Cloning And Expression Patterns Of Gelatinase Gene And Inhibitors Gene In Grass Carp

Grass carp （Ctenopharyngodon idella） is one of the most impotant freshaquaculture fishes in China. Bacterial diseases are serious problems in freshwater fishbreeding process, which is a limiting factor to fresh water breeding. Use of molecularfor beeding can promote the sustainable development of freshwater aquaculture, socomprehensive in-depth study of the pathogen pathogenesis and fish immune-relatedgenes. Gelatinase gene is a kind of important immune factor, it consists of matrixmetalloproteinase-2gene and matrix metalloproteinase-9gene. In this thesis, the grasscarp MMP-2,9, TIMP-2b and HSP60genes were cloning and characterized. Analysis oftheir physicochemical properties, amino acid structure, compare their homology, byformalin inactivated Aeromonas hydrophila liquid on grass carp, study them expressionchange at different developmental process, different organizations and by A. hydrophilaafter infectionin grass carp, and obtained the following results.1. Using degenerated PCR amplification strategy, MMP-2, MMP-9and HSP60cDNA fragments were cloned from the grass carp, A959bp fragment of TIMP-2b genewas obtained from a normalized full-length cDNA library of head kidney tissues ofgrass carp. After RACE PCR,3102bp，2880bp，2434bp，1224bp full-length cDNAof MMP-2, MMP-9, HSP60and TIMP-2b are obtained, which containing the openreading frame （ORF） encoded658aa （1974bp），675aa （2025bp），575aa （1728bp），218aa （657bp） respectively. The first29,20,24residues for MMP-2, MMP-9andTIMP-2b were predicted to be a signal peptideres pectively.2. BLAST rearch against the databases revealed that the deduced amino acidsequence shows high similarity to other similar protein sequence. The grass carpMMP-2shares94%amino acid identity with MMP-2in common carp. The grass carp MMP-9shares89%amino acid identity with MMP-9in common carp. The grass carpTIMP-2b shares83.9%amino acid identity with TIMP-2b in zebrafish. The grass carpHSP60shares96.9%amino acid identity with TIMP-2b in Tanichthys albonubes. Aphylogenetic tree was constructed, based on the deduced full-length amino acidsequences alignment by the neighbor joining （NJ） algorithm and Maximum Likelihood（ML） method embedded in MEGA5.0. Phylogenetic analysis results also showed thatgrass carp clustered with other teleost, which supports the traditional taxonomicrelationships.3. Using BLAST program searches the predicted amino acid sequences showed:The MMP-2and MMP-9protein contains four conserved domain structures including aN-terminal signal sequence, a propeptide domain, three repeats of fibronectin-typeⅡdomain inserted in the catalytic domain and C-terminal hemopexin-like domain. TheTIMP-2b protein encodeda signal peptide, an N-and a C-terminal domain. CiHSP60protein has a conserved ATP-binding/Mg²⁺binding site, a hinge region, ringoligomerization interface sites and stacking interaction sites.4. MMP-2, MMP-9, TIMP-2b and HSP60mRNA contents in tissues were detectedon the samples from three unchallenged fish to illustrate the spatial expression patternsof4genes in grass carp using qRT-PCR. Various tissues were tested, including blood,brain, muscle, trunk kidney, liver, head kidney, skin, spleen, heart, gill, intestine and fin.Total RNA was extracted as described above. Testing results showed that,4genes wasdetected in all tissues are expressed, suggesting that they may play a broad role in grasscarp. MMP-2mRNA was lower expressed in blood, hear kidney and fin. MMP-9andTIMP-2mRNA were highest expression in blood, head kidney, trunk kidney and spleen.HSP60mRNA was highest expression in blood and liver. Fluorescent real-timequantitative RT-PCR was used toexamine the expression after challenge with A.hydrophila. CiMMP-2expression levels increased in grass carp infected with A.hydrophila in the spleen and head kidney tissues at4h, and significantlydown-regulated at1day. A clear time-dependent expression pattern of MMP-9wasfound after the bacterialchallenge, and the mRNA expression reached a maximum levelat4hour and7days post-challenge. TIMP-2b mRNA expression level upon challengewith A. hydrophila, its expression was significantly up-regulated in all tissues. A cleartime-dependent expression pattern of HSP60was found after the bacterial challenge, and the mRNA expression reached a maximum level at three days post challenge, andreturned to control levels after seven days.5. Fluorescent real-time quantitative RT-PCR was used to examine theembryogentic expression patterns. We detected the expressionof4genes in eggs,suggesting that they may be matrilineal origin. Grass carp MMP-9and TIMP-2b geneshave similar expression profiles, expression is elevated in the late.Grass carp MMP-2and HSP60genes have similar expression profiles, higher expression in the earlyembryo development.Taken together the above results, Grass carp by A. hydrophila invasion, MMP/TIMP balance system is chaotic, this may lead to disease occurrence and worsening.The expression of grass carp MMP-2, MMP-9, TIMP-2b and HSP60are modulated by A.hydrophila infection, suggesting its importance in both immune.4genes have alreadyexists in early embryonic development, suggesting its importance in developmentalprocesses.Overall, all these researches would benefit the isolation of important immunefactors, selection of base population and protection of germplasm resources, which willcontribute towards improving the quality and quanlity of aquaculture.