科研之友
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摘要
Andisols, developed from late-Quaternary tephra (volcanic ash) deposits and dominated by the nanocrystalline aluminosilicate, allophane, contain large stores of organic matter and are potential reservoirs for DNA. However, DNA recovery from Andisols and other allophane-bearing soils has been difficult and inefficient because of strong chemical bonding between DNA and both allophane and organic matter, and also because much DNA can be encased and physically protected in nanopores in allophane nano/microaggregates. We have therefore developed a new two-step DNA isolation method for allophanic soils and buried paleosols, including those low in clay, which circumvents these problems. The method centres on (1) releasing mainly microbial DNA, and extra cellular (unbound) DNA, using an alkaline phosphate buffer ("Rai's lysis buffer") that blocks re-adsorption sites on the allophanic materials, and (2) the novel application of acidified ammonium oxalate (Tamm's reagent) to dissolve the allophane and to release DNA which had been chemically-bound and also which had been protected within nanopores. Ammonium oxalate has not previously been applied to soil DNA extraction. DNA yields up to 44.5 mu g g(-1) soil (oven-dry basis) were obtained from three field-moist natural allophanic soil samples from northern New Zealand using this two-step method. Following extraction, we evaluated different DNA purification methods. Gel electrophoresis of the extracted DNA followed by gel purification of the DNA from the agarose gel, despite some DNA loss, was the only purification method that removed sufficient humic material for successful DNA amplification using the polymerase chain reaction (PCR) of multiple gene regions. Sequencing of PCR products obtained from a buried allophanic paleosol at 2.2-m depth on a sandy Holocene tephra yielded endemic and exotic plants that differed from the European grasses growing currently on the soil's surface. This difference suggests that the DNA extraction method is able to access (paleo)environmental DNA derived from previous vegetation cover. Our DNA extraction and purification method hence may be applied to Andisols and allophane-bearing paleosols, potentially offering a means to isolate paleoenvironmental DNA and thus facilitate reconstruction of past environments in volcanic landscapes, datable using tephrochronology, and also aid biodiversity understanding of andic soils and paleosols.
- 出版日期2016-7-15
