Molecular Cloning Of G-type Lysozyme And Caspase Family Gene From Blout Snout Bream (Megahbrama Amblycephala) And It's Expression Analysis Under Ammonia-N Stress

Megahbrama amblycephala is an important herbivorous economic fish in China,and the overtop content of ammonia nitrogen in aquaculture water is an important factor which seriously restricted it's development of high density cultivation.Lysozyme acts as an important responsive molecular component of innate immunity,caspase 9 and caspase 8 are key proteases of apoptotic pathway,while ammonia-N stress is an important abiotic stress of aquaculture and environment.However whether or not the lysozyme or caspase family had been involved in the process of ammonia-N stress,the specific molecular mechanisms and how to participate in the regulation of its molecular mechanisms are unclear.In this study,the cDNA encoding g-type lysozyme,caspase 9 and caspase 8 of blout snout bream M.amblycephala was cloned by rapid amplification of cDNA ends(RACE)approach,and analysised their expression under ammonia-N stress and recovery by Quantitative real-time PCR analysis(Applied Biosystems 7500 Real Time PCR System),The main results are as follows:1.Molecular cloning of g-type lysozyme and it's expression analysis under ammonia-N stressThe full length cDNA of g-type lysozyme is 713 bp,consisting of a 5' untranslated region of 71 bp,a 3' untranslated region of 90 bp,and an open reading frame of 558 bp.Based on amino similarity comparison,the three catalytic sites and four substrate binding sites are highly conserved among different fish,and the phylogenetic tree analysis indicated that g-type lysozyme of M.amblycephala has close relationgship with Ctenopharyngodon idella.Quantitative real-time PCR analysis showed that the g-type lysozyme gene was expressed in most tissues of M.amblycephala with highest expression in intestinal.After ammonia-N challenge and recovery,the relative expression level of g-type lysozyme in liver and brain were higher than control group at 12 h,24 h,48 h,72 h post-stress,followed by a gradual decreasing at 72 h recovery.By contrast,the relative expression level of g-type lysozyme in gill were lower than control group from 3 h to 72 h post-stress,but increaing peak at 72 h recovery.It's speculuted that g-type lysozyme gene involved in molecular processes of ammonia-N stress,the result of this study can provide theoretical support for further understanding of molecular mechanism in response to environmental stress of M.amblycephala.2.Molecular cloning of caspase 9 and it's expression analysis under ammonia-N stressThe full length cDNA of caspase 9 is 1613 bp,consisting of a 5' untranslated region of 185 bp,a 3' untranslated region of 96 bp,and an open reading frame of 1332 bp which encoding 443 amino acids.Among these amino acids there is one cysteine active site and one histidine active site what are critical to activation of caspase 9 protease.Based on amino similarity comparison by DNAMAN and phylogenetic tree analysis by MEGA 5.0,the result showed the average homology is 77.74%,it's indicated that caspase 9 gene is highly conserved among different fish,and has close relationgship with Cyprinus carpio.Quantitative real-time PCR analysis(Applied Biosystems 7500 Real Time PCR System)showed that the caspase 9 gene was expressed in most tissues of M.amblycephala with highest expression in brain and then liver,kidney,intestine,gill and lowest expression in muscle.Under ammonia-N stress,the relative expression level of caspase 9 in liver and brain showed similar pattern,a "up-down-up" trend with highest expression in 72h ammonia-N stress,and after 72h recovery,it's expression fell.These results speculut that caspase 9 gene is associated with ammonia-N stress and participate in molecular processes of ammonia-N stress in M.amblycephala,and the result of this research can provide theoretical support for further understanding of molecular mechanism in response to ammonia-N stress of M.amblycephala.3.Molecular cloning of caspase 8 and it's expression analysis under ammonia-N stressThe length cDNA of caspase 8 is 2034 bp,consisting of a 5' untranslated region of 102 bp,a 3' untranslated region of 462 bp,and an open reading frame of 1470 bp encoding 489 amino acids.Based on amino similarity comparison,the average homology is 59.56%,and phylogenetic tree analysis indicated that caspase 8 gene is highly conserved in different fish,and has close relationgship with Cyprinus carpio and Danio rerio.Quantitative real-time PCR analysis showed that the caspase 8 gene was expressed in most tissues of M.amblycephala with highest expression in brain and loweer expression in muscle and gill.Under ammonia-N stress,the relative expression level of caspase 8 in liver and brain showed similar pattern,with highest expression in 72h ammonia-N stress,and after 72h recovery,if s expression fell.It's speculuted that caspase 8 gene involved in molecular processes of ammonia-N stress.