巴西固氮螺菌Yu62在玉米根的定植

将GFPmut2质粒中的gfp基因(编码绿色荧光蛋白)克隆到载体pVK100中,构建成重组质粒pVK1001。将pVK1001通过电转化方法导入到联合固氮菌巴西固氮螺菌Yu62中,获得GFP)标记的巴西固氮螺菌Yu62菌株。用标记菌株接种限菌培养条件下生长的玉米(农大3318)幼苗,在接种后8d、12d,用激光共聚焦扫描显微镜进行观测,结果表明巴西固氮螺菌Yu62菌株能定植于玉米根部皮层的薄壁细胞间隙。用扫描电镜和超薄切片电镜观察表明,大多数细菌主要定植于根表,少数菌可进入玉米根组织内。     

巴西固氮螺菌Yu62的EGFP标记及其在小麦体内的定殖研究

以质粒pEGFP-C1为模板,采用PCR方法特异性扩增增强型绿色荧光蛋白(EGFP)基因全长序列,将其与原核表达载体pVK-100连接,构建成重组载体pVK-EGFP.利用电转化法将重组载体导入巴西固氮螺菌Yu62中,得到EGFP标记菌株.用EGFP标记菌接种小麦'小偃107'种子,室内限菌条件下培养10 d后,用荧光显微镜观测标记菌在小麦体内的定殖规律并观察接菌植株的田间生长状况.结果显示,巴西固氮螺菌Yu62能定殖于小麦根毛区、茎组织的细胞间隙等部位,而且接菌小麦'小偃107'植株在根系发育、株高、分蘖数等方面比对照有较明显的优势.研究表明,巴西固氮螺菌Yu62能够定殖于小麦根茎内,并具有促进植物生长的作用.     

巴西固氮螺菌中P_Ⅱ和P_Z在固氮调节中的不同作用

在巴西固氮螺菌 (Azospirillumbrasilense)中 ,glnB和glnZ是两个高度同源基因 ,分别位于 3 7kb EcoRI+PstI和 3 7kb SalI的两个不同的染色体片段上。用卡那霉素盒 (Kmr cas sette)插入法 ,对glnB和glnZ分别进行定位诱变 ,并获得相应的突变株 ,即glnB- 和glnZ- 。研究表明 ,glnB- 突变株丧失固氮酶活性 ,表现为Nif- ,而glnZ- 象野生型菌株一样具有固氮酶活性。为了进一步研究这两个基因的功能 ,将glnB和glnZ分别构建在pVK1 0 0载体上形成重组质粒pVK -Ⅱ和pVK -Z ,对glnB- 和glnZ- 突变株进行互补实验 ,进一步证明了glnB与固氮酶活有直接相关性 ,而glnZ无此作用。同时 ,通过三亲接合法将pVK -Ⅱ和pVK -Z分别转移到巴西固氮螺菌野生型Yu62和具有一定抗铵能力的draT- 突变株中 ,使glnB和glnZ的拷贝数增加 ,进一步比较它们的固氮酶活性。结果表明多拷贝的glnB基因 ,能显著提高固氮酶活性 ,而多拷贝的glnZ对固氮酶活性无影响。同时 ,将pVK Ⅱ和pVK -…     

肺炎克氏杆菌的nifA基因产物在巴西固氮螺菌中的功效

通过三亲本杂交将质粒pCK3{携带改变了启动子的肺炎克氏杆菌(Klebsiella pneuma-niae)nifA 基因]引入巴西固氮螺菌(Azospirillum brasilense)Yu62菌株中,由此获得的转移接合子巴西固氮螺菌Yu62-4菌株在6．0 mmol/L以上NH+4浓度下,能表现出微弱的固氮酶活性(相当于无NH+4时活性的0．3-0．5％),而野生型Yu62则全部丧失固氮酶活性。固氯酶的丙烯酰胺凝胶电泳和铁蛋白的免疫杂交实验表明,转移接合子Yu62-4在高NH+4(50mmol／L)下,虽有铁蛋白合成,但合成量比无NH+4时少得多,而且有一部分铁蛋白未被共价修饰;野生型菌株Yu62在此NH+4浓度下无铁蛋白合成。实验结果表明：外源(来自肺炎克氏杆菌)的基因产物在巴西固氮螺菌Yu62中不能有效地解除NH+4对该菌固氮酶合成的阻遏作用。本文分析了出现这种现象的原因。     

肺炎克氏杆菌NifA对巴西固氮螺菌nifH启动子的转录激活作用

用PR方法克隆了巴西固氮螺菌Yu62 nifH的启动子片段,DNA序列分析表明菌株Yu62与标准菌株sp7之间的DNA序列差异很小。利用启动子探针质粒载体pcBl82,构建了3个不同的nifH：：lacz转录融合质粒,在大肠杆菌中分别测定肺炎克氏杆菌NifA对它们的转录激活作用。结果表明巴西固氮螺菌nifH启动子的转录是依赖于NifA的,缺失了上游激活序列的启动子不能被NifA激活转录,肺炎克氏杆菌NifA对其自身nifH及巴西固氮螺菌nifH启动子的转录激活作用并无很大差异。     

巴西固氮螺菌Yu62nifA基因克隆、测序及功能分析

用TD-PCR法克隆了巴西固氮螺菌(Azospirillun brasilense)Yu62的nifA基因.序列分析表明它与巴西固氮螺菌Sp7的nifA序列高度同源(96.5%),其编码的产物NifA蛋白与Sp7菌株NifA的氨基酸序列同源性为97.6%.该基因可以完全互补巴西固氮螺菌Sp7 nifA-突变株的Nif-表型.研究了NH4+和O2对Yu62 nifA基因的表达及NifA活性的影响.结果表明mfA基因在Yu62菌株中是部分组成型表达的,氨和氧不能完全阻遏其表达,在5mmol/LNH4Cl与微氧(0.5%O2)条件下表达最高;NifA蛋白在0.4%～0.5%O2时活性最高,氧分压降低和提高都使NifA活性下降,1mmol/L NH4Cl足以抑制NifA的活性.     

巴西固氮螺菌(Azospirillum brasilense)Yu62的质粒及nifHDK基因的定位

对从北京郊区玉米根际分离到的巴西固氮螺菌(Azospirillum brasilente)Yu 62的质粒进行了分离和检测,发现检测方法不同,得到的质粒数目也不同,用Kado法只发现一个大质粒,以pABm 1表示,其分子量约为1 20 Md;而用改良的Eckhardt方法,则发现有5个大质粒,分别以p&Bm l、pABm 2、pABm 3、pABm 4和pABm 5表示,它们的分子量分别约为120 Md、330 Md、360 Md、480 Md和900 Md,部是巨型质粒,用多种方法检测均未发现小质粒。应用生物素标记的孩酸杂交技术制备了Biotin-nifHDK探针,并对Yu62菌株的质粒及染色体片段进行了Southern印迹杂交和斑点杂交,杂交结果表明nifHDK基因定位于染色体上。     

巴西固氮螺菌中吸氢酶基因同源性的分子检测

通过TTC(2 ,3,5 氯化三苯基氮唑 )实验筛选出 12株能产生吸氢酶蛋白 (Hup+ )的巴西固氮螺菌 (Azospir rilumbrasilense)菌株。用Qiagen柱分离提纯含豌豆根瘤菌的hup基因片段的质粒 pHVT10 9和pHVT115 ,并用地高辛标记法标记pHVT115 ,与 12株Hup+ 巴西固氮螺菌的总DNA进行斑点杂交 ,结果显示pHVT115所含的吸氢酶基因 (hup基因 )片段与巴西固氮螺菌的大部分菌株的hup基因同源性不强。这一结果表明hup基因在固氮生物中存在着遗传多样性 ,异源hup基因探针不一定都适宜于探测hup基因。     

固氮作用

862711.巴西固氮特螺菌SP7的glnA基因的克隆和特性〔会,英〕/Fogher,C.…了Abs.6th Internatl.Sym.Nitrogen Fixa-t ion。一1985。一8一12 用广谱寄主范围的装配型质粒pVK100构建了巴西固氮螺菌(A:ospirillom brasi-lense)Sp7的总DNA基因文库。分离T能与该菌谷氨酞胺营养缺陷型突变株7029互补的克隆,称为     

巴西固氮螺菌中PⅡ和Pz在固氮调节中的不同作用

在巴西固氮螺菌(Azospirillum brasilense)中,glnB和glnZ是两个高度同源基因,分别位于3.7kb/EcoRI+PstI和3.7kb/SalI的两个不同的染色体片段上.用卡那霉素盒(Kmr-cas-sette)插入法,对glnB和glnZ分别进行定位诱变,并获得相应的突变株,即glnB-和glnZ-.研究表明,glnB-突变株丧失固氮酶活性,表现为Nif-,而glnZ-象野生型菌株一样具有固氮酶活性.为了进一步研究这两个基因的功能,将glnB和glnZ分别构建在pVK100载体上形成重组质粒pVK-Ⅱ和pVK-Z,对glnB-和glnZ突变株进行互补实验,进一步证明了glnB与固氮酶活有直接相关性,而glnZ无此作用.同时,通过三亲接合法将pVK-Ⅱ和pVK-Z分别转移到巴西固氮螺菌野生型Yu62和具有一定抗铵能力的draT-突变株中,使glnB和glnZ的拷贝数增加,进一步比较它们的固氮酶活性.结果表明多拷贝的glnB基因,能显著提高固氮酶活性,而多拷贝的glnZ对固氮酶活性无影响.同时,将pVK-Ⅱ和pVK-Z分别转移到nifA-突变株中,结果表明glnB和glnZ均不能恢复nifA-的固氮酶活性.     

巴西固氮螺菌Yu62由水稻和烟草根部向茎、叶的迁移运动(英文)

巴西固氮螺菌(Azospirillum brasilence)是重要的植物促生内生菌之一。用gfp基因标记固氮螺菌后接种无菌的水稻和烟草幼苗的根部,限菌培养一定时间后,用共聚焦激光显微镜观察,结果表明:除了根内部有发荧光的螺菌定殖外,螺菌还分布在茎、叶的表皮细胞,皮层细胞和维管系统组织的细胞和细胞间隙。从根、茎、叶器官分离固氮螺菌,都存在有较高的螺菌群体密度。这一结果证明螺菌在植物内存在着从根部向茎、叶顶端的迁移现象。这一发现为研究巴西固氮螺菌在宿主植物体内的迁移运动的机制、与植物细胞间的分子相互作用及其对植物的促生作用奠定了生态学和细胞形态学的基础,也为实际应用提供了进一步的科学依据,具有重要的科学和实践意义。     

巴西固氮螺菌Yu62由水稻和烟草根部向茎、叶的迁移运动

巴西固氮螺菌(Azospirillrm brasilence)是重要的植物促生内生菌之一.用gfp基因标记固氮螺菌后接种无菌的水稻和烟草幼苗的根部,限制培养一定时间后,用共聚焦激光显微镜观察,结果表明:除了根部有发荧光的螺菌定殖外,螺菌还分布在茎、叶的表皮细胞,皮层细胞和维管系统组织的细胞间隙.从根、茎、叶器官分离固氮螺菌,都存在有较高的螺菌群体密度.这一结果证明螺菌在植物内存在着从根部向茎、叶顶端的迁移现象.这一发现为研究巴西固氮螺菌在窠主植物体内的迁移运动的机制、与植物细胞间的分子相互作用及其对植物的促生作用奠定了生态学和细胞形态学的基础,也为实际应用提示了进一步的科学依据,具有重要的科学和实践意义.     

豌豆根瘤中细菌的扫描电镜观察

用扫描电镜观察了豌豆根瘤的侵染细胞.结果表明,在这些细胞中有大量的细菌,它们主要是杆状细菌,其次是球形、Y形和T形细菌,其它形状的细菌很少.除了细菌形状不同外,还有一些细菌比较特殊,如有的细菌较长,菌体出现部分收缩并形成一个或一个以上的收缩环,其形状类似一条莲根;有的细菌很大,它的体积是普通细菌的2倍或2倍以上;有的细菌粗细不均匀,端部膨大,呈棒槌状.侵染细胞中有许多小泡,它们大小不同,呈球形.它们存于细菌之间,其中一些小泡还位于细菌的表面上,而且附近细菌的表面有时还有各种隆起.     

Unique advantages of using low temperature scanning electron microscopy to observe bacteria

Summary Electron microscopy (EM) has greatly helped to elucidate our understanding of bacterial structure and function. However, several recent studies have cautioned investigators about artifacts that result from the use of conventional EM preparation procedures. To avoid these problems, the use of low temperature scanning electron microscopy (LTSEM) was evaluated for examining frozen, fully hydrated specimens. Spinach leaves (Spinacia oleracea L. cv. New Jersey), which were naturally infected or inoculated with bacteria, were used as the experimental material. 1 cm segments of the infected leaves were plunge frozen in liquid nitrogen, transferred to a cryochamber for sputter coating and then moved onto a cryostage in an SEM. After observation, some of the frozen, hydrated leaf segments were transferred onto agar medium to determine whether preparation for LTSEM was nondestructive to the bacteria. The other tissue segments were chemically fixed by freeze-substitution. The results indicated that after cryopreparation and observation in the LTSEM: (i) viable bacteria, which were recovered from the leaf sample, could be cultured on agar medium for subsequent study, and (ii) the frozen samples could be freeze substituted and embedded so that transmission electron microscopic (TEM) observations could be carried out on the same specimen. In conclusion, frozen, hydrated leaf tissue infected with bacteria can be observed using LTSEM and then can be either processed for TEM observation to obtain further structural details or recovered to culture the pathogenic bacteria for supplementary studies.Abbreviations EPS extracellular polysaccharide - EM electron microscopy - LTSEM low temperature scanning electron microscopy - SEM scanning electron microscopy - TEM transmission electron microscopy - TSA tryptic soy agar - TSB tryptic soy broth Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Er,Cr:YSGG激光照射对根管封闭效果影响的实验研究

探讨常规根管预备后使用不同能量参数Er,C r:YSGG激光进行根管内照射对根管封闭效果的影响,为此激光在根管治疗领域的临床应用提供重要的实验依据。选择新拔除的单根管牙80颗,分成4组,每组20颗牙。常规根管预备后,分别使用1 W、2 W、3 W Er,C r:YSGG激光于有水条件下进行根管内照射,另选一组不做任何处理,作为阴性对照。扫描电子显微镜下观察根管内壁形态学改变后,常规根管充填,检测根管微渗漏情况。采用单因素方差分析及q检验进行统计学处理。结果可见:(1)形态学观察:与对照组相比,激光照射均可有效去除根管内壁的玷污层及碎屑;牙本质小管口清晰可见;未见熔融及炭化等热损伤改变;其中3 W激光处理后根管内壁形态最为理想。(2)微渗漏检测:与对照组相比,各激光组根管微渗漏深度均显著减少(P<0.05);但各激光组间比较无显著性差异(P>0.05)。综上,使用Er,C r:YSGG激光进行根管内照射可达到较为理想的根管清洁目的,显著改善根管充填后的封闭效果,因此有望在临床上应用提高根管治疗的成功率。     

Bacillus cereus B-02对Botrytis cinerea拮抗机理的研究

采用电子显微镜(扫描、透射)和激光扫描共聚焦显微镜,从细胞形态学和生理学水平上研究蜡样芽孢杆菌Bacillus cereus B-02过滤液对灰葡萄孢菌Botrytis cinerea的拮抗机理。结果表明,处理菌丝表面形态受到严重破坏,发生强烈变形;菌丝细胞核、线粒体和细胞壁等亚细胞结构发生了明显改变,细胞内出现大量无膜透明内含物,并产生较大液泡。此外,处理菌丝DNA、线粒体膜电位和活性氧荧光强度均低于对照组,且差异极显著;说明B-02菌株对病原真菌菌丝细胞DNA的合成、线粒体膜电位和活性氧水平有重要影响。     

Bacillus cereus B-02对Botrytis cinerea 拮抗机理的研究

采用电子显微镜(扫描、透射)和激光扫描共聚焦显微镜,从细胞形态学和生理学水平上研究蜡样芽孢杆菌Bacillus cereus B-02过滤液对灰葡萄孢菌Botrytis cinerea的拈抗机理.结果表明,处理菌丝表面形态受到严重破坏,发牛强烈变形;荫丝细胞核、线粒体和细胞壁等哑细胞结构发生了明显改变,细胞内出现大量无膜透明内含物,并产生较人液泡.此外,处理菌丝DNA、线粒体膜电位和活性氧荧光强度均低于对照组,且差异极显著;说明B-02菌株对病原真菌菌丝细胞DNA的合成、线粒体膜电位和活性氧水平有重要影响.     

Elemental composition of strawberry plants inoculated with the plant growth‐promoting bacterium Azospirillum brasilense REC3, assessed with scanning electron microscopy and energy dispersive X‐ray analysis

The elemental composition of strawberry plants (Fragaria ananassa cv. Macarena) inoculated with the plant growth‐promoting bacterium Azospirillum brasilense REC3, and non‐inoculated controls, was studied using scanning electron microscopy (SEM) and energy dispersive X‐ray (EDS) analysis. This allowed simultaneous semi‐quantification of different elements in a small, solid sample. Plants were inoculated and grown hydroponically in 50% or 100% Hoagland solution, corresponding to limited or optimum nutrient medium, respectively. Bacteria‐inoculated plants increased the growth index 45% and 80% compared to controls when grown in 100% and 50% Hoagland solution, respectively. Thus, inoculation with A. brasilense REC3 in a nutrient‐limited medium had the strongest effect in terms of increasing both shoot and root biomass and growth index, as already described for Azospirillum inoculated into nutrient‐poor soils. SEM‐EDS spectra and maps showed the elemental composition and relative distribution of nutrients in strawberry tissues. Leaves contained C, O, N, Na, P, K, Ca and Cu, while roots also had Si and Cl. The organic fraction (C, O and N) accounted for over 96.3% of the total chemical composition; of the mineral fraction, Na had higher accumulation in both leaves and roots. Azospirillum‐inoculated and control plants had similar elemental quantities; however, in bacteria‐inoculated roots, P was significantly increased (34.33%), which constitutes a major benefit for plant nutrition, while Cu content decreased (35.16%).     

In Situ Localization of Azospirillum brasilense in the Rhizosphere of Wheat with Fluorescently Labeled, rRNA-Targeted Oligonucleotide Probes and Scanning Confocal Laser Microscopy

The colonization of wheat roots by Azospirillum brasilense was used as a model system to evaluate the utility of whole-cell hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes for the in situ monitoring of rhizosphere microbial communities. Root samples of agar- or soil-grown 10- and 30-day-old wheat seedlings inoculated with different strains of A. brasilense were hybridized with a species-specific probe for A. brasilense, a probe hybridizing to alpha subclass proteobacteria, and a probe specific for the domain Bacteria to identify and localize the target bacteria. After hybridization, about 10 to 25% of the rhizosphere bacteria as visualized with 4(prm1),6-diamidino-2-phenylindole (DAPI) gave sufficient fluorescence signals to be detected with rRNA-targeted probes. Scanning confocal laser microscopy was used to overcome disturbing effects arising from autofluorescence of the object or narrow depth of focus in thick specimens. This technique also allowed high-resolution analysis of the spatial distribution of bacteria in the rhizosphere. Occurrence of cells of A. brasilense Sp7 and Wa3 was restricted to the rhizosphere soil, mainly to the root hair zone. C-forms of A. brasilense were demonstrated to be physiologically active forms in the rhizosphere. Strain Sp245 also was found repeatedly at high density in the interior of root hair cells. In general, the combination of fluorescently labeled oligonucleotide probes and scanning confocal laser microscopy provided a very suitable strategy for detailed studies of rhizosphere microbial ecology.