| Summary: | 碩士 === 東吳大學 === 微生物學系 === 94 === In the studies of prawns, the expression pattern and regulation mechanism of a
specific gene is more often being observed by means of injecting immunostimulants,
while the changes of the entire gene expression pattern is less being noticed.
In this study, Demethylated nucleic acid CpG ODN 2006 and the
lipopolysaccharide (LPS) of the Gram-Negative bacteria cell wall were the two
immunostimulants used to inject into freshwater prawn Macrobranchium rosenbergii.
The hemocytes were extracted out at 1, 6 and 12 hours after the injection. Different
gene expression pattern between the hemocytes of injected and non-injected prawns
were screened via suppression subtractive hybridization (SSH). There are positively
regulated and negatively regulated genes. A subtractive library was constructed after
subcloning these target fragments and transforming them into competent cell. Three
hundred and fifteen positively regulated and 224 negatively regulated clones were
screened out using methods such as Polymerase Chain Reaction (PCR), Northern blot
and restriction enzyme map. Among them, 46 positively regulated and 29 negatively
regulated gene fragments were identified, respectively.
The positively regulated genes could be classified into 6 different categories
based on their physiological functions, including defense-related molecules,
respiration and antioxidation-related enzymes, metabolism or rRNA-related molecules,
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and those with unknown functions and others. Among the positively regulated gene
fragments, 13 of them were further confirmed under different stimulants and different
time points by Northern blot with respective probes. And the results have shown that
the correlation between the gene expression and different stimulants at selected time
points was identical to the previously described subtractive genes expression.
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