.)、β-1,3－葡聚糖酶（β－1,3－Glucanase,Glu.)和萝卜抗真菌蛋白（Rs-AFP2)是植物体内正常的表达产物,它们对防御植物的真菌病害具有重要的作用。基于它们在功能上具有协同作用,本研究利用基因工程技术构建了几丁质酶和抗真菌蛋白、几丁质酶和葡聚糖酶双价表达载体,通过农杆菌直接转化技术将双价表达载体转入农杆菌EHA105,最后采用PCR、DNA dot blotting技术对所获得的农杆菌工程菌株进行了鉴定分析。     

拮抗多种植物病原真菌Streptomyces triostinicus C2的分离与鉴定北大核心CSCD

旨在分离筛选获得对植物病原真菌具有抑制作用的拮抗菌。以水稻纹枯病菌为指示菌,采用平板对峙培养法进行拮抗菌的分离筛选;根据菌体形态学观察、生理生化特征以及16S r DNA序列分析,对拮抗菌进行菌种鉴定。结果显示,从采集自云南、广东、安徽、湖北、江西等地的18个土样中分离筛选到一株对水稻纹枯病菌具有良好抑制效果的菌株C2,该菌株初步鉴定为Streptomyces triostinicus,并暂命名为链霉菌C2;进一步测定链霉菌C2的抗菌谱发现,它对水稻纹枯病菌、水稻稻瘟病菌、葡萄炭疽病菌、西瓜枯萎病菌和橘青霉等多种植物病原真菌均具有良好的抑制作用,其抑菌率分别为49.78%、56.62%、80.96%、18.59%和94.10%。链霉菌C2是一株植物病原真菌广谱拮抗菌。     

荧光蛋白在双色蜡蘑中的构建与表达

菌根是真菌与植物之间形成的互惠互利的营养共生体,对生态环境有重大的意义。外生菌根真菌与植物互作机制以及真菌基因功能的深入研究都需要对菌根真菌进行遗传转化,本研究以外生菌根真菌模式生物双色蜡蘑(Laccaria bicolor)为研究对象,选择细胞核中的核小体蛋白H₂B基因为目的基因,以pCEBN为表达载体,融合红色荧光蛋白,最终构建在真菌中表达的双元载体,使用根瘤农杆菌介导转化法转化双色蜡蘑菌丝,随后利用PCR对真菌转化子进行验证后,通过激光共聚焦显微镜观察到菌丝细胞核中的红色荧光,成功将融合荧光蛋白转化菌根真菌,为后续研究菌根真菌中基因的亚细胞定位提供了实验平台。结果表明,利用双元载体和农杆菌转化方法,建立了高效的双色蜡蘑转化体系(93.33%),在激光共聚焦显微镜下观察到菌丝细胞核中红色荧光信号,验证了融合荧光蛋白在双色蜡蘑中的成功表达。本研究成功地构建了菌根真菌中的核小体蛋白和红色荧光蛋白融合表达的真菌转化体系。     

萝卜抗真菌蛋白1(Rs-AFP1)在大肠杆菌中的融合表达及其对大丽轮枝菌抑制活性的研究(英文)

利用聚合酶链式反应 (PCR)获得了萝卜 (RaphanussativusL .)抗真菌蛋白 1(Rs_AFP1)基因编码区核苷酸序列。将整个阅读框架片段和去除了N_端信号肽序列的片段分别装入原核表达载体pET_32b( )中 ,在大肠杆菌中表达 ,发现带有信号肽的Rs_AFP1不能在大肠杆菌中表达 ,而当这一序列去除后 ,表达出约 2 7kD的Rs_AFP1的融合蛋白。用凝血酶处理融合蛋白以去除N_端His.tag的部分序列 ,然后用处理后的融合蛋白进行了抑制真菌生长的实验。结果表明 ,在加入 0 .3g/L的Rs_AFP1的融合蛋白的培养液中 ,大丽轮枝菌 (VerticilliumdahliaeKleb .)的生长受到抑制 ,分别比加入对照细菌蛋白和PBS下降 5 7.5 %和 6 9.8% ;孢子的萌发也受到抑制。显然 ,细菌表达的融合蛋白对大丽轮枝菌的生长有抑制作用。     

欧美107杨树提取物体外对植物病原真菌的抑制活性

制备了欧美107杨树枝条和叶的乙醇粗提物及不同极性溶剂的萃取部分,并测定了它们对植物病原真菌的抑制活性。枝条和叶的乙醇粗提物对棉花枯萎病菌、小麦纹枯病菌、番茄早疫病菌、番茄枯萎病菌、黄瓜枯萎病菌、小麦赤霉以及玉米弯孢等7种植物病原真菌均具有一定的抑制作用。而枝条的乙醇粗提物对杨树溃疡病菌的菌丝生长有一定的促进作用。抗菌活性成分主要集中在乙酸乙酯萃取部分。     

小麦Fe超氧歧化酶基因的原核表达分析

采用RT-PCR技术分离小麦Fe超氧歧化酶基因(FeSOD)的ORF全长cDNA,然后构建其原核表达载体,并对其表达的诱导时间、IPTG浓度、温度进行优化,以期获得较大量的重组蛋白。结果表明:实验获得了小麦FeSOD基因的ORF全长（600 bp）,ORF全长与原核表达载体pET-Dute1相连接构建了原核表达载体pET-FeSOD,将pET-FeSOD导入宿主菌Rosetta(DE3)中,经SDS-PAGE电泳结果显示,可以高效表达融合蛋白且表达的蛋白均主要以包涵体的形式存在;重组质粒表达出25.8 kD的融合蛋白,除去载体pET-Duet1自身表达的3.0 kD蛋白后,与FeSOD编码的约为22.8 kD蛋白的大小一致;对诱导表达条件的优化结果显示,融合蛋白 pET-FeSOD最佳的诱导表达条件为:0.5 mmol/L的IPTG浓度,37 ℃诱导5 h。该研究结果为进一步深入研究该基因的特性与功能奠定了基础。     

麻疯树核糖体失活蛋白Curcin2的原核可溶性表达及其抗真菌活性研究

Curcin2是麻疯树幼苗在真菌侵染、干旱及高低温胁迫下诱导产生的一种核糖体失活蛋白.采用分子克隆的方法,从麻疯树基因组中扩增到Curcin2成熟肽编码基因,分别连接到质粒载体pGEX-6p-1、pMAL-c5E上,转化大肠杆菌最终获得重组菌PGC(pGEX-6p-1-curcin2)和PMC(pMAL-c5E-curcin2).在不同温度、IPTG浓度、时间诱导下,Curcin2在重组菌PGC中均以包涵体形式表达,在重组菌PMC中主要以可溶性融合蛋白形式表达,且蛋白质的表达量与诱导条件相关.重组菌PMC表达可溶性curcin2的优化条件为:温度28℃,IPTG 0.3mmol/L,时间8h,此条件下目的蛋白占总蛋白表达量的30.6％,1L培养物中可获得19.74mg电泳纯的重组蛋白.重组蛋白可被MBP TrapTM HP柱亲和纯化并与curcin抗体发生抗原抗体反应.体外抗真菌活性实验表明,纯化后的curcin2融合蛋白有抑制真菌生长作用,且对小麦赤霉、油菜菌核的抑制作用强于curcin.此蛋白的获得为其相关功能的研究奠定了基础.     

抗真菌蛋白研究进展

对几种抗真菌蛋白 :病程相关蛋白、防卫素、核糖体失活蛋白、几丁质连接蛋白、蛋白酶抑制剂等的类型、特征、抗菌菌机理进行了阐述 ,着重讨论了病程相关蛋白和核糖体失活蛋白的最新研究进展 ,并且讨论了抗真菌蛋白在植物真菌病害综合防治中的应用前景。     

解淀粉芽孢杆菌NK10.BAhjaWT抑真菌作用的研究

芽孢杆菌属以多产抗菌素闻名。通过筛选几十株不同来源的芽孢杆菌, 获得1株具有强抑真菌活性的芽孢杆菌。经过16S rDNA检测与Biolog分析, 确定此株菌为解淀粉芽孢杆菌。本实验对摇瓶发酵的条件进行了优化, 经过对发酵上清液硫酸铵盐析、透析、真空冷冻干燥获得粗提蛋白。并对粗蛋白的热稳定性、pH稳定性、抗蛋白酶水解能力、离子稳定性以及抑真菌谱进行了研究, 最后使用扫描电镜对抑真菌机制进行了探索。     

解淀粉芽孢杆菌NK10.BAhjaWT抑真菌作用的研究

芽孢杆菌属以多产抗菌素闻名.通过筛选几十株不同来源的芽孢杆菌,获得1株具有强抑真菌活性的芽孢杆菌.经过16S rDNA检测与Biolog分析,确定此株菌为解淀粉芽孢杆菌.本实验对摇瓶发酵的条件进行了优化,经过对发酵上清液硫酸铵盐析、透析,真空冷冻干燥获得粗提蛋白.并对粗蛋白的热稳定性、pH稳定性、抗蛋白酶水解能力、离子稳定性以及抑真菌谱进行了研究,最后使用扫描电镜对抑真菌机制进行了探索.     

Investigation of aryl isonitrile compounds with potent,broad-spectrum antifungal activity

Invasive fungal infections present a formidable global public health challenge due to the limited number of approved antifungal agents and the emergence of resistance to the frontline treatment options, such as fluconazole. Three fungal pathogens of significant concern are Candida, Cryptococcus, and Aspergillus given their propensity to cause opportunistic infections in immunocompromised individuals. New antifungal agents composed of unique chemical scaffolds are needed to address this public health challenge. The present study examines the structure-activity relationship of a set of aryl isonitrile compounds that possess broad-spectrum antifungal activity primarily against species of Candida and Cryptococcus. The most potent derivatives are capable of inhibiting growth of these key pathogens at concentrations as low as 0.5 µM. Remarkably, the most active compounds exhibit an excellent safety profile and are non-toxic to mammalian cells even at concentrations up to 256 µM. The present study lays the foundation for further investigation of aryl isonitrile compounds as a new class of antifungal agents.     

Synthesis and antifungal activity of functionalized 2,3-spirostane isomers

Invasive fungal infections are a major complication for individuals with compromised immune systems. One of the most significant challenges in the treatment of invasive fungal infections is the increased resistance of many organisms to widely used antifungals, making the development of novel antifungal agents essential. Many naturally occurring products have been found to be effective antimicrobial agents. In particular, saponins with spirostane glycosidic moieties—isolated from plant or marine species—have been shown to possess a range of antimicrobial properties. In this report, we outline a novel approach to the synthesis of a number of functionalized spirostane molecules that can be further used as building blocks for novel spirostane-linked glycosides and present results from the in vitro screenings of the antifungal potential of each derivative against four fungal species, including Candida albicans, Cryptococcus neoformans, Candida glabrata, and the filamentous fungus Aspergillus fumigatus.     

青霉TS67菌株活性产物的抗真菌作用

本文初步探讨了青霉TS67(Penicillum sp.)的发酵活性产物对植物病原真菌的抑制作用机理,实验结果表明,用其50%的发酵液分别处理玉蜀黍平脐蠕孢菌(Bipolarismaydis)和大豆尖孢镰刀菌(Fusarium oxysporum)120 h后,菌丝生长的抑制率分别为77.78%和70.30%,对孢子产生的抑制率分别达58.8%和73.5%:同时发现用50%发酵液处理病原茵的无性繁殖孢子12 h后,孢子萌发抑制率分别达78.3%和62.O%.经显微镜观察抗菌活性物质处理后的菌丝体,发现菌丝体表面瘤状畸形、菌丝生长顶端不规则膨胀、内部发生原生质浓缩,初步推测青霉TS67主要通过影响植物病原真菌的细胞壁而实现抑制作用.     

Isolation and characterization of Neosartorya fischeri antifungal protein (NFAP)

A novel 6.6 kDa antifungal peptide (NFAP) from the culture supernatant of the mold, Neosartorya fischeri (anamorf: Aspergillus fischerianus), and its encoding gene were isolated in this study. NFAP is a small, basic and cysteine-rich protein consisting of 57 amino acid residues. It shows 37.9-50% homology to similar proteins described in literature from Aspergillus clavatus, Aspergillus giganteus, Aspergillus niger, and Penicillium chrysogenum. The in silico presumed tertiary structure of NFAP, e.g. the presence of five antiparallel β-sheet connected with filaments, and stabilized by three disulfide bridges, is very similar to those of the defensin-like molecules. NFAP exhibited growth inhibitory action against filamentous fungi in a dose-dependent manner, and maintained high antifungal activity within broad pH and temperature ranges. Furthermore, it exhibited relevant resistance to proteolysis. All these characteristics make NFAP a promising candidate for further in vitro and in vivo investigations aiming at the development of new antifungal compounds.     

Antifungal activity of substituted aurones

Novel antifungals are in high demand as there is a growing resistance to antifungals currently in use. In particular, opportunistic fungal infections caused by Candida spp. are on the rise with infections by this genus accounting for the most severe fungal infections following chemotherapy, implantation procedures, and in patients with HIV/AIDS. A series of simple aurone analogs were synthesized and screened for antifungal activity versus Candida spp. Several compounds displayed activity at 100 μM, with two having IC₅₀ values below 20 μM for three species of Candida. One of the compounds tested here also exhibits anti-biofilm activity for mid-maturation growth.     

In vitro susceptibilities of clinical yeast isolates to three antifungal agents determined by the microdilution method

A comparative evaluation of the in vitro susceptibilities of 597 clinical yeast isolates to amphotericin B, fluconazole, and 5-fluorocytosine (5FC) was conducted. The broth macrodilution reference method of the National Committee for Clinical Laboratory Standards (NCCLS, M27-P) was adapted to the microdilution method. Microdilution endpoints for amphotericin B were scored as the lowest concentration in which a score of 0 (complete absence of growth) was observed and for 5FC and fluconazole as the lowest concentration in which a score of 2 (prominent decrease in turbidity; MIC-2) was observed compared to the growth control. The MIC values were read after 24 and 48 h incubation. A broad range of MIC values was observed with each antifungal agent. Amphotericin B was very active (MIC₉₀1.0 µg/ml) against all of the yeast isolates with the exception ofC. lusitaniae (MIC₉₀2.0 µg/ml). Fluconazole was most active againstC. parapsilosis (MIC₉₀ of 1.0 µg/ml) and least active againstC. krusei (MIC₉₀ of 32 µg/ml). 5FC was most active againstC. albicans, C. parapsilosis, C. tropicalis, andT. glabrata (MIC₉₀1.0 µg/ml) and was least active againstC. krusei andC. lusitaniae (MIC₉₀16 µg/ml). These data indicate that the microdilution method, performed in accordance with M27-P, provides a means of testing larger numbers of yeast isolates against an array of antifungal agents and allows this to be accomplished in a reproducible and standardized manner. Given these results, it appears that the microdilution method may be a useful alternative to the macrodilution reference method for susceptibility testing of yeasts.