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8 d、12 d,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。     

巴西固氮螺菌的周质蛋白与宿主相关性的研究

本文报道用一种微量的方法研究从小麦、玉米、水稻、谷子等作物根系分离到的30株巴西固氮螺菌的周质蛋白。结果表明:尽管经形态、生理生化等多项指标鉴定确认它们同属于Azospirillum brasilcnse (巴西固氮螺菌),但是来源于不同植物根表的菌株间的周质蛋白扫描图谱有明显的差异,而来自同一种作物根表的菌株间的周质蛋白图谱却很相似。从而表明,巴西固氮螺菌具有宿主特异性。     

巴西固氮螺菌的周质蛋白与宿主相关性的研究*

本文报道用一种微量的方法研究从小麦、玉米、水稻、谷子等作物根系分离到的30株巴西固氮螺菌的周质蛋白。结果表明:尽管经形态、生理生化等多项指标鉴定确认它们同属于Azospirillum brasilcnse (巴西固氮螺菌),但是来源于不同植物根表的菌株间的周质蛋白扫描图谱有明显的差异,而来自同一种作物根表的菌株间的周质蛋白图谱却很相似。从而表明,巴西固氮螺菌具有宿主特异性。     

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。     

固氮螺菌与植物的相互关系研究进展

固氮螺菌与植物的相互关系研究进展阎大来,何路红,李季伦（北京农业大学农业生物技术国家重点实验室,北京１０００９４）固氮螺菌（Ａｚｏｓｐｉｒｉｌｌｕｍ）首先是从巴西热带禾本科牧草俯仰马唐（Ｄｉｇｉｔａｒｉａｄｅｃｕｍｂｅｎｓ）根际分离得到的￣［１,２］...     

糖蜜草(Melinis minutiflora Beauv.)内生固氮菌分离鉴定

糖蜜草（Melinis minutiflora Beauv．）是热带地区的一种优良牧草。采用选择性培养基在厌氧和好氧两种培养条件下,从糖蜜草根、茎中都可分离得到具有较强固氮酶活性的菌株。通过SDS-PAGE全细胞蛋白电泳技术快速聚类分析表明,来源于糖蜜草中的菌株为同一类群。16S rDNA序列分析和总DNA的G＋c％含量进一步确定糖蜜草中所分离的菌株属于固氮螺菌属（Azospirillum）,与产脂固氮螺菌（Azospirillum lipoferum）亲缘关系较近。BIOLOG板测定结果显示,糖蜜草菌株TMCY243对多种碳源具有很强的适应性,与产脂固氮螺菌（A．lipoferum）的模式菌株DSM 1691存在着较大的差异。以上结果表明,糖蜜草内生固氮菌为固氮螺菌属的一个新类群。     

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.     

Colonization of Maize and Rice Plants by Strain Bacillus megaterium C4

Bacillus megaterium C4, a nitrogen-fixing bacterium, was marked with the gfp gene. Maize and rice seedlings were inoculated with the, GFP-labeled B. megaterium C4 and then grown in gnotobiotic condition. Observation by confocal laser scanning microscope showed that the GFP-labeled bacterial cells infected the maize roots through the cracks formed at the lateral root junctions and then penetrated into cortex, xylem, and pith, and that the bacteria migrated slowly from roots to stems and leaves. The bacteria were mainly located in the intercellular spaces, although a few bacterial cells were also present within the xylem vessels, root hair cells, epidermis, cortical parenchyma, and pith cells. In addition, microscopic observation also revealed clearly that the root tip in the zone of elongation and differentiation and the junction between the primary and the lateral roots were the two sites for the bacteria entry into rice root. Therefore, we conclude that this Gram-positive nitrogen-fixer has a colonization pattern similar to those of many Gram-negative diazotrophs, such as Azospirillun brasilense Yu62 and Azoarcus sp. As far as we know, this is the first detailed report of the colonization pattern for Gram-positive diazotrophic Bacillus.     

东乡野生稻可培养内生细菌群落组成及多样性

内生细菌对宿主植物生长发育和生理代谢具有重要影响, 揭示其群落组成和多样性具有重要的生物学和生态学意义。本研究采用可培养法从东乡野生稻(Oryza rufipogon)不同组织中分离获得94株内生细菌, 基于16S rRNA基因序列比对和系统发育分析将其归属于变形菌门、放线菌门和厚壁菌门3门14科17属, 其中芽孢杆菌属(Bacillus)和微小菌属(Microbacterium)为优势属, 分别占总株数的27.7%和20.2%。不同组织中内生细菌的分布和多样性存在差异, 根中内生细菌数量最多(34株, 36.2%), 叶次之(32株, 34.0%), 茎最少(28株, 29.8%); 根中内生细菌Shannon-Wiener多样性指数(H′ = 2.52)、Simpson优势度指数(D = 0.88)和Pielou均匀度指数(E = 0.72)均高于茎与叶, 根和茎的Jaccard相似性系数(C = 0.29)低于根和叶(C = 0.47)以及茎和叶(C = 0.45)。研究结果表明, 东乡野生稻内生细菌具有丰富的物种多样性, 并且不同组织部位的内生细菌组成存在差异, 根中内生细菌多样性最丰富。     

Cd、Zn、Pb及其相互作用对烟草、小麦的影响

烟草对Cd、Zn、Pb是一种敏感性作物,Cd、Zn、Pb对烟草的影响比对小安、水稻都大。 Cd、Zn、Pb在烟草各器官中的累积随土壤Cd、Zn、Pb浓度的增高而增大。 Cd、Zn、Pb在烟草各器官中含量的次序为:茎叶>根>籽粒。它们在根中受阻,而较易转移到茎叶和籽粒中。 烟草对Pb的吸收比对Zn和Cd明显地低。Pb仍然是一种低吸收性元素。 土壤Zn增加,减低了烟草对Cd的吸收,而土壤Pb的增加,则促进了烟草对Cd的吸收。     

巴西固氮螺菌Yu62 draTG基因及其下游区域的定位诱变分析

用卡那霉素盒（Ｋｍ－ｃａｓｓｅｔｔｅ）插入法,对巴西固氮螺菌（Ａｚｏｓｐｉｒｉｌｌｕｍｂｒａｓｉｌｅｎｓｅ）Ｙｕ６２的ｄｒａＴＧ基因及其下游区域进行了诱变,并获得相应的突变株,研究表明ｄｒａＴ变突株的固氮酶活性不再受铵抑制,而ｄｒａＧ突变株在有铵时则丧失固氮酶活性,但当铵耗尽后却不能使像野生型菌株那样恢复活性,ｄｒａＴＧ下游区域突变株ＹＺ４（突变位点距ｄｒａＧ约２ｋｂ）在无氮及限铵条件下,其固氮酶     

Characteristics of infection of plants and their cultured tissue with cyanobacterial-bacterial symbiotic associations]

The infection of tobacco, nightshade, rice plants, and their tissue cultures with the cyanobacteria-bacteria symbiotic associations (CBSA) isolated from natural syncyanoses (the ferns Azolla pinnata and Azolla sp. and the cycad Encephalartos ferox) was studied. The inoculation of the intact plants or their cuttings with CBSA led to the colonization of the plant roots, stems, and leaves by cyanobacteria and their bacterial symbionts (referred to as satellite bacteria, SB). The sites of the long-term contact of plant organs with cyanobacteria were characterized by the formation of copious slime. On the roots of infected plants, one could observe the callus growth of cortical parenchyma cells and the formation of pseudonodules, in which SB cells gradually accumulated. In mixed cultures of plant callus tissues and the CBSA isolated from the ferns A. pinnata and Azolla sp., the callus tissue specifically influenced the growth of the CBSA components, causing (depending on the plant species and strain) either their balanced growth, or their cyclic growth, or the predominant growth of one of the CBSA components (either cyanobacteria or satellite bacteria). This phenomenon is proposed to be used for the dissociation of stable multicomponent natural symbiotic complexes and the selection of their particular components.     

Biochemical Characteristics of Rice Endophytic DiazotrophicBacteria and Their Infection to Tobacco and Maiz

Some biochemical properties of rice endophytic diazotrophic bacteria N1 ( Bacillus pumilus), N2 ( Enterobacter cloacae) and N3 ( Enterococcus gallinarum) were characterized in this study. The plasmids harboring the reporter gene lacZ fused to the promoters of nifH and nifHDK, and the reporter gene gfp as well, were first transformed into rice endophytic diazotrophic bacteria respectively through triparental mating. After the conjugated bacteria were inoculated into tobacco Yunyan 85 and maize Jitian 6, the staining of β-galactosidase was carried out on the tobacco and maize roots and the observation was made under the confocal laser scanning microscope, transmission electron microscope and scanning electron microscope. The results showed that the rice endophytic diazotrophic bacteria were not only present in epidermal cells, cortex cells and intercellular spaces of tobacco and maize roots, but also found in vascular tissue cells of maize stem also, indicating that the bacteria had migrated from the roots into the stem. Data showed that the the fresh weight of maize plants inoculated with rice endophytic diazotrophic bacteria N3 was 18.52% more than that of the control.