Legumes acquire access to atmospheric nitrogen through nitrogen fixation by rhizobia in root nodu... more Legumes acquire access to atmospheric nitrogen through nitrogen fixation by rhizobia in root nodules. Rhizobia are soil dwelling organisms and there is a tremendous diversity of rhizobial species in different habitats. From the legume perspective, host range is a compromise between the ability to colonize new habitats, where the preferred symbiotic partner may be absent, and guarding against infection by suboptimal nitrogen fixers. Here, we investigate natural variation in rhizobial host range across Lotus species. We find that Lotus burttii is considerably more promiscuous than Lotus japonicus, represented by the Gifu accession, in its interactions with rhizobia. This promiscuity allows Lotus burttii to form nodules with Mesorhizobium, Rhizobium, Sinorhizobium, Bradyrhizobium, and Allorhizobium species that represent five distinct genera. Using recombinant inbred lines, we have mapped the Gifu/burttii promiscuity QTL to the same genetic locus regardless of rhizobial genus, suggesti...
Sinorhizobium fredii HH103-Rifr, a broad host range rhizobial strain, induces nitrogen-fixing nod... more Sinorhizobium fredii HH103-Rifr, a broad host range rhizobial strain, induces nitrogen-fixing nodules in Lotus burttii but ineffective nodules in L. japonicus. Confocal microscopy studies showed that Mesorhizobium loti MAFF303099 and S. fredii HH103-Rifr invade L. burttii roots through infection threads or epidermal cracks, respectively. Infection threads in root hairs were not observed in L. burttii plants inoculated with S. fredii HH103-Rifr. A S. fredii HH103-Rifr nodA mutant failed to nodulate L. burttii, demonstrating that Nod factors are strictly necessary for this crack-entry mode, and a noeL mutant was also severely impaired in L. burttii nodulation, indicating that the presence of fucosyl residues in the Nod factor is symbiotically relevant. However, significant symbiotic impacts due to the absence of methylation or to acetylation of the fucosyl residue were not detected. In contrast S. fredii HH103-Rifr mutants showing lipopolysaccharide alterations had reduced symbiotic c...
Characterization of monogenic symbiotic mutant phenotypes found by forward genetic screens has be... more Characterization of monogenic symbiotic mutant phenotypes found by forward genetic screens has been instrumental for establishing the framework of genes coordinating nodule formation in the model legumes Lotus japonicus and Medicago truncatula. To complement this approach it is now important to investigate more complex traits governing symbiosis. We are in the process of transforming the genomic resources of the model legume L. japonicus to allow QTL and genome-wide association studies of multigene inherited traits to be combined with efficient validation by reverse genetics. These approaches require a complete and well-annotated genome, a catalog of natural variation, and a comprehensive mutant collection. We have produced accurate gene models using a hierarchical selection scheme that combines mRNA-seq data, ab initio modeling and homology information. Using these models, we have annotated the effects on gene structure and coding sequence of SNPs detected by full genome re-sequenc...
Characterization of monogenic symbiotic mutant phenotypes found by forward genetic screens has be... more Characterization of monogenic symbiotic mutant phenotypes found by forward genetic screens has been instrumental for establishing the framework of genes coordinating nodule formation in the model legumes Lotus japonicus and Medicago truncatula. To complement this approach it is now important to investigate more complex traits governing symbiosis. We are in the process of transforming the genomic resources of the model legume L. japonicus to allow QTL and genome-wide association studies of multigene inherited traits to be combined with efficient validation by reverse genetics. Our results and experiences with genome assembly and annotation, SNP marker generation, QTL and association analysis, as well the development of a sequenced insertion mutant population will be discussed. The reference genome sequence is being updated by integrating Illumina reads with existing shotgun and BAC Sanger data. By sequencing 30 additional Lotus accessions, a dense set of markers has been generated, a...
International Journal of Systematic and Evolutionary Microbiology, 2010
A nitrogen-fixing bacterium, designated strain S27T, was isolated from rhizosphere soil of Sophor... more A nitrogen-fixing bacterium, designated strain S27T, was isolated from rhizosphere soil of Sophora japonica. Phylogenetic analysis based on a fragment of the nifH gene and the full-length 16S rRNA gene sequence revealed that strain S27T is a member of the genus Paenibacillus. High levels of 16S rRNA gene sequence similarity were found between strain S27T and Paenibacillus durus DSM 1735T (97.3 %), Paenibacillus sabinae DSM 17841T (96.9 %), Paenibacillus forsythiae DSM 17842T (96.7 %) and Paenibacillus zanthoxyli DSM 18202T (96.6 %). However, DNA–DNA hybridization values between strain S27T and the four type strains were 37.64 %, 23.12 %, 25.6 % and 34.99 %, respectively. Levels of 16S rRNA gene sequence similarity between strain S27T and the type strains of other recognized members of the genus Paenibacillus were below 96.5 %. The DNA G+C content of strain S27T was 46.0 mol%. The major fatty acids were anteiso-C15 : 0, C16 : 0 and iso-C16 : 0. The major isoprenoid quinone was MK-7. ...
International Journal of Systematic and Evolutionary Microbiology, 2010
A nitrogen-fixing bacterium, designated strain Be17T, was isolated from rhizosphere soil of Begon... more A nitrogen-fixing bacterium, designated strain Be17T, was isolated from rhizosphere soil of Begonia semperflorens planted in Beijing Botanical Garden, PR China. Phylogenetic analyses based on a segment of the nifH gene sequence and a full-length 16S rRNA gene sequence revealed that strain Be17T was a member of the genus Paenibacillus. High levels of 16S rRNA gene sequence similarity were found between strain Be17T and Paenibacillus graminis RSA19T (97.9 %), Paenibacillus sonchi LMG 24727T (97.8 %), Paenibacillus riograndensis CECT 7330T (96.2 %) and Paenibacillus borealis DSM 13188T (96.1 %), respectively. Levels of 16S rRNA gene sequence similarity between strain Be17T and the type strains of other recognized members of the genus Paenibacillus were below 96.0 %. However, the DNA–DNA hybridization values between strain Be17T and P. graminis RSA19T, P. sonchi LMG 24727T and P. riograndensis CECT 7330T were 47.9 %, 38.7 % and 37.5 %, respectively. The DNA G+C content of strain Be17T w...
The ethylene-forming enzyme gene (efe) from Pseudomonas syringae pv. glycinea was transferred int... more The ethylene-forming enzyme gene (efe) from Pseudomonas syringae pv. glycinea was transferred into Pseudomonas putida KT2440 by recombination at five of the seven 16S rDNA sites. PCR analysis demonstrated that strains DC1, DC2 and DC3 contained three, four and five copies of efe, respectively. In contrast to the parent strain which produced ethylene at 14.7 mg h(-1) g(-1) dry weight, strains DC1, DC2 and DC3 produced ethylene at 36.2, 47.2 and 53.8 mg h(-1) g(-1) dry weight, respectively. Quantitative PCR showed that efe mRNA levels increased with increasing efe copy numbers. When additional copies of efe were introduced into strain DC3 via the broad-host-range plasmid pBBR1MCS2 an ethylene production rate of 80.2 mg h(-1) g(-1) dry weight was obtained and 489 mg ethylene was produced in 24h corresponding to a conversion rate of 21.7 mg ethylene g(-1) glucose. Our results indicated that P. putida KT2440 could be genetically modified to be a promising biomaterial producer.
Pleurotus eryngii was transformed using a polyethylene glycol-mediated method. A plasmid, pEPUGH,... more Pleurotus eryngii was transformed using a polyethylene glycol-mediated method. A plasmid, pEPUGH, containing a reporter gene (enhanced green fluorescent protein gene, egfp) and a positive selectable marker gene (hygromycin phosphotransferase gene, hph) was constructed. The fused egfp-hph gene was placed under the control of the strong and constitutive native gpd promoter from P. eryngii. The recombinant plasmid was used to transform of P. eryngii protoplasts. Successful transformation was demonstrated by molecular analyses. Moreover, the mycelia of the transformants showed green epipolic dispersion on fluorescence microscopy. About 90-210 transformants were produced per μg plasmid DNA per 10(7) viable protoplasts.
ABSTRACT Large-scale cultivation of Chroogomphus rutilus is too inefficient to be commercially fe... more ABSTRACT Large-scale cultivation of Chroogomphus rutilus is too inefficient to be commercially feasible. In addition, isolating C. rutilus mycelia in the wild is difficult. Thus, determining the natural habitat of its fruiting body is important. The present study focused on the ecology of the C. rutilus habitat to facilitate its large-scale cultivation. A culture-independent molecular approach—a powerful technology for microbiological ecology studies—was used to investigate the diversity of soil fungal communities in samples surrounding C. rutilus from the Beijing region of China. Metagenomic DNA was isolated from soil samples collected around C. rutilus, and an internal transcribed spacer (ITS) gene library was constructed. Subsequently, polymerase chain reaction products were digested with HinfI, HaeIII, MspI, TaqI, or MboI. Clones were selected and sequenced based on their restriction fragment length polymorphisms. The diversity of the fungi represented by their ITS sequences was analyzed. Our results indicate the presence of numerous fungi in the C. rutilus habitat. This study is the first demonstration of the fungal ecology surrounding C. rutilus using a culture independent method.
Legumes acquire access to atmospheric nitrogen through nitrogen fixation by rhizobia in root nodu... more Legumes acquire access to atmospheric nitrogen through nitrogen fixation by rhizobia in root nodules. Rhizobia are soil dwelling organisms and there is a tremendous diversity of rhizobial species in different habitats. From the legume perspective, host range is a compromise between the ability to colonize new habitats, where the preferred symbiotic partner may be absent, and guarding against infection by suboptimal nitrogen fixers. Here, we investigate natural variation in rhizobial host range across Lotus species. We find that Lotus burttii is considerably more promiscuous than Lotus japonicus, represented by the Gifu accession, in its interactions with rhizobia. This promiscuity allows Lotus burttii to form nodules with Mesorhizobium, Rhizobium, Sinorhizobium, Bradyrhizobium, and Allorhizobium species that represent five distinct genera. Using recombinant inbred lines, we have mapped the Gifu/burttii promiscuity QTL to the same genetic locus regardless of rhizobial genus, suggesti...
Sinorhizobium fredii HH103-Rifr, a broad host range rhizobial strain, induces nitrogen-fixing nod... more Sinorhizobium fredii HH103-Rifr, a broad host range rhizobial strain, induces nitrogen-fixing nodules in Lotus burttii but ineffective nodules in L. japonicus. Confocal microscopy studies showed that Mesorhizobium loti MAFF303099 and S. fredii HH103-Rifr invade L. burttii roots through infection threads or epidermal cracks, respectively. Infection threads in root hairs were not observed in L. burttii plants inoculated with S. fredii HH103-Rifr. A S. fredii HH103-Rifr nodA mutant failed to nodulate L. burttii, demonstrating that Nod factors are strictly necessary for this crack-entry mode, and a noeL mutant was also severely impaired in L. burttii nodulation, indicating that the presence of fucosyl residues in the Nod factor is symbiotically relevant. However, significant symbiotic impacts due to the absence of methylation or to acetylation of the fucosyl residue were not detected. In contrast S. fredii HH103-Rifr mutants showing lipopolysaccharide alterations had reduced symbiotic c...
Characterization of monogenic symbiotic mutant phenotypes found by forward genetic screens has be... more Characterization of monogenic symbiotic mutant phenotypes found by forward genetic screens has been instrumental for establishing the framework of genes coordinating nodule formation in the model legumes Lotus japonicus and Medicago truncatula. To complement this approach it is now important to investigate more complex traits governing symbiosis. We are in the process of transforming the genomic resources of the model legume L. japonicus to allow QTL and genome-wide association studies of multigene inherited traits to be combined with efficient validation by reverse genetics. These approaches require a complete and well-annotated genome, a catalog of natural variation, and a comprehensive mutant collection. We have produced accurate gene models using a hierarchical selection scheme that combines mRNA-seq data, ab initio modeling and homology information. Using these models, we have annotated the effects on gene structure and coding sequence of SNPs detected by full genome re-sequenc...
Characterization of monogenic symbiotic mutant phenotypes found by forward genetic screens has be... more Characterization of monogenic symbiotic mutant phenotypes found by forward genetic screens has been instrumental for establishing the framework of genes coordinating nodule formation in the model legumes Lotus japonicus and Medicago truncatula. To complement this approach it is now important to investigate more complex traits governing symbiosis. We are in the process of transforming the genomic resources of the model legume L. japonicus to allow QTL and genome-wide association studies of multigene inherited traits to be combined with efficient validation by reverse genetics. Our results and experiences with genome assembly and annotation, SNP marker generation, QTL and association analysis, as well the development of a sequenced insertion mutant population will be discussed. The reference genome sequence is being updated by integrating Illumina reads with existing shotgun and BAC Sanger data. By sequencing 30 additional Lotus accessions, a dense set of markers has been generated, a...
International Journal of Systematic and Evolutionary Microbiology, 2010
A nitrogen-fixing bacterium, designated strain S27T, was isolated from rhizosphere soil of Sophor... more A nitrogen-fixing bacterium, designated strain S27T, was isolated from rhizosphere soil of Sophora japonica. Phylogenetic analysis based on a fragment of the nifH gene and the full-length 16S rRNA gene sequence revealed that strain S27T is a member of the genus Paenibacillus. High levels of 16S rRNA gene sequence similarity were found between strain S27T and Paenibacillus durus DSM 1735T (97.3 %), Paenibacillus sabinae DSM 17841T (96.9 %), Paenibacillus forsythiae DSM 17842T (96.7 %) and Paenibacillus zanthoxyli DSM 18202T (96.6 %). However, DNA–DNA hybridization values between strain S27T and the four type strains were 37.64 %, 23.12 %, 25.6 % and 34.99 %, respectively. Levels of 16S rRNA gene sequence similarity between strain S27T and the type strains of other recognized members of the genus Paenibacillus were below 96.5 %. The DNA G+C content of strain S27T was 46.0 mol%. The major fatty acids were anteiso-C15 : 0, C16 : 0 and iso-C16 : 0. The major isoprenoid quinone was MK-7. ...
International Journal of Systematic and Evolutionary Microbiology, 2010
A nitrogen-fixing bacterium, designated strain Be17T, was isolated from rhizosphere soil of Begon... more A nitrogen-fixing bacterium, designated strain Be17T, was isolated from rhizosphere soil of Begonia semperflorens planted in Beijing Botanical Garden, PR China. Phylogenetic analyses based on a segment of the nifH gene sequence and a full-length 16S rRNA gene sequence revealed that strain Be17T was a member of the genus Paenibacillus. High levels of 16S rRNA gene sequence similarity were found between strain Be17T and Paenibacillus graminis RSA19T (97.9 %), Paenibacillus sonchi LMG 24727T (97.8 %), Paenibacillus riograndensis CECT 7330T (96.2 %) and Paenibacillus borealis DSM 13188T (96.1 %), respectively. Levels of 16S rRNA gene sequence similarity between strain Be17T and the type strains of other recognized members of the genus Paenibacillus were below 96.0 %. However, the DNA–DNA hybridization values between strain Be17T and P. graminis RSA19T, P. sonchi LMG 24727T and P. riograndensis CECT 7330T were 47.9 %, 38.7 % and 37.5 %, respectively. The DNA G+C content of strain Be17T w...
The ethylene-forming enzyme gene (efe) from Pseudomonas syringae pv. glycinea was transferred int... more The ethylene-forming enzyme gene (efe) from Pseudomonas syringae pv. glycinea was transferred into Pseudomonas putida KT2440 by recombination at five of the seven 16S rDNA sites. PCR analysis demonstrated that strains DC1, DC2 and DC3 contained three, four and five copies of efe, respectively. In contrast to the parent strain which produced ethylene at 14.7 mg h(-1) g(-1) dry weight, strains DC1, DC2 and DC3 produced ethylene at 36.2, 47.2 and 53.8 mg h(-1) g(-1) dry weight, respectively. Quantitative PCR showed that efe mRNA levels increased with increasing efe copy numbers. When additional copies of efe were introduced into strain DC3 via the broad-host-range plasmid pBBR1MCS2 an ethylene production rate of 80.2 mg h(-1) g(-1) dry weight was obtained and 489 mg ethylene was produced in 24h corresponding to a conversion rate of 21.7 mg ethylene g(-1) glucose. Our results indicated that P. putida KT2440 could be genetically modified to be a promising biomaterial producer.
Pleurotus eryngii was transformed using a polyethylene glycol-mediated method. A plasmid, pEPUGH,... more Pleurotus eryngii was transformed using a polyethylene glycol-mediated method. A plasmid, pEPUGH, containing a reporter gene (enhanced green fluorescent protein gene, egfp) and a positive selectable marker gene (hygromycin phosphotransferase gene, hph) was constructed. The fused egfp-hph gene was placed under the control of the strong and constitutive native gpd promoter from P. eryngii. The recombinant plasmid was used to transform of P. eryngii protoplasts. Successful transformation was demonstrated by molecular analyses. Moreover, the mycelia of the transformants showed green epipolic dispersion on fluorescence microscopy. About 90-210 transformants were produced per μg plasmid DNA per 10(7) viable protoplasts.
ABSTRACT Large-scale cultivation of Chroogomphus rutilus is too inefficient to be commercially fe... more ABSTRACT Large-scale cultivation of Chroogomphus rutilus is too inefficient to be commercially feasible. In addition, isolating C. rutilus mycelia in the wild is difficult. Thus, determining the natural habitat of its fruiting body is important. The present study focused on the ecology of the C. rutilus habitat to facilitate its large-scale cultivation. A culture-independent molecular approach—a powerful technology for microbiological ecology studies—was used to investigate the diversity of soil fungal communities in samples surrounding C. rutilus from the Beijing region of China. Metagenomic DNA was isolated from soil samples collected around C. rutilus, and an internal transcribed spacer (ITS) gene library was constructed. Subsequently, polymerase chain reaction products were digested with HinfI, HaeIII, MspI, TaqI, or MboI. Clones were selected and sequenced based on their restriction fragment length polymorphisms. The diversity of the fungi represented by their ITS sequences was analyzed. Our results indicate the presence of numerous fungi in the C. rutilus habitat. This study is the first demonstration of the fungal ecology surrounding C. rutilus using a culture independent method.
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Papers by Haojie Jin