Cloning And Expression Analysis Of Antioxidant Genes Involving In The Immune System In Chinese Shrimp Fenneropenaeus Chinensis

Shrimp cultivation has been suffering serious problems due to the outbreak of diseases in the whole world. It is an emergent and necessary work for us to explore the immune system of shrimps and to find effective methods to control the shrimp diseases. It has been proved that one important immune defense reaction of crustacean hemocytes is phagocytosis when the organism is attacked by microorganisms or viruses. During the course of phagocytosis, the host glycolytic reactions get activated which in turns increase the consumption of oxygen and induce the production of a mass of reactive oxygen species (ROS). At the same time, the host also starts other immune response to defense the infection of pathogenys. All these immune response will need more ATP to support the energy which will also result in more ROS production from the electron transport chain. Though ROS can kill foreign invaders, the mass accumulation of these reactive molecules in organisms will cause serious cell damage. So the rapid elimination of these excessive ROS is essential for the proper functioning of cells and the survival of shrimps. In the thesis, four key genes of antioxidant enzymes (mitochondrial superoxide dismutase, cytosolic superoxide dismutase, catalase and peroxiredoxin) related to the immune system in Chinese shrimp Fenneropenaeus chinensis have been clone and analyzed.Two superoxide dismutase genes, mitochondrial MnSOD (mMnSOD) and cytosolic MnSOD (cMnSOD), were cloned from hemocytes of Chinese shrimp by 3'and 5'rapid amplification of cDNA ends (RACE) PCR. The full-length mMnSOD cDNA consists of 1185 bp with a 660 bp open reading frame, encoding 220 amino acids. The deduced amino acid sequence contains a putative signal peptide of 20 amino acids. Sequence comparison showed that the mMnSOD of F. chinensis shares 88% and 82% identity with that of Macrobrachium rosenbergii and Callinectes sapidus, respectively. mMnSOD transcripts were detected in hepatopancreas, hemocytes, lymphoid organ, intestine, ovary, muscle and gill by Northern blotting. RT-PCR analysis indicated that level of mMnSOD transcripts increased significantly in shrimp hemocytes and hepatopancreas after 3h post-injection of WSSV. In addition, a fusion protein containing mMnSOD was produced in vitro. LC–ESI–MS analysis showed that two peptide fragments of the recombinant protein were identical to the corresponding sequence of M. rosenbergii mMnSOD, and the enzyme activity of the refolded recombinant protein was also measured. The full-length cMnSOD cDNA consists of 1284 bp with an 861 bp open reading frame, encoding 287 amino acids. Sequence comparison showed that the cMnSOD of F. chinensis shares 98% and 94% identity with that of Penaeus monodon and Litopenaeus vannamei, respectively. The transcripts of cMnSOD gene were detected in all tissue tested. RT-PCR analysis indicated that cMnSOD transcripts increased to 3.5 fold of nomal level after 23h post-injection in hepatopancreas and increased to 2.5 fold of nomal level after 59h post-injection change in hemocytes.A partial fragment of catalase gene was cloned from hepatopancreas of Chinese shrimp by RACE and degenerate primer designed according to the sequence homolog with catalase genes from other species. Sequence comparison showed that the deduced amino acid of the partial fragment of catalase gene from F. chinensis shares 95% and 73% identity with that of Macrobrachium rosenbergii and Haliotis discus, respectively. Northern blotting analysis showed that Catalase transcripts were abundant in hepatopancreas, hemocytes, lymphoid organ, intestine and ovary, but were not abundant in muscle and gill. Real-time PCR analysis indicated that catalase gene transcripts increased significantly in shrimp hemocytes and hepatopancreas after 23h and 37h post-injection of WSSV, respectively.A Prx gene was firstly isolated in the crustacean, Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA consists of 942bp with a 594 bp open reading frame, encoding 198 amino acids. The molecular mass of the deduced amino acid is 22041.17 Da with an estimated pI of 5.17. Sequence comparison showed that Prx of F. chinensis shares 77%, 76% and 73% identity with that of Aedes aegypti, Branchiostoma belcheri tsingtaunese and Drosophila melanogaster, respectively. Northern blot analysis revealed the presence of Prx transcripts of F. chinensis in all tissues examined. Real-time PCR analysis indicated that the Prx showed different expression profiles in shrimp hemocytes and hepatopancreas after artificial infection with Vibrio anguillarum. In addition, a fusion protein containing Prx was produced in vitro. LC–ESI–MS analysis showed that four peptide fragments of the recombinant protein were identical to the corresponding sequence of F. chinensis Prx. And the purified recombinant proteins were shown to reduce H2O2 in the presence of dithiothreitol.