Identification of genes that are differentially expressedin hemocytes of Macrobranchium rosenbergiifollowing stimulus of immunostimulants

• Main Authors: Hsin-ju Sung, 宋昕如
• Other Authors: Hung-hung Sung
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/04004731636992291363

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, 3 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.