Study On Glutamate Dehydrogenase Gene Of The Economic Macro-decapods (Crustacean) Species

Free amino acid (FAA) play a vital role in cell volume and osmoregulation process of organisms. Among these FAAs, glutamate is a most important one. But little information is known about glutamate dehydrogenase (GDH), which is the key enzyme in the process of glutamate metabolism. Meanwhile, as a major group of crustacean, decapods species have various salinity adaptability. So the study of relationship between GDH gene and salinity variation has a significant meaning for further understanding in osmoregulation and polarization evolution of decapoda.The GDH gene of Litopenaeus vannamei was analyzed using methods of bioinformatics after fragments of GDH cDNAs from 5 economic animals of decapods were sequenced in this study. Then the expression profiles of GDH mRNA during acute and chronic salinity stress in L.vannamei were investigated. Results of current study can provide basic data for understanding mechanism of osmoregulation and new approach for culture of economic crustaceans.1. Characterization of the glutamate dehydrogenase genes in five economic animals of Decapods, CrustaceanConventional PCR technical was used for amplification of GDH cDNA fragments of L. vannamei, Macrobrachium rosenbergii, Cherax quadricarinatus, Eriocheir sinensis, and Scylla paramamosain. The alignment of the five GDH amino acid sequences with those of other species showed some highly conservative regions. A highly homology region was detected in all eukaryotic GDH sequences. Other two conservative regions existed in sequences of vertebrates and invertebrates, respectively. Accroding to the existing literatures, GDH in eukaryotic organisms can divided into two families (GDH-1 and GDH-2) by their differences in structure.2. Bioinformatics analysis for glutamate dehydrogenase gene in L.vannameiFurther bioinformatics analysis for GDH-B in L.vannamei was carried out and 20α-helices and 14β-strands were found in the secondary structure of GDH-B. However, 14α-helices and 10β-strands were discovered in its three-dimensional structure that made by Swiss-MODEL. Two conservative amino acid regions located in twoβ-strands concentration regions. Meanwhile, we found a Glu/Leu/Phe/Val dehydrogenases active site encoding VpfGGAKaGlkiNP at th 170-183 aa of GDH-B. The deduced amino acids composition of GDH-B in L.vannamei is analysised using Bioedit and the results showed glycine, alanine and glutamate which related with osmoregulation are abundant in this protein.3. Expression profiles of glutamate dehydrogenase gene during acute salinity stress in L.vannameiA semi-quantitative real-time PCR assay was developed to estimate the expression profiles of two GDH genes in muscle and gills of L. vannamei, during acute salinity stress. The results showed that GDH-A mRNA expression reached the peak level 24 hr after exposure both at hypo-and hyperosmotic stress. The relative amount of GDH-A mRNA in gills is higher than that in muscle and the difference are most obvious at 24 hr exposure. The maximum mRNA level of GDH-B attained at 48 hr both in gill and muscle, but no significant change can been observed in diffetent exposure time. While the relative amount of GDH-B mRNA in gill reached the peak point at 96 hr exposure and significantly higher than the expression levels of other sampling times. The results suggest GDH-A has a closer relationship with osmoregulation in acute salinity stress for the short response time in gills and GDH-B may paly its role in the complex process of metabolism.4. Expression profiles of glutamate dehydrogenase gene during chronic salinity stress in L.vannameiA semi-quantitative real-time PCR assay was developed to estimate the expression profiles of two GDH genes in muscle and gills of L. vannamei, during chronic salinity stress. The results showed a significant low expression level of GDH-A in muscle and gills at 5%o rather than 20%o and 32%o, but no remarkable tissue specific was detected. For GDH-B, a significant high expression level in muscle but gills can be observed. As muscle is a main site for accumulation of amino acid, it can be speculated that GDH-B is important for synthetic of glutamate, while GDH-A's role in osmoregulation is not apparent for long-term salinity stress.