| Large yellow croaker belongs to perciformes, Sciaenidae, which was widely distributed in the east China sea, the south China sea and southern yellow sea. Large yellow croaker has become the most quantity of production in marine cage culture, with huge economic value. In the recent years, disease caused by vibrio genera showed outbreak trend. The disease has high mortality and spread speed, causing serious effect in large yellow croaker culture. The complement system is an important component in fish innate immunity which can recognize and facilitate the elimination of pathogens, activating immune cell, modulating the adaptive immune response. In the immune tissues, about half of the complement component exist as regulating factor and form protease complexes against invading pathogens, which plays a key role in complement activation. In this thesis, the Large yellow croaker complement regulator factors CFH, CFHR1, CFHR2 genes were identificated and characterized, the full length of complement component CFH(L.c-cfh), complement component CFHR1(L.c-cfhr1), complement component CFHR2(L.c-cfhr2) cDNAs were identificated, respectively. We also study complement regulator factors actute-phase response under Vibrio alginolyticus exposure as well as gene information and function. The result of research as follows:1. The large yellow croaker full genome sequencing sketches was used for three complement regulator factors gene cDNAs sequences: L.c-cfh full length is 1332 bp, encoding 443 aa. molecular weight is 49.96 KDa and the theoretical PI is 6.66, the number of GeneBank(KP710858). L.c-cfhr1 full length is 411 bp, encoding 137 aa. molecular weight is 15.69 KDa and the theoretical PI is 4.75, the number of GeneBank(KP710860). L.c-cfhr2 full length is 1170 bp, encoding 389 aa. molecular weight was 43.53 KDa and the theoretical PI was 5.61, the number of GeneBank(KP710859).2. Amino acid sequence structure analysis showed that complement regulator factor has the typical characteristics of RCA protein family: L.c-cfh contain five conserved CCP domains, with one N-glycosylation(Asn237) site; L.c-cfhr1 contain one conserved CCP domains, without N-glycosylation site; L.c-cfhr2 contain four conserved CCP domains, with three N-glycosylation sites(Asn90, Asn112 and Asn 148).3. Real-time PCR analysis showed that L.c-cfh was expressed in liver, spleen, kidney, intestine, brain, gill, heart, and muscle, the Liver displayed the most abundant expression level among the tested tissues. genes expressions were significantly up-regulated after challenge with Vibrio alginolyticus. In liver, the expression level of L.c-cfh was highest at the post- infection 24 h stage. then expression level although retreat, but still much higher than the control group. In the spleen, L.c-cfh expression were set to rise after the fall, at 12 h post infection quantity rose abruptly peak(p<0.05), then expressing quantity was lower but still significantly higher than the control group(p<0.05). CFH expression levels increased little in the kidney, reaching the highest point at 48 h.4. Real-time PCR analysis showed that expression level of CFHR1 was higher in the liver and heart, while weakly in the brain. Lc-cfhr1 expression growth in liver was not obvious(P>0.05), from 12 to 24 hours it had a marked increase(P<0.05), and to reach the peak. the CFHR1 expression decreased from 24 h to 48 h before increasing from 0h to 24 h in the spleen. There was a slight fluctuation before 24 h and then increased 6.2 fold compared with control group.5. The expression level of CFHR2 in eight tissues from high to low: liver, kidney, intestine, gill, muscle, heart and spleen. In liver, expression level grew gradual from 0 to 12 h after challenge with Vibrio alginolyticus(P>0.05), while with a rapid growth till 24 h, reaching the highest point. Expression of CFHR2 increased steadily in the spleen, reached 1.3 fold that of control group at 6h, then decreased a little before keeping 2 fold after 24 h. In the kidney, there was a slowly up-regulation from 0h to 36 h, then increased sharply to the 7.4 fold compared with control group.6. Mammalian species formed distinct monophyletic groups and were phylogenetically separate from fish group, analysis revealed that mammalian species and bony fish had different evolution patterns, which is consistent with the classical taxonomy.7. The paper proved the existence of large yellow croaker complement regulatory proteins, that fish has a set of relatively complete complement regulation system, providing data support for final interpretation of the immune regulation mechanism of large yellow croaker, as well as for the basis study of CFH gene families in the innate immune function and its evolution. |
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