Biomedical Engineering Reference
In-Depth Information
suggest that in the present work we studied fragment of CDA gene of the
fungus S. nodorum.
Other strains pathogens of wheat: Tilletia caries , Ustilago tritici , Bipolaris
sorokiniana , Fusarium culmorum , Septoria tritici and nonspecifi c insects
pathogen agent Beauveria bassiana , chosen from the collection of the our
laboratory were used to determine primers specifi city CDA -F and CDA -R
for DNA of the S. nodorum fungus. To analyze the effi ciency of application
of the primers for the CDA gene, as DNA probes, it is necessary to use more
additional markers that are amplifi ed in almost all the forms of fungi. For
monitoring the effi ciency of DNA extraction primers to highly conservative
site of internal transcribed spacer ( ITS) that had the following sequence
(table) is localized between ribosomal DNA genes encoding the small and
large subunits fungal ribosomes were used (White et al. 1990).
Before PCR analysis, the concentrations of all RNA samples were
equalized. cDNA synthesis was performed with primers and an M-MVL
reverse transcriptase as recommended by the manufacturer (Fermentas,
Lithuania). Electrophoresis of obtained amplicons was performed in
7% polyacrylamide gel. Before amplifi cation the concentration of all the
samples was equalized after determining the optical density of DNA in
the samples using BioSpec-mini DNA-RNA-Protein analyzer device
(Shimadzu, Japan). To visualize DNA, after electrophoresis, the gel was
being incubated for 10 minutes in a solution of ethidium bromide (0.5 ug/
ml) and was examined in transilluminator Gel Doc XR (Bio Rad, USA).
DNA marker GeneRulerTM 100 bp DNA Ladder («Fermentas», Lithuania)
to determine the sizes of amplicons were used.
To control the expression of the CDA gene in wheat leaves infected by
different strains of the fungus, during RT-PCR we used «house-keeping»
β-tubulin ( TUB ) gene (table) (Fraaije et al. 1999) since its transcriptional
activity is constant. Biochemical and molecular biological analysis was
carried out in fi ve repeats. Statistical processing was done using software
from Statsoft (Statistica 6.0).
Analysis of the septoria glume blotch development caused by
S. nodorum on wheat leaves showed its symptoms appear on the fourth
day in the form of brown patch ( Figure 2). Infection with strains 6VD and
4VD resulted in narrow brown necrosis on the leaves on the 4 day of the
experiment. In contrast—the strains of 9MN and Bas1 during the same
period of the experiment formed a clearly visible rounded brown patch.
There were also differences in the rate of development of septoria glume
blotch on segments of wheat leaves infected by various strains of fungus
and in the character of manifestations of infectious patch. On the seventh
day after infection, the size glume blotch patch on the leaves infected by
strains 9MN and Bas1, was twofold larger than with the infection of slightly
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