碩士 / 東吳大學 / 微生物學系 / 90 / In insect and crustacean, propheoloxidase (proPO) activating system plays an important role of the defense and recognition functions. The previous studies have proven that the PO activity is related to the defense function of the giant freshwater prawn Macrobranchium rosenbergii. However the enzyme activity was easily influenced by the environmental conditions. Therefore, the PO activity is not able to be used as a parameter for activation of proPO system.
In this study, proPO cDNA of hemocyte of M. rosenbergii was cloned and sequenced. In future, the proPO gene can be used to further study the regulation of gene expression and to clarify the relationship between prawn defense and the expression of the proPO system.
In the experiment, cDNA of the mRNA isolated from hemocyte of M. rosenbergii was cloned into λgt11 to construct a cDNA library. The monoclonal antibody(mAb) was used to screen the library of the proPO. The result showed that the protein produced byλgt11 could be recognized by mAb. Therefore, the proPO specific mAb can not be used to screen the proPO cDNA. Futhermore, two sets of primers were design based on sequences of proPO cDNA of Penaeus monodon and Pacifasticus leniusculus, and hemocyanin cDNA of M. rosenbergii. Partial proPO cDNA was first amplified by RT-PCR using primer 1 and 2, then re-amplified by Nested-PCR using primer 3 and 4.After amplification, a 1458 bp fragment was isolated and sequenced. Using the BLAST algorithm, the nucleotide sequence of this fragment is homologous to the proPO cDNA of P. monodon with 99% similarities, of Penaeus japonicus with 82% similarities and of P. leniusculus proPO with 87% similarities. In addition, the DNA fragment from M. rosenbergii contains the six histidine residues within the two copper binding sites of proPO highly conserved in all arthropod proPOs. Therefore, we suggest that the 1458 bp cDNA fragment should be a partial proPO cDNA of M. rosenbergii.
| Identifer | oai:union.ndltd.org:TW/090SCU00381013 |
| Date | January 2002 |
| Creators | Hsin Hua Lee, 李欣樺 |
| Contributors | Hung Hung Sung, Kaun Yu Lu, 宋宏紅, 路光予 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 77 |
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