SPP 1090 Böden als Quelle und Senke von CO 2
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SPP 1090 Böden als Quelle und Senke von CO 2 Contact: Prof. Dr. François Buscot, Institute of Ecology, Department of Environmental Sciences, Friedrich- Schiller-University of Jena, Dornburger Str. 159, D-07743 Jena / Mail: [email protected] Diversity of basidiomycete laccase genes in soil samples Patricia Luis 1,2 , Grit Walther 1 , Francis Martin 2 and François Buscot 1 1 Friedrich Schiller University of Jena, Institute of Ecology, Department of Environmental Sciences 2 Centre INRA of Nancy, UMR INRA/UHP 1136 “Interactions Arbres/Micro-organismes” Summary Introduction and goal Fungi are one of the major organism groups involved in formation and decomposition of soil organic matter (SOM) By producing oxidative exo-enzymes without substrate specificity, they fully mineralize organic compounds or recombine organic radicals in stable polymers Among the exo-enzymes, phenol oxidases (laccases) are produced by the broadest range of fungi. Therefore, they were chosen as model to develop a technique to monitor fungi with an oxidative potential in soils, without taking them in culture Design of degenerated primers specific for basidiomycetes Vertical diversity of laccase genes in 2 forest soils Comparing laccase-genes of soil & fruit-bodies Conclusions • The optimized primers allow to amplify approximately 200 bp fragments of laccase genes in a broad range of Basidiomycetes • Analyses on soils revealed a high soil and horizon specificity of the fungal laccase genes • Ectomycorrhizal fungi seems to have a wider vertical distribution compared to the others functional fungi groups • First RT-PCR on soil RNA followed by cDNA amplification was realized. Additional sample analyses and sequencing should confirm of the effectiveness of the method I II III IV 200bp 1200bp Cu2 Nested PCR Cu4 PCR Cu3 Nested PCR Cu2 PCR Cu1 PCR DNA Regions encoding Cu- binding sites Degenerated primers tested Degenerated primers used for PCR amplifications 200bp The primers appear adequate to specifically amplify laccase genes from basidiomycetes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 9: Neurospora crassa 10: Aspergillus nidulans 11: Cladosporium sp. 12: Cryphonectria parasitica 13: Podospora anserina 14: Morchella esculenta 15: Cenococcum geophilum 2: Pycnoporus cinnabarinus 3: Ganoderma lucidum 4: Pleurotus ostreatus 5: Trametes versicolor 6: Lentinula edodes 7: Hypholoma sp. 8: Gymnopus fusipes Assessment of the primer pair specificity on fungal culture strains, 100bp DNA ladder (1), negative control (16) basidiomycetes ascomycetes 0 1 2 3 4 5 6 7 8 lac1 lac2 lac3 lac4 lac5 lac6 lac7 lac8 lac9 lac10 lac11 lac12 lac13 lac14 lac15 lac16 lac17 lac18 lac19 lac20 lac21 O f-h Ahe B hs-sv Frequences (number) Frequences (number) Laccase genes Laccase genes 0 1 2 3 4 5 6 7 8 9 lac1 lac2 lac3 lac4 lac5 lac6 lac7 lac8 lac9 lac10 lac11 lac12 lac13 lac14 lac15 lac16 lac17 lac18 lac19 lac20 lac21 lac22 lac23 O f-h Ah Bv Podzolic forest soil (Waldstein) Podzolic forest soil (Waldstein) Brown forest soil (Steigerwald) Brown forest soil (Steigerwald) % of laccase diversity % of Corg 19.0% 3.38% 1.19% Of-h 60.0% 52.0% 40.0% 50.0 % 76.9 % 60.8 % Ah 40.0% 46.1% 36.8% 46.6% 42.1 % 46.6% Bv 28.0 % 45.4 % 29.5 % 35.7 % 42.1 % 45.4 % ( N u m b e r o f d i f f e r e n t l a c c a s e s / n u m b e r o f c l o n e s s e q u e n c e d ) Comparison Podzolic\Brown forest soil Brown forest soil replicates For both soils, the diversity is stronger in O horizons (highest concentration of SOM) and generally decreases with the depth Higher diversity in O horizons Stronger horizon specificity in the Podzol Small number of common laccases between soils Replicate heterogeneity Diversity decreases with the depth The primers detect laccases in a broad range of Basidiomycota of all functional groups (saprophytes, pathogens & mycorrhiza) Laccase gene sequences are species specific Most fungi possess a family of laccase genes Sequence and genera clades rarely overlap Diversity of laccase genes is higher than the one of fungi Off 70 soil sequences, only 18 correspond to laccases of the collected fruit- bodies (12 saprophytic, 1 pathogen & 5 ectomycorrhizal fungi) Fruit bodies don’t reflect correctly the soil fungi community (seasonal fruiting) Unknown groups of laccase genes (bold bars) were detected, especially in mycorrhizal fungi Ectomycorrhizal fungi seem to have a wide vertical distribution First extraction of soil RNA & PCR on cDNA Analysis of fungal laccase gene expression in soils seems feasible Soil RNA extraction PCR on cDNA obtained from soil RNA The protocol developed gives high concentrations of soil total RNA. After purification, the remaining concentration allows to perform a RT- PCR 100pb DNA ladder (1 & 6), total RNA obtained from soil before (2 & 3) and after purification (4 & 5) Amplification products from cDNA gave fragments of the expected size (142pb) 100pb DNA ladder (1 & 10), PCR products obtained on cDNA which are respectively synthesized with 15 amplification cycles (2 & 3), 18 cycles (4 & 5), 21 cycles (6 & 7) and 24 cycles (8 & 9). 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10
description
Brown forest soil (Steigerwald). Frequences (number). Podzolic forest soil (Waldstein). I II III IV. 200bp. 1200bp. 200bp. DNA. Cu1 PCR. Cu4 PCR. Cu2 Nested PCR. Cu3 Nested PCR. Cu2 PCR. Regions encoding Cu- binding sites. Degenerated primers tested. Laccase genes. - PowerPoint PPT Presentation
Transcript of SPP 1090 Böden als Quelle und Senke von CO 2
SPP 1090 Böden als Quelle und Senke von CO2
Contact: Prof. Dr. François Buscot, Institute of Ecology, Department of Environmental Sciences, Friedrich-Schiller-University of Jena, Dornburger Str. 159, D-07743 Jena / Mail: [email protected]
Diversity of basidiomycete laccase genes in soil samples
Patricia Luis1,2, Grit Walther1, Francis Martin2 and François Buscot1
1 Friedrich Schiller University of Jena, Institute of Ecology, Department of Environmental Sciences
2 Centre INRA of Nancy, UMR INRA/UHP 1136 “Interactions Arbres/Micro-organismes”
Summary
Introduction and goal Fungi are one of the major organism groups involved in formation and
decomposition of soil organic matter (SOM)
By producing oxidative exo-enzymes without substrate specificity, they fully mineralize organic compounds or recombine organic radicals in stable polymers
Among the exo-enzymes, phenol oxidases (laccases) are produced by the broadest range of fungi. Therefore, they were chosen as model to develop a technique to monitor fungi with an oxidative potential in soils, without taking them in cultureDesign of degenerated primers specific for
basidiomycetes
Vertical diversity of laccase genes in 2 forest soils
Comparing laccase-genes of soil & fruit-bodies
Conclusions• The optimized primers allow to amplify approximately
200 bp fragments of laccase genes in a broad range of Basidiomycetes
• Analyses on soils revealed a high soil and horizon specificity of the fungal laccase genes
• Ectomycorrhizal fungi seems to have a wider vertical distribution compared to the others functional fungi groups
• First RT-PCR on soil RNA followed by cDNA amplification was realized. Additional sample analyses and sequencing should confirm of the effectiveness of the method
I II III IV200bp 1200bp
Cu2 Nested PCRCu4 PCR
Cu3 Nested PCR
Cu2 PCR
Cu1 PCRDNA
Regions encoding Cu- binding sites
Degenerated primers tested
Degenerated primers used for PCR amplifications
200bp
The primers appear adequate to specifically amplify laccase genes from basidiomycetes
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
9: Neurospora crassa 10: Aspergillus nidulans11: Cladosporium sp.12: Cryphonectria parasitica13: Podospora anserina14: Morchella esculenta15: Cenococcum geophilum
2: Pycnoporus cinnabarinus 3: Ganoderma lucidum 4: Pleurotus ostreatus 5: Trametes versicolor 6: Lentinula edodes 7: Hypholoma sp. 8: Gymnopus fusipes
Assessment of the primer pair specificity on fungal culture strains, 100bp DNA ladder (1), negative control (16)
basidiomycetes ascomycetes
0
1
2
3
4
5
6
7
8
lac1 lac2 lac3 lac4 lac5 lac6 lac7 lac8 lac9 lac10 lac11 lac12 lac13 lac14 lac15 lac16 lac17 lac18 lac19 lac20 lac21
Of-h
Ahe
Bhs-sv
Fre
qu
en
ces
(n
um
ber
)F
req
ue
nce
s (
nu
mb
er)
Laccase genesLaccase genes
0
1
2
3
4
5
6
7
8
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lac1 lac2 lac3 lac4 lac5 lac6 lac7 lac8 lac9 lac10 lac11 lac12 lac13 lac14 lac15 lac16 lac17 lac18 lac19 lac20 lac21 lac22 lac23
Of-h
Ah
Bv
Podzolic forest soil (Waldstein)Podzolic forest soil (Waldstein)
Brown forest soil (Steigerwald)Brown forest soil (Steigerwald)
% o
f la
cca
se
div
ers
ity
% of Corg 19.0% 3.38% 1.19%
Of-h
60.0%
52.0%
40.0%
50.0 %
76.9 %
60.8 %
Ah
40.0%
46.1%
36.8%
46.6%
42.1 %
46.6%
Bv
28.0%
45.4%
29.5%
35.7%
42.1%
45.4%
(Nu
mb
er o
f d
iffe
ren
t la
ccas
es/n
um
ber
of
clo
nes
seq
uen
ced
)
Comparison Podzolic\Brown forest soil
Brown forest soil replicates
For both soils, the diversity is stronger in O horizons (highest concentration of SOM) and generally decreases with the depth
Higher diversity in O horizons
Stronger horizon specificity in the Podzol
Small number of common laccases between soils
Replicate heterogeneity
Diversity decreases with the depth
The primers detect laccases in a broad range of Basidiomycota of all functional groups (saprophytes, pathogens & mycorrhiza)
Laccase gene
sequences are species specific
Most fungi possess a
family of laccase genes
Sequence and genera
clades rarely overlap
Diversity of
laccase genes is higher than the one of fungi
Off 70 soil sequences,
only 18 correspond to laccases of the collected fruit-bodies(12 saprophytic, 1 pathogen & 5 ectomycorrhizal fungi)
Fruit bodies don’t
reflect correctly the soil fungi community (seasonal fruiting)
Unknown groups of
laccase genes (bold bars) were detected, especially in mycorrhizal fungi
Ectomycorrhizal
fungi seem to have a wide vertical distribution
First extraction of soil RNA & PCR on cDNA
Analysis of fungal laccase gene expression in soils seems feasible
Soil RNA extraction
PCR on cDNA obtained from soil RNA
The protocol developed gives high concentrations of soil total RNA. After purification, the remaining concentration allows to perform a RT-PCR
100pb DNA ladder (1 & 6), total RNA obtained from soil before (2 & 3) and after purification (4 & 5)
Amplification products from cDNA gave fragments of the expected size (142pb)
100pb DNA ladder (1 & 10), PCR products obtained on cDNA which are respectively synthesized with 15 amplification cycles (2 & 3), 18 cycles (4 & 5), 21 cycles (6 & 7) and 24 cycles (8 & 9).
1 2 3 4 5 6
1 2 3 4 5 6 7 8 9 10
