Agriculture Reference
In-Depth Information
detection and quantification of mRNAs present in low amounts in environmental
samples including soils (Pfaffl and Hageleit
2001
). However, this method requires
previous knowledge of the sequence of the mRNA of interest.
7.5.6 Denaturing Gradient Gel Electrophoresis
Differences in the melting behavior of small DNA fragments (200-700 bp) that
differ in as little as a single base substitution can be detected by DGGE (Muyzer
et al.
1993
). The denaturants used are heat (a constant temperature of 60
C) and a
fixed ratio of formamide (ranging from 0 to 40 %) and urea (ranging from 0 to 7 M).
The benefit of this approach is that a molecular fingerprint of the community
structure is generated for each soil. In fact, each band in each lane of the gel
theoretically represents a different bacterial species. In addition, this technique
enables the excision and subsequent sequencing of bands, allowing species identi-
fication using existing databases. The structure of the bacterial communities asso-
ciated with the root endosphere and in the plant rhizosphere can be dissected by 16S
rRNA gene-based PCR-DGGE (denaturing gradient gel electrophoresis) analysis.
Naik et al. (
2008
) performed molecular phylogenetic analyses by aligning the
sequences of 16S rRNA using the multiple sequence alignment program
CLUSTAL W. The aligned sequences were then checked for gaps manually,
arranged in a block of 600 bp in each row, and saved as molecular evolutionary
genetics analysis (MEGA) format in software MEGA v3.0. The pair-wise evolu-
tionary distances were computed using the Kimura 2-parameter model. A DGGE
technique has also been developed for analyzing the diversity of the PQQ biosyn-
thetic gene pqqC, a gene which has been found as a good molecular marker for
investigations of natural populations of P-solubilizing pseudomonads (Naik
et al.
2008
).
7.5.7 Temperature Gradient Gel Electrophoresis
Variations are known to exist in the genetic microdiversity within the species of
Bacillus
and
Paenibacillus
(McSpadden Gardener and Driks
2004
). Wieland
et al. (
2001
) studied the spatiotemporal variation among the microbial communities
from soil, rhizosphere, and rhizoplane with respect to crop species (clover, bean,
and alfalfa), soil type, and crop development following a comparative study of 16S
rRNA sequences employing TGGE. According to their study, the type of plant
species had profound effects on microbial community dynamics, with the effect of
soil type typically exceeding that of plant type. Plant development had only minor
habitat-dependent effect, and insignificant variations were observed in time-
dependent shifts among the microbial communities compared to the soil type or
plant type in all the habitats under study. Systematic community shifts could not be
