CDNA Cloning And Expression Analysis Of The Enzyme Genes Related To Ammonia Metabolism In Fenneropenaeus Chinensis And Study On Effect Of These Enzymes In The Detoxification Of Ammonia Following Ambient Ammonia Stresses

Fenneropenaeus chinensis, is an important mariculture species in Chinese Huanghai sea andBohai sea. It has many outstanding features, such as growing faster, resisting low temperatureand being good quality. A new species of Fenneropenaeus chinensis named "Huanghai NO.3"has been selected in our library and has stronger stress (pH、ammonia)resistance than other F.chinensis.The concentration of ammonia nitrogen is the important index in measuring water quality. Itis well known that excessive amounts of ammonia in the aquaculture environment has asignificant effect on physiology and even leads to death except of pathogen infection. Glutamicoxalacetic transaminase, glutamate dehydrogenase and glutamine synthetase play importantroles in the processes of amino acid catabolism and the ammonia nitrogen transformation andexcretion, so, in this paper we successfully cloned the above enzyme genes (including aspartateaminotransferase and glutamate dehydrogenase) cDNA of F. chinensis and investigated theireffects on ammonia detoxification. This will give a theoretical foundation for the research ofammonia metabolism of F. chinensis and an experiment foundation for the research ofstress-resistance mechanism of "Huanghai NO.3".The study consists of three parts:The first part: Cloning of aspartate aminotransferase gene from F. chinensis and the effectof ambient ammonia stresses on its activity and gene expression. The activity of GOT wasanalyzed in the gill, hepatopancreas of F. chinensis, after exposure to different ammoniaconcentrations(4mg/L,16mg/L). The results showed that the activity of GOT was inhibited inexperimental groups most of the time.We successfully cloned the aspartate aminotransferase cDNA of F. chinensis (FcGOT).The FcGOT cDNA was1910bp in length, which contained an open reading frame (ORF) of1284bp, encoding a427amino-acid polypeptide. FcGOT protein exhibited the typical AAT likefamily members, including a Lys catalytic residue and10pyridoxal-5′-phosphate binding sitessuggesting that it belongs to the AAT-Ⅰsuperfamily. Homology analysis revealed that theamino acid sequence of FcGOT was highly conserved with its homologs in other arthropod.The similarities of FcGOT with the GOT of Procambarus clarkia and Planococcus citri were78%and73%, respectively. Phylogenetic analysis showed that FcGOT was in the same branchwith that of P. clarkia.The tissues expression analysis showed that FcGOT transcripts was detected in all testedtissues, including hepatopancreas, gill, muscle, hemocytes, heart and lymph, and the highest expression level of FcGOT was in hepatopancreas. To study the biological functions of FcGOTin F.chinensis, the hepatopancreas and gill were drawn in1h,6h,12h,24h,48h,72h after exposedto ammonia nitrogen of different concentrations. The expression of FcGOT gene wassignificantly different compared with control group (P<0.05). The results implied that FcGOTmight play an important role in the prawn ammonia metabolism and be involved in responses toacute ammonia stresses.The second part: Cloning of glutamate dehydrogenase gene from F. chinensis and the effectof ambient ammonia stresses on its activity and gene expression. The activity of GDH wasanalyzed in muscle of F. chinensis, after exposure to different ammoniaconcentrations(4mg/L,16mg/L). Our results show that the activity of GDH was stimulated byammonia stresses and the activity in16mg/L group was higher than that in4mg/L group.We successfully cloned the glutamate dehydrogenase cDNA of F. chinensis (FcGDH). TheFcGDH cDNA was1779bp in length, which contained an open reading frame (ORF) of1659bp, encoding a552amino-acid polypeptide. Homology analysis revealed that the amino acidsequence of FcGdh was highly conserved with its homologs in other arthropod. The similaritiesof FcGDH with the GDH of Litopenaeus Vannamei was98%. Phylogenetic analysis showedthat FcGDH was in the same branch with that of L. Vannamei, and then in the branch with thatof Eriocheir sinensis, Drosophila melanogaster and Aedes aegypti in sequence.The tissues expression analysis showed that FcGDH transcripts was detected in all testedtissues, including hepatopancreas, gill, hemocytes, muscle, lymph, stomach and ntestine, andthe highest expression level of FcGDH was in muscle. Muscle is the major tissue for proteindeposition and possibly represents the main pool of amino acids. The metabolism of mostamino acids also occurs in this tissue. To study the biological functions of FcGDH inF.chinensis, the muscle and gill were drawn after exposed to ammonia nitrogen of differentconcentrations. The expression of FcGDH gene was significant changed in these tissues andup-regulation at a later stage, significantly different compared with control group (P<0.05). Theresults implied that FcGDH might play an important role in the process of ammoniadetoxification.The third part: The effect of ammonia stress on glutamine synthetase activity amd geneexpression in F. chinensis. The activity of GS was significantly up regulated in hepatopancreasand muscle following ammonia stresses and the activity in16mg/L group was higher than thatin4mg/L group. In addition, the activity increased as stress time went on.The FcGS gene expression was obviously increased in hepatopancreas and muscle followingdifferent concentration ammonia stresses. On the whole, the mRNA expression of FcGS inmuscle was a little more than that in hepatopancreas, which is in agreement with the result ofmeasuring GS activity. The results suggested that GS have a positive response to ambientammonia stress and supported the role of GS in the production of glutamine to reduce the toxicity of ammonia.