DGGE method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil

Watanabe, Takeshi; Asakawa, Susumu; Nakamura, Asumi; Nagaoka, Kazunari; Kimura, Makoto

doi:10.1016/s0378-1097(04)00045-x

Cited by 190 publications

(120 citation statements)

References 43 publications

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“…Aliquots of 1 µl of the extracted DNA from the in vitro contents were used as template for the first amplification in a total reaction of 50 µl containing 2.5 µl dNTPs (2mM each), 5.0 µl of primers (10µM) 348aF and 915aR (Watanabe et al, 2004), 5.0 µl Buffer (10x), 5.0 µl MgCl 2 (25mM), 2.0 µl BSA (10 mg/ml), 0.6 µl Taq DNA polymerase, and 23.9 µl Milli-Q water. PCR started at 94°C for 10 min and was carried out in 40 cycles each consisting of 1 min at 94°C, 1 min at 61°C, and 2 min at 72°C, followed by a final extension period of 8 min at 72°C.…”

Section: Nested Pcr For Dgge Of Methanogenic Archaeamentioning

confidence: 99%

“…Aliquots of 2 µl were applied in a second PCR, which contained the same reagent amount as before except for 23.0 µl Milli-Q water and 5 µl of the primers 0357F-GC and 691R (Watanabe et al, 2004). The amplification conditions were 94°C for 10 min, 35 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 2 min, and a final extension at 72°C for 8 min.…”

Section: Nested Pcr For Dgge Of Methanogenic Archaeamentioning

confidence: 99%

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Evaluation of the effects of tropical tanniferous plants on rumen microbiota using qRT PCR and DGGE analysis

Longo¹,

Abdalla²,

Liebich³

et al. 2013

CZECH J ANIM SCI

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Tanniferous forages may have bacteriostatic and/or bactericidal effect on different rumen microbial populations. We investigated the influence of the tropical tanniferous plants Styzolobium aterrimum (STA), Styzolobium deeringianum (STD), Leucaena leucocephala (LEU), and Mimosa caesalpiniaefolia (MIC) containing 20, 64, 56, and 105 g condensed tannis (CT)/kg dry matter (DM) and Cynodon spp. cv. Tifton 85 (CYN) as control on Fibrobacter succinogenes and methanogenic microbes in rumen liquor from sheep using the in vitro gas production technique (Hohenheim gas test). The relative gene expression of F. succinogenes at t ½ (time point when 50% of the maximal gas production has been reached) analyzed by quantitative PCR was 0.20-and 0.28-fold lower than the control when LEU and STA was applied and 0.91-and 0.85-fold lower with MIC and STD. Methanogenic population was 0.29-and 0.58-fold reduced with STA and LEU compared to the control, but 5.50-and 1.43-fold higher with MIC and STD. At 24 h, F. succinogenes was reduced for all legumes as well as methanogenic bacteria, except for MIC which increased 4.15-fold. Denaturing gradient gel electrophoresis (DGGE) of the methanogenic community resulted in different band patterns: CYN presented some strong bands, which became weaker in the analyzed treatments. Some bands appeared weaker, especially in MIC and STD, but not in STA and LEU. MIC seemed to increase the total number of weak bands. Overall, the tannin-rich plants negatively affected the F. succinogenes population and caused changes in the structure of the methanogenic community.

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Section: Nested Pcr For Dgge Of Methanogenic Archaeamentioning

confidence: 99%

Section: Nested Pcr For Dgge Of Methanogenic Archaeamentioning

confidence: 99%

Evaluation of the effects of tropical tanniferous plants on rumen microbiota using qRT PCR and DGGE analysis

Longo¹,

Abdalla²,

Liebich³

et al. 2013

CZECH J ANIM SCI

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“…PCR products used for DGGE were amplified by primer pairs 1106F-GC/1378R (Watanabe et al 2004), targeting methanogenic archaeal 16S rRNA gene. The reaction was performed in a 50 μL mixture containing 6 μL of 10 × HotStar-Taq buffer, 0.5 μL of each 20 μmol/L primer, 2.5 U HotStar-Taq polymerase (TaKaRa) and 1 μL DNA template.…”

Section: Methodsmentioning

confidence: 99%

Nitrate addition inhibited methanogenesis in paddy soils under long-term managements

Wang¹,

Xu²,

Lichu³

et al. 2018

Plant Soil Environ.

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Rice fields are a major source of atmospheric methane (CH4). Nitrate has been approved to inhibit CH4 production from paddy soils, while fertilization as well as water management can also affect the methanogenesis. It is unknown whether nitrate addition might result in shifts in the methanogenesis and methanogens in paddy soils influenced by different practices. Six paddy soils of different fertilizer types and groundwater tables were collected from a long-term experiment site. CH4 production rate and methanogenic archaeal abundance were determined with and without nitrate addition in the microcosm incubation. The structure of methanogenic archaeal community was analysed using the PCR-DGGE (polymerase chain reaction denaturing gradient gel electrophoresis) and pyrosequencing. The results showed that nitrate addition significantly decreased the CH4 production rate and methanogenic archaeal abundance in all six paddy soils by 70–100% and 54–88%, respectively. The quantity, position and relative intensity of DGGE bands exhibited differences when nitrate was added. Nitrate suppressed the growth of methanogenic archaeal species affiliated to Methanosaetaceae, unidentified Euryarchaeota, Thaumarchaeota and Methanosarinaceae. The universal inhibition of nitrate addition on the methanogenesis and methanogens can be adopted as a practice of mitigating CH4 emission in paddy soils under different fertilization and water managements.

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“…Therefore, universal DGGE methanonenic and archaeal primers were used in the amplification reactions. The universal archaeal primers PARCH340f and PARCH519r based on the E. coli 16S rRNA gene sequence and methanogenic primers 0357F and 0691R were used to amplify 179 and 334 bp fragments (Watanabe et al, 2004), respectively. The GC clamp, 5"-CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG-3" as described by Chan et al (2001), was included on the 5 end of the forward primer PARCH340f and 0357F to enable the separation of the fragments using DGGE.…”

Section: Pcr-based Dgge Fingerprinting Of Methanogensmentioning

confidence: 99%

Exploitation of molecular genetics in microbial degradation and decolorization of industrial waste water effluent

Shah¹

2015

Afr. J. Biotechnol.

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Activated sludge samples were used to analyse microbial community structures from the anoxic and aerobic zones of a laboratory-scale modified Ludzack-Ettinger system. Fluorescent in situ hybridization and denaturing gradient gel electrophoresis were applied for analysis. With the help of DNA specific fluorochrome DAPI, approximately 75 to 80% of total cells were detected, hybridised with a specific eubacterial probe for the anoxic and aerobic zones. Results corroborate the dominance of the alpha and gamma subclasses of the Proteobacteria in the anoxic zone whilst the aerobic zone was dominated with the beta subclass of the Proteobacteria. Genetic diversity of the microbial community present in each of the anoxic and aerobic zones was employed by the DGGE technique. Results were obtained from the application of fluorescent in situ hybridisation (FISH) and PCR-DGGE yields a more precise understanding of the microbial community structure and genetic diversity present in domestic wastewater of a laboratory scale treatment process. Nitrogen mass balances indicated an upset in the nitrogen levels for wastewater batches two and seven. The carbon mass balance fell in the range of 92.4 and 105.9% and the nitrogen mass balance fell in the range of 98.4 and 160.0%.

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DGGE method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil

Cited by 190 publications

References 43 publications

Evaluation of the effects of tropical tanniferous plants on rumen microbiota using qRT PCR and DGGE analysis

Evaluation of the effects of tropical tanniferous plants on rumen microbiota using qRT PCR and DGGE analysis

Nitrate addition inhibited methanogenesis in paddy soils under long-term managements

Exploitation of molecular genetics in microbial degradation and decolorization of industrial waste water effluent

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