一株水稻促生长内生真菌的绿色荧光蛋白基因标记与示踪

将绿色荧光蛋白基因(gfp)转入到碱蓬内生真菌JP4-1中并检测菌株在水稻幼苗中的定殖情况。采用PEG-Ca Cl2介导的原生质体转化方法将携带gfp基因的p CT74质粒与菌株基因组整合获得转化子,用转化子侵染水稻幼苗,荧光显微镜下示踪JP4-1菌株及其侵染特性。转化子经连续传代6次仍能发出绿色荧光且荧光强度良好,能够稳定遗传;经PCR验证gfp基因已成功转入JP4-1菌株和水稻幼苗植株内并表达。转化可获得稳定表达GFP的JP4-1转化子,JP4-1菌株可定殖于水稻幼苗的根、茎、叶,定殖位置为细胞间隙,其促生作用与野生型菌株无明显差别。     

用gfp基因标记法研究大豆根瘤菌在大豆根部定殖结瘤情况

采用三亲本杂交的方法,将绿色荧光蛋白基因(gfp)转入高效、抗逆、广适应性的快生大豆根瘤菌Sinorhizobium frediiCCBAU 01287中,获得含gfp基因的转基因菌株CBAU 01287(G);平板传代和共生检测表明:外源质粒在CCBAU 01287中能够自我复制,稳定遗传。进一步研究表明,gfp标记菌CCBAU 01287(G)可用于实时监测根瘤菌在大豆根部的早期定殖情况和定殖密度的测定;标记菌株对大豆的生长及生物量的积累与出发菌株的效果无显著差异。     

水稻内生成团泛菌YS19与菠萝泛菌YJ76共培养提高环境胁迫生存适应性

成团泛菌(Pantoea agglomerans)YS19和菠萝泛菌(Pantoea ananatis)YJ76是从水稻(Oryza sativa)“越富”品种中分离得到的2株优势内生固氮细菌。利用YS19和YJ76共培养方式模拟菌体栖息微环境,探索潜在的种间生物效应,发现适量的YJ76能够促进YS19共质体的形成并且YJ76细胞可以参与到YS19的共质体结构中,YS19?YJ76接种量比例为10?3时促进效果最为显著。相比之下,当使用吲哚产量显著下降的YJ76突变株Δzwf与YS19共培养时,上述促进效应均显著减弱。与单独培养相比,共培养显著提高了两个菌株对重金属Zn²⁺、四环素、紫外线以及干燥的耐受能力,YS19分别提高了63.9%、56.0%、57.4%、72.0%,YJ76分别提高了147.9%、172.7%、266.3%、348.6%。结果表明水稻的两种主要内生菌YS19和YJ76在共栖息时表现为强烈的协同作用,而YJ76产生的吲哚信号分子在这个过程中起到重要调节作用。     

生防放线菌Ahn75的荧光标记及其在水稻中的定殖

【背景】目前gfp标记基因已成为研究靶标微生物与宿主之间互作的一种重要工具。利用gfp基因标记生防菌株,可以对生防菌株的生存及定殖能力进行有效追踪。【目的】对生防放线菌Ahn75进行荧光标记,探讨其在水稻中的定殖规律,为研究Ahn75的稻瘟病防治机制奠定基础。【方法】首先通过电激转化将含绿色荧光标记基因(gfp)的质粒pIJ8655导入大肠杆菌ET12567中,然后采用接合转移的方法将gfp整合到Ahn75基因组上;通过平板对峙试验检验Ahn75-GFP在标记绿色荧光后对稻瘟病病原菌的抑菌活性;采用喷施孢子液的方式将带荧光标记的Ahn75-GFP定殖水稻,并利用荧光显微镜观察生防菌在水稻中的定殖情况;对定殖水稻中的内生菌进行重分离,探究菌株在水稻组织中的分布规律。【结果】PCR扩增和荧光观察表明,绿色荧光标记基因成功整合到生防放线菌Ahn75中。通过平板对峙试验,发现Ahn75-GFP对稻瘟病病原菌抑菌活性与原始菌株没有显著差别。在荧光显微镜下,可以观察到Ahn75-GFP能稳定定殖于水稻的根、茎、叶等组织中,而水稻内生菌重分离试验表明该菌株在茎中的定殖力最强。【结论】获得一株绿色荧光标记生防菌株Ahn75-GFP,结果显示该菌株定殖水稻效果良好,这对于研究Ahn75的稻瘟病防治具有重要意义。     

板栗疫病菌绿色荧光与红色荧光蛋白共定位载体的构建

低毒病毒-板栗疫病菌组合是研究病毒与宿主相互作用的一个优秀的模式系统.我们构建了含绿色荧光蛋白基因gfp的载体pCPXHY2GFP与含红色荧光蛋白基因rfp的载体pCPXG418RFP,并用于转化野生型菌株EP155,获得了以潮霉素为筛选标记、表达绿色荧光蛋白的转化株pCPXHY2GFP/EP155和以G418为筛选标记、表达红色荧光蛋白的转化株pCPXG418RFP/EP155.将载体pCPXG418RFP转化pCPXHY2GFP/EP155,获得的转化株能观察到绿色荧光蛋白与红色荧光蛋白共定位的现象.板栗疫病菌绿色荧光与红色荧光共定位载体pCPXHY2GFP与pCPXG418RFP的构建,为深入研究病毒与宿主相互作用的分子机制提供了强有力的研究材料.     

成团泛菌YS19 symplasmata结构对菌体抵抗逆境的作用

成团泛菌（Pantoea agglomerans）YS19是从水稻“越富”品种中分离出的优势内生菌,其所形成的共质体（symplasmata）是一种与生物薄膜（biofilm）类似的多细胞聚集体结构,但细胞间联系比biofilm更加紧密。研究symplasmata结构对成团泛菌YS19抵抗逆境的贡献,有助于阐释内生菌与植物的相互作用的适应性。比较研究了symplasmata结构与散生菌体对于蔗糖渗透压冲击、重金属离子和干燥处理的抵抗能力差异,结果表明,与以散生状态存在的菌体相比,在面临逆境时形成symplasmata结构的菌体抗逆存活能力显著增强。     

绿色荧光蛋白基因原核表达载体的构建及其在昆虫肠道短短芽孢杆菌和苏云金芽孢杆菌中的表达

【目的】构建带有苏云金芽孢杆菌cry3a基因非芽孢依赖启动子和绿色荧光蛋白基因gfp(Green Fluorescent Protein)的原核表达载体,并转化从桑粒肩天牛幼虫肠道分离的两株常驻细菌短短芽孢杆菌CQUBb和苏云金芽孢杆菌CQUBt,以检测cry3a启动子在昆虫肠道常驻菌中的启动子活性,获得GFP标记菌株,为常驻菌在昆虫幼虫肠道中的定殖情况和杀虫工程菌的构建奠定基础。【方法】采用重叠延伸PCR将cry3a基因启动子和gfp基因进行融合,并与pHT304载体连接构建重组质粒pHT3AG,获得的重组质粒以电脉冲转化肠道常驻菌短短芽孢杆菌CQUBb和苏云金芽孢杆菌CQUBt,于可见光和荧光显微镜下观察荧光并通过SDS-PAGE分析重组菌株的蛋白表达情况,然后对重组菌株进行生长动力学分析和稳定性测试。【结果】重组菌在营养期大量组成型表达GFP,经电泳分离在凝胶上出现约29kDa的特异蛋白条带;重组菌生长曲线与出发菌没有显著差异,说明外源质粒未对宿主菌的生长带来明显不利影响;抗性条件下传代30次后两菌株外源质粒稳定性仍可达95%、67%;两个菌株比较,CQUBb比CQUBt质粒转化率高、重组菌GFP表达时间长、表达量大,并且重组菌株稳定性好。【结论】成功地将cry3a基因核心启动子和gfp基因转入桑粒肩天牛幼虫肠道常驻菌,实现了该启动子在Bt之外的菌株中发挥作用,构建了两个GFP标记菌株;重组基因工程菌株表达量大,稳定性好,可以用作昆虫肠道内微生态研究和芽孢杆菌表达系统以及杀虫菌株的构建。     

内生菌与植物的相互作用：促生与生物薄膜的形成

植物内生菌由于其独特的生态学地位而广受关注,近年来有关植物内生菌与宿主相互作用的研究取得了很大进展.本文综述了植物内生菌通过分泌促生物质、拮抗病原菌等实现与宿主共生互作,同时植物为内生菌提供适宜的黏附表面,使其形成以生物薄膜(biofilm)为主要形式的多细胞聚集体结构以更好地适应周围的生存环境,从而更加高效地对植物产生促生作用.本文论述了内生菌在与植物的互作中形成的多细胞聚集结构在抵抗非生物胁迫方面的独特生理及生态学意义,结合水稻内生成团泛菌YS19形成多细胞聚集体symplasmata现象及其生物学效应,对未来有关植物内生菌的研究方向提出了一些看法.     

植物内生菌定殖检测技术及其应用

自然环境中内生菌定殖于植物体内,对宿主植物产生多种有益效应,但是内生菌定殖情况难以检测,相关研究不够深入系统。目前在该领域使用较为广泛的检测技术包括：荧光标记、抗生素标记、荧光定量PCR和高通量测序等。内生菌通过孔隙伤口和降解细胞壁等方式侵染植物,通过种子垂直传递核心内生菌。对植物内生菌定殖的侵染方式、定殖方法和检测技术进行了归纳和整理,介绍了内生菌多种侵染和迁移途径,总结了目前内生菌定殖在生物防治、促进植物生长和污染修复等方面的功能,综述了多种检测方式在应用中的特点,以期为内生菌定殖植物的相关研究及其应用提供参考依据。     

用于植物病原细菌标记的pBB-GFP载体构建及应用

扩增青枯劳尔氏菌RipAK基因启动子序列,与lacZ基因融合得到p HM1:P_(RiPAK)LacZ。携带pHM1:P_(RiPAK)LacZ的青枯劳尔氏菌在营养丰富和基本培养基中都有LacZ活性,表明RipAK启动子可以推动lacZ基因的表达。为构建用于标记植物病原细菌的绿色荧光表达载体,把RipAK启动子和gfp基因克隆到质粒pBBR1MCS-5,使得gfp基因在RipAK启动子的驱动下表达;构建的表达载体pBB-GFP在大肠杆菌中即可表达绿色荧光蛋白。pBB-GFP载体能有效标记青枯劳尔氏菌、番茄细菌性斑点病菌和柑橘溃疡病菌,在荧光显微镜下观察到3种植物病原细菌呈短杆状,青枯劳尔氏菌还可形成多个菌体串联的线状结构。荧光标记对3种病原菌在寄主植物上的致病力没有影响,将标记菌株分别滴加在寄主植物叶片的创伤处,可观察到大量的绿色荧光聚集。本研究构建的pBB-GFP载体能用于多种植物病原细菌的绿色荧光标记,标记后的病原细菌在液体培养及侵染寄主植物过程中都能观察到荧光。     

Rice endophyte Pantoea agglomerans YS19 forms multicellular symplasmata via cell aggregation

Pantoea agglomerans is characterized by the formation of multicellular symplasmata. One unanswered question regarding this bacterium is how these structures are formed. In this study, the rice diazotrophic endophyte P. agglomerans YS19 was selected for exploration of this theme. YS19 was labeled with green fluorescent protein and the resulting recombinant YS19::gfp was observed to grow only slightly more slowly (a decrease of 5.5%) than the wild-type strain, and to show high GFP label stability (label loss rate 8.9218 x 10(-6) per generation, nearly reaching the generally accepted spontaneous mutation rate for most bacteria). YS19::gfp resembled the wild-type YS19 in symplasmata formation and growth profiles. Based on associated cultivation of both strains by mixing their individually cultivated single cells, symplasmata were formed and composed of both YS19::gfp and YS19, suggesting that YS19 formed symplasmata via aggregation, not proliferation, of the original single cells.     

Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum sp. isolated from wild rice species.

Nitrogen-fixing bacteria were isolated from the stems of wild and cultivated rice on a modified Rennie medium. Based on 16S ribosomal DNA (rDNA) sequences, the diazotrophic isolates were phylogenetically close to four genera: Herbaspirillum, Ideonella, Enterobacter, and Azospirillum. Phenotypic properties and signature sequences of 16S rDNA indicated that three isolates (B65, B501, and B512) belong to the Herbaspirillum genus. To examine whether Herbaspirillum sp. strain B501 isolated from wild rice, Oryza officinalis, endophytically colonizes rice plants, the gfp gene encoding green fluorescent protein (GFP) was introduced into the bacteria. Observations by fluorescence stereomicroscopy showed that the GFP-tagged bacteria colonized shoots and seeds of aseptically grown seedlings of the original wild rice after inoculation of the seeds. Conversely, for cultivated rice Oryza sativa, no GFP fluorescence was observed for shoots and only weak signals were observed for seeds. Observations by fluorescence and electron microscopy revealed that Herbaspirillum sp. strain B501 colonized mainly intercellular spaces in the leaves of wild rice. Colony counts of surface-sterilized rice seedlings inoculated with the GFP-tagged bacteria indicated significantly more bacterial populations inside the original wild rice than in cultivated rice varieties. Moreover, after bacterial inoculation, in planta nitrogen fixation in young seedlings of wild rice, O. officinalis, was detected by the acetylene reduction and (15)N(2) gas incorporation assays. Therefore, we conclude that Herbaspirillum sp. strain B501 is a diazotrophic endophyte compatible with wild rice, particularly O. officinalis.     

Characterization of a small cryptic plasmid from endophytic Pantoea agglomerans and its use in the construction of an expression vector

A circular cryptic plasmid named pPAGA (2,734 bp) was isolated from Pantoea agglomerans strain EGE6 (an endophytic bacterial isolate from eucalyptus). Sequence analysis revealed that the plasmid has a G+C content of 51% and contains four potential ORFs, 238(A), 250(B), 131(C), and 129(D) amino acids in length without homology to known proteins. The shuttle vector pLGM1 was constructed by combining the pPAGA plasmid with pGFPmut3.0 (which harbors a gene encoding green fluorescent protein, GFP), and the resulting construct was used to over-express GFP in E. coli and P. agglomerans cells. GFP production was used to monitor the colonization of strain EGE6gfp in various plant tissues by fluorescence microscopy. Analysis of EGE6gfp colonization showed that 14 days after inoculation, the strain occupied the inner tissue of Eucalyptus grandis roots, preferentially colonizing the xylem vessels of the host plants.     

Rice endophyte Pantoea agglomerans YS19 promotes host plant growth and affects allocations of host photosynthates

AIMS: The aims of the study were to identify the effects of rice endophyte Pantoea agglomerans YS19 on host plant growth and allocations of photosynthates. METHODS AND RESULTS: Endophytic diazotrophic YS19 showed nitrogen-fixing activity in N-free medium, and produced four categories of phytohormones which were indole-3-acetic acid, abscisic acid, gibberellic acid and cytokinin in Luria-Bertani medium. Inoculation of YS19 improved the biomass of the 12-day-cultivated host rice seedlings by 63.4% on N-free medium or by 18.7% on N-supplemented medium. Spraying of YS19 cell culture onto the rice plants at the premilk stage enhanced the transportation of the photosynthetic assimilation product from the source (flag leaves) to the sink (stachys) significantly. The formation of the plant sink was obviously inhibited when YS19 cell culture was applied at the late milk stage. CONCLUSIONS: This research suggests that endophyte YS19 promotes host rice plant growth and affects allocations of host photosynthates. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggested that YS19 possesses the potential for increasing rice production in field application. Meanwhile, a suitable plant growth stage must be selected for the foliar spraying of YS19 cell culture.     

Endophytic Colonization and In Planta Nitrogen Fixation by a Herbaspirillum sp. Isolated from Wild Rice Species

Nitrogen-fixing bacteria were isolated from the stems of wild and cultivated rice on a modified Rennie medium. Based on 16S ribosomal DNA (rDNA) sequences, the diazotrophic isolates were phylogenetically close to four genera: Herbaspirillum, Ideonella, Enterobacter, and Azospirillum. Phenotypic properties and signature sequences of 16S rDNA indicated that three isolates (B65, B501, and B512) belong to the Herbaspirillum genus. To examine whether Herbaspirillum sp. strain B501 isolated from wild rice, Oryza officinalis, endophytically colonizes rice plants, the gfp gene encoding green fluorescent protein (GFP) was introduced into the bacteria. Observations by fluorescence stereomicroscopy showed that the GFP-tagged bacteria colonized shoots and seeds of aseptically grown seedlings of the original wild rice after inoculation of the seeds. Conversely, for cultivated rice Oryza sativa, no GFP fluorescence was observed for shoots and only weak signals were observed for seeds. Observations by fluorescence and electron microscopy revealed that Herbaspirillum sp. strain B501 colonized mainly intercellular spaces in the leaves of wild rice. Colony counts of surface-sterilized rice seedlings inoculated with the GFP-tagged bacteria indicated significantly more bacterial populations inside the original wild rice than in cultivated rice varieties. Moreover, after bacterial inoculation, in planta nitrogen fixation in young seedlings of wild rice, O. officinalis, was detected by the acetylene reduction and ¹⁵N₂ gas incorporation assays. Therefore, we conclude that Herbaspirillum sp. strain B501 is a diazotrophic endophyte compatible with wild rice, particularly O. officinalis.     

In vitro symplasmata formation in the rice diazotrophic endophyte Pantoea agglomerans YS19

Pantoea (formerly Enterobacter) agglomerans YS19 is an endophytic diazotrophic bacterium isolated from rice (Oryza sativa cv. Yuefu) grown in temperate climatic regions in west Beijing (China). The bacterium forms aggregate structures called `symplasmata'. A symplasmatum is a multicellular aggregate structure in which several (at least two) to hundreds of individual cells tightly bind together. The studies on the symplasmata formation of YS19 showed that there were two growth stages for YS19, including the single cell stage existing before exponential growth phase and the symplasmata forming stage starting at the early stationary growth phase in liquid GY (glucose yeast extract) medium or at the end of the exponential growth phase in liquid LB (Luria-Bertani) medium. There was a correlation between symplasmata formation and bacterial growth phase. When the medium was acidified, the cell growth rate was affected by the low pH of the medium, but the time required for symplasmata formation was not influenced by it. YS19 also formed symplasmata on agar medium, where more symplasmata were formed than in liquid medium. The volume of individual constitutional cells of symplasmata was sharply decreased by more than a half in comparison with that of the single cells existing before symplasmata formation. On all the media tested, YS19 formed symplasmata in most of the cell growth phases. The genome DNA/DNA homology between P. agglomerans YS19 and type strain P. agglomerans JCM1236^T (ATCC27155^T) was determined as 90.1%, confirming its membership of P. agglomerans. In order to investigate the phylogenetic relationships of YS19 at the intraspecific, intrageneric and super-generic level, the 16S rDNA similarities between strain YS19 and 17 other strains of Pantoea and 4 representatives of the closely related genera were analyzed. All the strains of Pantoea were clustered into 5 groups, and YS19 was clustered in a unique branch. The 16S rDNA similarity between YS19 and type strain JCM1236^T was 93.9%, much lower than the generally accepted value (=97%) for members of the same species, indicating that the 16S rDNA of YS19 has a distinct molecular characteristic.     

Differences between Pseudomonas syringae pv. syringae B728a and Pantoea agglomerans BRT98 in epiphytic and endophytic colonization of leaves

The leaf colonization strategies of two bacterial strains were investigated. The foliar pathogen Pseudomonas syringae pv. syringae strain B728a and the nonpathogen Pantoea agglomerans strain BRT98 were marked with a green fluorescent protein, and surface (epiphytic) and subsurface (endophytic) sites of bean and maize leaves in the laboratory and the field were monitored to see if populations of these strains developed. The populations were monitored using both fluorescence microscopy and counts of culturable cells recovered from nonsterilized and surface-sterilized leaves. The P. agglomerans strain exclusively colonized epiphytic sites on the two plant species. Under favorable conditions, the P. agglomerans strain formed aggregates that often extended over multiple epidermal cells. The P. syringae pv. syringae strain established epiphytic and endophytic populations on asymptomatic leaves of the two plant species in the field, with most of the P. syringae pv. syringae B728a cells remaining in epiphytic sites of the maize leaves and an increasing number occupying endophytic sites of the bean leaves in the 15-day monitoring period. The epiphytic P. syringae pv. syringae B728a populations appeared to originate primarily from multiplication in surface sites rather than from the movement of cells from subsurface to surface sites. The endophytic P. syringae pv. syringae B728a populations appeared to originate primarily from inward movement through the stomata, with higher levels of multiplication occurring in bean than in maize. A rainstorm involving a high raindrop momentum was associated with rapid growth of the P. agglomerans strain on both plant species and with rapid growth of both the epiphytic and endophytic populations of the P. syringae pv. syringae strain on bean but not with growth of the P. syringae pv. syringae strain on maize. These results demonstrate that the two bacterial strains employed distinct colonization strategies and that the epiphytic and endophytic population dynamics of the pathogenic P. syringae pv. syringae strain were dependent on the plant species, whereas those of the nonpathogenic P. agglomerans strain were not.