碩士 / 國立高雄海洋科技大學 / 海洋生物技術研究所 / 100 / Abstract
Prophenoloxidase (proPO) and cytosolic manganese superoxide dismutase (cMnSOD) play crucial roles in crustacean innate immunity. In the present study, both of above genes were cloned from hemocytes of the red claw crayfish Cherax quadricarinatus using reverse transcription- polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) method, and then total hemocyte counts (THCs), gene expression and enzyme activities were characterized after injection of white spot syndrome virus (WSSV) or Aeromonas hydrophila. The full length cDNA of proPO is 2930 bp and encodes 665 amino acids, wherease the cMnSOD cDNA is 1096 bp in length and encodes 287 amino acids. The calculated molecular mass of translated protein of proPO and cMnSOD is 75.8 kDa and 31.3 kDa with an estimated pI of 6.08 and 5.82, respectively. Phylogenetic analysis of amino acid sequences showed that C. quadricarinatus proPO and cMnSOD are more closely related to the proPO and cMnSOD of other crayfish than to that of penaeids, crabs, lobsters, or freshwater prawns. Tissue distributation analysis revealed that the proPO is primarily expressed in hemocytes, gill and heart, though the higher expression levels of cMnSOD showed in hemocytes, gill and hepatopancrease. The crayfish artificially infected with WSSV all died within 4 days generally. However, there was no mortality in crayfish when infected with A. hydrophila. THCs significantly decreased in crayfish at 48 h and 72 h after the infection of WSSV compared to the PBS group. In contrast, THCs of crayfish after A. hydrophila challenge were recovered at 48 h and 72 h from the significantly lower level at 24 h. The expression level of proPO and cMnSOD transcripts in hemocytes varied greatly after the challenge of WSSV or A. hydrophila. After WSSV challenge, the expression of both genes were significantly up-regulated at 24 h, but down-regraulated at other time points including 6 h, 12 h, 48 h and 72 h. While after the infection of A. hydrophila, both genes transcripts only significantly increased at 12 h and 24 h compared with the PBS group. There were similiar responses for the enzyme activity toward WSSV and A. hydrophila infection. The PO activities per hemocyte significantly increased from 24 h to 72 h compared to the PBS group, and cMnSOD exhibited higher level later at 48 h and 72 h post virus and bacteria infection.
| Identifer | oai:union.ndltd.org:TW/100NKIMT270009 |
| Date | January 2012 |
| Creators | Yen-Ting Liu, 劉彦廷 |
| Contributors | Jyh-Ming Tsai, 蔡志明 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 54 |
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