Denaturing Gradient Gel Electrophoresis(DGGE) Separation Of DNA Fragments And Its Application To The Study Of Soil Microbial Composition

DNA has become a new target for analytical chemistry with the development in biological research.DNA sequencing is an important task that provides the premise of effective DNA separation from a complex sample.Denaturing gradient gel electrophoresis(DGGE)can separate DNA by discriminating DNA-base composition.It is one of the most widely used approaches for DNA separation,and has great advantages in time consumption,cost,and sample quantity.Soil microorganisms fulfill important ecosystem functions,including organic matter decomposition,nutrient cycling,and element transformation,so they are able to maintain and improve soil quality.Thus,soil microorganisms are important in alleviating food,energy,resource,and environment crises.As a basic life characteristic of soil ecosystem,soil microbial composition analysis has a high demand for analytic technologies.DNA can be detected through DGGE by separating PCR-amplifying DNA fragments.DDGE can also be used to analyze DNA composition and sequence as it manifest in organisms.A soil microbial community comprises of extremely complex groups,and thus high DGGE resolution for DNA fragments is essential to DNA sequencing.In the thesis,the method for 16S rRNA gene fragment separation using DGGE was established and applied to analyze soil bacteria composition.The recovery of soil bacteria composition was assessed,and it is suggested first that soil-salt content does not affect the dominant composition of a bacterial community in the field condition.In addition,the method for nitrogen-cycling genes separation using denaturing gradient procedure was also established and applied to analyze nitrogen-cycling microflora in crust and meadow soils under different use.It is suggested first that the crust soil microbial community comprise of species from Proteobacteria.The change from meadow to forest had small effects on microbial community composition and increased soil nutrients,while the change from meadow to farmland and paddy field led to significant shifts and loss of soil nutrients.The detailed contents are described in the following:In Chapter 1,three methods for detecting microbial composition,including theory,application,advantages and limitation,and diversity and distribution of bacteria and organisms involved in nitrogen cycling,were described.Horqin Sandy Land and related plantations were introduced.In Chapter 2,the method for 16S rRNA gene fragment separation using DGGE was established,with soil crusts as materials.Soil crust DNA was extracted from biological soil crusts under an age sequence of Caragana microphylla,Hedysarum fruticosum,Artemisia halodendron,and Salix gordejevii,using soil genome extraction kit.Soil crust DNA was also used as templates to amplify fragments of bacterial and cyanobacterial 16S rRNA gene by bacterial-and cyanobacterial-specific primers.Fourteen sequenced DNA fragments(14 bands)from 16S rRNA gene amplicon were separated by DGGE under 30-60%denaturing gradient,and 80 V and 9 h optimal electrophoresis conditions.These bands were reproducible and prominent,and distance among bands was appropriate for removal.Six cyanobacterial DNA fragments were obtained under 60 V and 10 h electrophoresis conditions for cyanobacterial 16S rRNA gene.Seven of fourteen DNA fragments were related to Escherichia genus,and others were affiliated with Bacillus,Paenibacillus,Shigella,and Pseudomonas.Six cyanobacterial DNA fragments belonged to Microcoleus,Leptolyngbya,and Haslea.DGGE plus sequence analysis can be used to analyze soil bacterial and cyanobacterial 16S rRNA gene compositions.In Chapter 3,DGGE was applied to analyze soil bacterial composition from different plantations by 16S rRNA gene separation.Thus,soil bacterial composition recovery and nutrients were investigated with the analysis of physical and chemical soil properties,biological activities,and DGGE.H.fruticosum,A.halodendron,and C.microphylla were representative native plants that exhibit strong adaptations to windy and sandy environments.They are widely used in revegetation programs to fix moving sand dunes in the semiarid Horqin Sandy Land of Northeast China.Soil samples were collected from the three plantations and moving sand dunes.Using DGGE,21,20,13,and 17 DNA fragments were separated from H.fruticosum,A.halodendron,C.microphylla,and moving sand dune,respectively,under 30%-60%denaturing gradient,180 V,and 5 h conditions.Distance among bands is appropriate for removal,and bands were sequenced.DNA fragments were identified by phylogenetic analysis.Results suggested that these sequences were closely related to Bacteroidetes,Actinobacteria,Acidobacteria,Gemmatimonadetes,and Proteobacteria,which were dominant in sandy land.H.fruticosum and C.microphylla produced more bands than moving sand dune,revealing that their establishment increased the number of bacterial species.However,A.halodendron had an inverse effect.Three plantations induced increases in soil nutrients and biological activities.Specifically,their soil organic matter,total N,P,and Mg,available N,P,and K,electrical conductivity,CBA,DNA content,and enzymatic activities(urease,protease,and phosphomonoesterase)increased.Therefore,restoring soil nutrients and microbiological properties is possible by vegetation reestablishment in severely degraded sandy lands.In Chapter 4,DGGE separation of 16S rRNA gene fragments and analysis of soil properties,enzyme activities,and community level physiological profiles were applied to analyze salinization level effects on bacterial community composition,soil properties,enzyme activities,and metabolic profiles.Soil samples with a gradient of salinization level(0.135%,0.375%,and 1.063%,respectively)produced 15 bands(DNA fragments)and had the same profiles,suggesting that soil-salt contents do not have an effect on bacterial composition.All bands were excised and sequenced.DNA identification results based on phylogenetic analysis prove that y-Proteobacteria were the dominant phylum.Soil-salt contents had great influence on soil properties,enzyme activities,and carbon metabolicactivities.Specifically,soil moisture,porosity,SOC,total N and P,available N and K,DNA content,microbial biomass,enzymatic activities(dehydrogenase,phosphomonoesterase,urease,and polyphenol oxidase),and metabolic function diversity all decreased significantly with an increase in salt content.Soil-bulk density,pH,and available P-concentration increased significantly.All of the aforementioned soil properties had significant linear relations with salt content(P<0.001 or P<0.05).An increase in soil-salt content restrains proliferation and metabolic activity of soil bacteria,but it does not affect the dominant composition of a bacterial community.In Chapter 5,DGGE separation of dinitrogenase reductase(nifH),nitrous oxide reductase(nosZ),and ammonia-oxidizing 16S rRNA gene fragments from soil crusts were established.Based on DGGE detection,total bacterial composition was also analyzed using 16S rRNA gene libraries.Soil samples were obtained from three plantations,namely,C.microphylla Lam.,H.fruticosum,and A.halodendron.Using DGGE,11 nifH,13 ammonia-oxidizing 16S rRNA genes,and 8 nosZ DNA fragments,which were all sequenced,were separated under 30-60%,30-55%,and 30-50%denaturing gradient and 150 V and 5 h,60 V and 11 h,and 150 V and 3.5 h electrophoresis conditions,respectively.DNA fragment phylogenetic identification indicated that most of nifH formed a distinct cluster that branched with known Azotobacter diazotrophs.Ammonia-oxidizing community comprised uncultured Nitrosomonas species and Nitrosospira members affiliated with Nitrosospira cluster 3.Although nosZ genes belong to three subclasses of Proteobacteria,almost half are closely affiliated with Pseudomonas of y-Proteobacteria.Results of 16S rRNA gene phylogenetic analysis indicate that phyla Proteobacteria,Acidobacteria,Bacteroidetes,and Actinobacteria were dominant groups.This study demonstrates that dominant phylum in BSCs was Proteobacteria.DGGE profiles showed that samples from three plantations displayed similar compositions of three genes,suggesting that plantation type does not have any influence on the dominant population of nitrogen-cycling bacterial communities.DGGE plus DNA sequencing proved that Proteobacteria were the most dominant phylum.In Chapter 6,DGGE separation of ammonia monooxygenase subunit A(amoA)and Cu-containing nitrite reductases(nirK)gene fragments were established.Then,they were combined with nifH,nosZ,and 16S rRNA gene fragments separation to investigate the effect of change in land use on compositions of nitrogen-fixing,ammonia-oxidizing,and denitrifying bacteria and total bacteria.Also,the effect of land use on soil properties was analyzed.Soil samples were collected from meadow grassland,woodland,upland,and paddy field.Using DGGE,18 ammonia-oxidizing bacterial,31 ammonia-oxidizing archaeal amoA,and 10 nirK DNA fragments,which were all sequenced,were separated under 30-60%,13-55%,and 15-60%optimal denaturing gradients and 80 V and 10 h,80 V and 8 h,and 150 V and 3 h electrophoresis conditions,respectively.Besides,DGGE separated nifH,nosZ,and 16S rRNA gene fragments from four samples.All DNA fragments were identified by phylogenetic analysis.Results show that Proteobacteria were the dominant phylum in microbial nitrogen-cycling microflora.Specifically,members of y-Proteobacteria are the main components of nitrogen-fixing community;ammonia-oxidizing community comprised of species related to Nitrosomonas nitrosa and Nitrosospira cluster 3 that belongs to ?-Proteobacteria and to Nitrososphaera,which belongs to archaea;members related to ?-,?-,and ?-Proteobacteria comprised the denitrifying community.DGGE profiles of 16S rRNA and nifH genes revealed that change in grassland use does not have an effect on total bacterial and nitrogen-fixing bacterial compositions.By contrast,ammonia-oxidizing bacterial and archaeal communities were altered significantly.The number of ammonia-oxidizing bacteria(band number)in woodland,upland,and paddy field were all lower than that of meadow grassland,especially the upland.The number of ammonia-oxidizing archaea in paddy field was the lowest and does not have any significant difference among the other three soils.The denitrifying community of wood and paddy differed from that of meadow.In addition,the contents of SOC,total N,and total and available P of woodland soil were higher than that of meadow grassland;except for available P,the aforementioned soil properties of upland and paddy field were lower than that of meadow grassland.The electrical conductivity of upland increased significantly compared with that of meadow grassland.The seventh chapter draws the conclusions as followed:the optimal DGGE profiles of 16S rRNA and nitrogen-cycling genes were obtained by adjusting enaturing gradient,voltage and electrophoresis time,and DGGE plus sequencing can be used to analyze bacteria and nitrogen-cycling organisms compositions in different soil types;y-Proteobacteria is the dominant group in arid and semiarid Horqin Sandy Land;the types of reestablished plantations and the salt level does not affect bacteria compositions in arid and semiarid soils;restoring soil nutrients and microbiological properties is possible by vegetation reestablishment in severely degraded sandy lands;the shift from meadow to forest does not influence on microbial community composition and increased soil nutrients,while the change from meadow to farmland and paddy field led to significant shifts and loss of soil nutrients.