慢生型大豆根瘤菌nfe基因的分子克隆

ｎｆｅＣ基因是从慢生型大豆根瘤菌中克隆到的,与竞争结瘤有关的基因。本研究从慢生型大豆根瘤菌菌株ＧＸ２０１的ｐＬＡＦＲ３为载体的基因文库中,筛选出与ｎｆｅ同源基因克隆。以转座子Ｔｎ５ｇｕｓＡ５诱变获得了ｇｕｓ基因表达的Ｔｎ５ｇｕｓＡ５插入突变质粒。     

苜蓿中华根瘤菌042B与公共结瘤基因nodABC有关的大片段物？…

将苜蓿中华根瘤菌０４２Ｂ总ＤＮＡ提取,用Ｓａｕ３ＡＩ部分酶切后回收２０－３０ｋｂ长的片段,以ｐＬＡＦＲ３为载体,构建基因文库。用苜蓿中华根瘤菌ｎｏｄＡＢＣ作探针,经Ｓｏｕｔｈｅｒｎ杂交获得了三个阳性克隆Ａ,Ｂ和Ｃ,将这３个阳性克隆所携带的质粒分别命名为ｐＸＣＡ,ｐＸＣＢ,ｐＸＣＣ。在辅助质粒ｐＲＫ２０１３的帮助下,将这３个质粒分别转入ｎｏｄＣ＾－突变株ＡＫ１６５７后接种苜蓿,携带ｐＸＣＢ质粒的接合     

海南根瘤菌S25的质粒生物学特性

根瘤菌Ｓ２５从海南岛山毛豆分离得到，在分类上比较独特。该菌株具有较强抗逆性，如耐酸，耐亚硝酸，对多种抗生素具有抗性。质粒检测，结果显示，它含有一条大允１５０ｋｂ的大质粒。以共同结瘤基因ｎｏｄＡＢＣ和固氮酶结构基因ｎｉｆＨＤＫ为探针与质粒进行分子杂交，结果表明，该质粒载有结瘤与因氮基因。     

根瘤菌结瘤基因与竞争结瘤基因的研究进展

根瘤菌的结瘤基因与竞争结瘤基因在根瘤菌与结瘤的过程中起着关键的作用。随着研究的深入,已经发现,分离和定位了数十个结瘤基因和几个与竞争结瘤有关的基因,本文将简要地介绍了这方面的研究成果。     

慢生型大豆根瘤菌putA基因的功能分析

重组质粒pGX300上的putA基因是从慢生型大豆根瘤菌GX201中克隆到的编码脯氨酸脱氢酶的基因。通过Tn5gusA5定位诱变方法构建了putA基因的突变株GX20120，实验表明，GX20120的结瘤时间推迟，竞争结瘤能力降低。     

Frankia共生固氮基因的分离与克隆

利用ＤＮＡ－ＤＮＡ杂交方法，分离细枝木麻黄的Ｆｒａｎｋｉａ菌株Ｃｏ０１的ｎｉｆ克隆ｐＣｃ１ＧＸ，赤杨内生菌株Ａｔ４的ｎｉｆ克隆ｐＡｔ１ＧＸ及沙棘的ＦｒａｎｋｉａＨｒ１８的ｎｉｆ克隆ｐＨｒ１８ＧＸ、ｐＨｒ１１－③ＧＸ。用基因功能互补法，从Ｆｒａｎｋｉａ菌株Ａｔ４的基因文库中分离到二个可能可互补豌豆根瘤菌ｎｏｄ基因功能的克隆ｐＡｔ２ＧＸ、ｐＡｔ３ＧＸ，并制作了ｐＡｔ２ＧＸ、ｐＡｔ３ＧＸ的亚克隆，予进一步实验，获得充分的证据证明ｐＡｔ２ＧＸ、ｐＡｔ３ＧＸ是否带有Ｆｒａｎｋｉａ菌株Ａｔ４的ｎｏｄ基因．     

根瘤菌结瘤基因的表达调控研究概况

根瘤菌结瘤基因的表达调控在根瘤菌与植物的共生结瘤过程中起着十分重要的作用。随着研究的深入,发现根瘤菌的结瘤过程不仅与根瘤菌结瘤基因表达调控有关,而且与寄主植物的信号分子如黄酮类物质有关。根瘤菌结瘤基因的表达调控有一个复杂的过程,本文将简要地介绍这方面的研究成果。     

GUS基因标记法对慢生花生根瘤菌竞争结瘤和接种效果的研究

应用GUS基因标记技术,可简便、快速、准确、原位、直观地确定标记花生根瘤菌株形成的根瘤,从而方便地研究标记菌株与土著根瘤菌的竞争结瘤能力。无氮水培试验表明,标记菌株gusA4-5、gusA2-9分别与土著菌混和接种占瘤率为71.4%、77.0%。盆栽试验表明,接种供试菌株Spr4-5、Spr2-9占瘤率分别为57.9%、63.0%,比对照极显著增产52.5%、22.7%;接种Spr4-5比Spr2-9极显著增产24.2%。初步说明两个供试菌株的竞争结瘤力比土著根瘤菌强,菌株Spr2-9强于Spr4-5;Spr4-5比Spr2-9有效性高,是结瘤适量,竞争结瘤能力强的高效菌株。     

慢生型大豆根瘤菌菌株GX201的脯氨酸脱氢酶基因(putA)的分子克隆

根据已报道的脯氨酸脱氢酶基因（ｐｕｔＡ）序列,通过ＰＣＲ方法,从慢生型大豆根瘤菌菌株ＧＸ２０１的总ＤＮＡ中扩增到—ＰＣＲ产物。序列分析表明,该ＰＣＲ产物的长度为４１８ｂｐ,与已报道的ｐｕｔＡ基因具有９３．５％的同源性。以广谱寄主范围质粒ｐＬＡＦＲ３为载体,在大肠杆菌ＤＨ５α中构建了ＧＸ２０１的基因文库,并以该ＰＣＲ产物为探针,从基因文库中筛选到一重组质粒ｐＧＸＮ３００。     

马占相思根瘤菌结瘤固氮特性的分析

相思属（Acacia）树种是速生的热带、亚热带豆科植物。对现在大面积种植的相思树品种——马占相思的根瘤菌进行分离和筛选，获得菌株若干株，对它们的结瘤固氮特性和对马占相思、大叶相思及厚荚相思生长的影响等进行了初步研究。结果表明。无氮条件下接种马占相思根瘤菌，能显著促进这3个树种的株重、株高和生物量的增长；苗圃接种马占相思根瘤菌。马占相思苗高增加27．6％，地径增加14．8％，植株鲜重增加32．6％，结瘤数增加83．8％。     

Frankia菌株At4的类nodD基因的克隆与研究

通过功能互补法从Frankia菌株At4的基因文库中筛选到可互补豌豆根瘤菌nodD基因功能的pAt2GX和pAt3GX,把pAt2GX和pAt3GX重新导入tmdD突变体中,接种豌豆苗,可观察到nodD突变体恢复了结瘤功能。扫描电镜观察、根瘤内生菌抗性标记检测及内生菌重组质粒的酶切分析均表明nadD突变体恢复结瘤功能是山于带有外源质粒pAt2GX和pAt3GX所致,这就证明了pAt2GX和pAt3GX带有类nodD基因功能的DNA片段。酶切分析及DNA-DNA杂交实验结果说明pAt2GX和pAt3GX不属于同—克隆,但这两个质粒的DNA之间有同源区。     

Characterization of local rhizobia in Thailand and distribution of malic enzymes

TWenty-six isolates were obtained from nodules of various legume plants (Glycine max, Vigna sinensis, Arachis hypogaea, Desmanthus virgatus, Acacia mangium, Centrosema pascuorum, Pterocarpus indicus, Xylia xylocarpa, and Sesbania rostrata) in Thailand. After confirming their nodulation and nitrogen-fixing abilities, they were identified by 16S rRNA gene analysis as Bradyrhizobium japonicum, Bradyrhizobium elkanii, Rhizobium leguminosarum, Rhizobium gallicum, and Rhizobium galegae. Using these local isolates, the distribution of the activities of both NAD⁺-dependent (DME: EC 1.1.1.39) and NADP⁺-dependent (TME: EC 1.1.1.40) malic enzymes was surveyed. The malic enzyme activities were present in all the isolated rhizobia and in other 17 local Bradyrhizobium strains in Thailand. In almost all the rhizobia, the DME activity predominated whereas the TME activity predominated only in the Rhizobium gallicum strains that were major symbionts of Sesbania rostrata. Southern hybridization analysis was performed to survey the distribution of the malic enzyme genes among the local rhizobia, which are similar to those of B. japonicum. DNA probes (ME1 for DME and ME2 for TME) were prepared by polymerase chain reaction (PCR) using degenerated primers from conserved regions of the protein sequences of bacterial malic enzymes. Southern blot analysis with ME1 as a probe showed a single band in about half of the isolates, especially in B. japonicum and R. leguminosarum strains, suggesting the wide distribution of such DME genes among local rhizobia. In contrast, Southern blot analysis with ME2 as a probe detected a single band only in five B. japonicum strains, suggesting that the TME genes, which are similar to those of B. japonicum, would be unique in a group of B. japonicum.     

Genetic diversity and relationship between Bradyrhizobium strains isolated from blackgram and cowpea

The genetic diversity of bradyrhizobial strains associated with blackgram and cowpea grown in two different agricultural soils (non-saline and saline) along the coastline of Tamil Nadu has been analysed. Phenotypically indistinguishable isolates were analysed for DNA polymorphism using random amplification of polymorphic DNA (RAPD) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of 16S rDNA and nifD. Although these bacteria belong to a group with a broad host range, RAPD analysis showed a considerable level of genetic diversity among the strains isolated from different host plants. Soil pH and salinity seem to have an effect on the selection of natural populations as revealed by PCR-RFLP of 16S rDNA. A combination of PCR-RFLP genotyping with nodulation studies indicates that monocropping of blackgram and the salinity of the soil have made ineffective rhizobia the dominant genotype, thereby creating an ecological burden on their other compatible hosts. A group of strains and a type strain sharing three different 16S PCR-RFLP types were shown to have the same set of symbiotic genes as inferred from the PCR-RFLP pattern of nifD. Another group of cowpea rhizobia that were found to be effective nitrogen fixers and sharing distinct 16S profiles were found to have a different set of symbiotic genes.     

Lentil (Lens culinaris L.) growth promoting rhizobacteria and their effect on nodulation in coinoculation with rhizobia

Lentil is cultivated in Chilean Mediterranean drylands, in areas with soils that are nutrient depleted and eroded. Inoculation of lentil with rhizobia in co-inoculation with growth promoting rhizobacteria would allow higher biomass and an opportunity for early nodulation and increased nitrogen fixation. The objective of this research was to select rhizosferic bacteria (PGPR) from lentils and to evaluate their effect on lentil nodulation in co-inoculation with rhizobia. Sixty three lentil rhizobacteria isolates where obtained from nine soils in the mediterranean area. These were fingerprinted through BOX-PCR reducing the number to 57 distinct strains. The strains were evaluated for ACCdeaminase activity, IAA production and compatibility with rhizobia. Seventeen strains showed ACC-deaminase activity, all of them synthesized IAA and 38 were compatible with the rhizobia. Ten selected strains were identified as Pseudomonas spp. through 16S rRNA sequencing. The strains were inoculated in lentil seedlings growing on seed germination pouches, to evaluate nodule formation. The strain LY50a increased early nodulation in 85% in comparison to the control inoculated with rhizobia (AG-84) only. In conclusion, bacteria from the rhizosphere from Mediterranean soils of Chile can be used as nodulation promoters in lentils.     

Colonization of rhizospheres and nodulation of two Vigna species by rhizobia inoculated onto seed: Influence of soil

Changes in the populations of Rhizobium strains CB756str, CB985 and CB1024strspc in the rhizospheres of cowpea (Vigna unguiculata) and black gram (V. mungo) grown at three sites were evaluated. The population dynamics of the three rhizobia varied with soil type but the strain responses on the two legumes were generally similar. Most noticeable was the ability of CB756str to grow in the sandy soil (Beerwah) but not in the heavy clays (Narayen and Emerald). In contrast, the levels of CB1024strspc and CB985 generally increased in the clay soils.Nodulation (% due to the inoculum strain) did not always reflect events within the rhizosphere. Although not suited to Narayen, CB756str formed a similar proportion of the nodule population of black gram as CB1024strspc but this may have been due to higher seed inoculum levels of CB756str. At Emerald nodulation by all three strains of rhizobia was poor regardless of the success in colonization of the rhizosphere. Successful competition for nodule sites by native rhizobia may contribute to this discrepancy between Narayen and Emerald although lower seed inoculum levels at Emerald may also have been important.Nodule decay was consistently associated with a large increase in the number of rhizobia per root system. This is likely to be important in the survival of strains into the following season.Comparisons of nodulation by parent and mutant rhizobia suggested that resistance to antibotics may have slightly reduced nodule forming ability for CB1024strspc on black gram at Emerald.     

Renodulation and characterization of Rhizobium isolates from cicer milkvetch (Astragalus cicer L.)

In 1993 and 1994, 12 bacterial isolates were isolated from root nodules of cicer milkvetch (Astragalus cicer). In the tests for nodulation of A. cicer by these bacterial isolates, five were found to form hypertrophic structures, while only two formed true nodules. These true nodules were formed in a sterilized soil system. This system might be able to act as a DNA donor to provide residual DNA to other microbes in the soil. The rhizobial isolates were thought to have lost genetic material crucial to nodulation during the isolation process. This hypothesis was supported by an experiment in which isolate B2 was able to nodulate A. cicer in vermiculite culture after being mixed with heat-killed rhizobia, Rhizobium leguminosarum bv. trifolii and R. loti. The nodulation would not occur in vermiculite culture system without the heat-killed rhizobia. Based on the biochemical data, the B2 and 9462L, which formed true nodules with A. cicer, were closely related. The rhizobia type cultures that nodulate A. cicer include Bradyrhizobium japonicum, Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viceae, and R. loti. All of these rhizobia were from different cross-inoculation groups. The B2 and 9462L isolates could only nodulate Medicago sativa, Phaseolus vulgaris, and Melilotus officinalis, but not these species within the genus from which they were isolated: Astragalus. The traditional cross-inoculation group concept obviously does not fit well in the classification of rhizobia associated with Astragalus. The rhizobia isolated from A. cicer can be quite different, and the rhizobia able to renodulate A. cicer also quite diverse. Received: 27 June 1996     

硼对大豆结瘤和固氮作用的影响——Ⅰ.对生长、营养元素吸收与分布和固氮量影响

在短期无氮营养液栽培(28天)条件下,研究缺硼和正常供硼处理对Bragg大豆品种及不结瘤突变体nod49和超结瘤突变体nts382生长、矿质元素吸收与分布及固氮量的影响。试验结果表明:1.在正常供硼的条件下,不结瘤突变体nod的植株生长量,株高、主根生长均高于超结瘤突变体及其亲本。缺硼处理时三种基因型大豆的生长均受到明显的抑制作用。无论是正常供硼还是缺硼处理,超结瘤突变体nts382根系的生长量均明显小于其它基因型。2.正常供硼处理时三种基因型大豆地上部、根及nts382的根瘤中硼的浓度和积累量均高于缺硼处理。缺硼处理,超结瘤突变体及其亲本地上部和根中大部分矿质元素浓度高于正常供硼处理,但积累总量低于正常供硼处理或变化不大。施硼处理的nts382和Bragg,根和根瘤中钙的浓度和积累量明显降低,而地上部变化不大,同时在nts382根瘤中,锌、锰、铁、铜浓度和含量也下降,而钼含量增加。3.施硼处理和Bragg和nts382地上部、根及根瘤中氮的浓度和含量均显著高于缺硼处理,其中超结瘤突变体中氮浓度和总量最高。在缺硼条件下,Bragg和超结瘤突变体能够结瘤,但是Bragg大豆根瘤的固氮作用完全受到抑制,而nts382根瘤还能保持较强的固氮能力。     

Cropping history affects nodulation and symbiotic efficiency of distinct hairy vetch (Vicia villosa Roth.) genotypes with resident soil rhizobia

Compatible rhizobia strains are essential for nodulation and biological nitrogen fixation (BNF) of hairy vetch (Vicia villosa Roth, HV). We evaluated how past HV cultivation affected nodulation and BNF across host genotypes. Five groups of similar HV genotypes were inoculated with soil dilutions from six paired fields, three with 10-year HV cultivation history (HV+) and three with no history (HV?), and used to determine efficiency of rhizobia nodulation and BNF. Nodulation was equated to nodule number and mass, BNF to plant N and Rhizobium leguminosarum biovar viceae (Rlv) soil cell counts using qPCR to generate an amplicon of targeted Rlv nodD genes. Both HV cultivation history and genotype affected BNF parameters. Plants inoculated with HV+ soil dilutions averaged 60 and 70 % greater nodule number and mass, respectively. Such plants also had greater biomass and tissue N than those inoculated with HV? soil. Plant biomass and tissue N were strongly correlated to nodule mass (r ²?=?0.80 and 0.50, respectively), while correlations to nodule number were low (r ²?=?0.50 and 0.31, respectively). Although hairy vetch rhizobia occur naturally in soils, past cultivation of HV was shown in this study to enhance nodulation gene-carrying Rlv population size and/or efficiency of rhizobia capable of nodulation and N fixation.     

硼对大豆结瘤和固氮作用的影响——Ⅰ.对生长、营养元素吸收与分布和固氮量影响

在短期无氮营养液栽培(28天)条件下,研究缺硼和正常供硼处理对Bragg大豆品种及不结瘤突变体nod49和超结瘤突变体nts382生长、矿质元素吸收与分布及固氮量的影响。试验结果表明:1.在正常供硼的条件下,不结瘤突变体nod的植株生长量,株高、主根生长均高于超结瘤突变体及其亲本。缺硼处理时三种基因型大豆的生长均受到明显的抑制作用。无论是正常供硼还是缺硼处理,超结瘤突变体nts382根系的生长量均明显小于其它基因型。2.正常供硼处理时三种基因型大豆地上部、根及nts382的根瘤中硼的浓度和积累量均高于缺硼处理。缺硼处理,超结瘤突变体及其亲本地上部和根中大部分矿质元素浓度高于正常供硼处理,但积累总量低于正常供硼处理或变化不大。施硼处理的nts382和Bragg,根和根瘤中钙的浓度和积累量明显降低,而地上部变化不大,同时在nts382根瘤中,锌、锰、铁、铜浓度和含量也下降,而钼含量增加。3.施硼处理和Bragg和nts382地上部、根及根瘤中氮的浓度和含量均显著高于缺硼处理,其中超结瘤突变体中氮浓度和总量最高。在缺硼条件下,Bragg和超结瘤突变体能够结瘤,但是Bragg大豆根瘤的固氮作用完全受到抑制,而nts382根瘤还能保持较强的固氮能力。