【作者】 熊泽泉；

【导师】 陈立侨； 曾嶒；

【作者基本信息】 华东师范大学 ， 动物学， 2010， 硕士

【摘要】 游离氨基酸在生物体的渗透调节过程中发挥着重要的作用,谷氨酸是其中最为重要的一种,而谷氨酸脱氢酶(Glutamate dehydrogenase, GDH)作为谷氨酸代谢过程中的关键酶之一,有关其基因的研究并不多见。同时,十足目动物作为甲壳纲中一个重要的经济类群,对盐度的适应范围多种多样。因此,研究十足目中主要的经济动物GDH基因及其与盐度变化的关系,将对深入了解游离氨基酸在渗透调节过程中的作用机制及十足目动物的分化演变有着重要的意义。本研究在获得甲壳纲十足目5种经济动物谷氨酸脱氢酶(GDH)序列片段的基础上,选择其中凡纳滨对虾的GDH-B基因序列进行生物信息学分析,并通过对凡纳滨对虾进行急性和长期的盐度胁迫,研究谷氨酸脱氢酶基因在不同盐度胁迫条件下的表达差异及变化规律,其中主要研究结果如下：1.十足目5种经济动物谷氨酸脱氢酶基因的定性研究采用常规PCR技术,扩增获得了凡纳滨对虾(Litopenaeus vannamei)、罗氏沼虾(Macrobrachium rosenbergii)、红螯螯虾(Cherax quadricarinatus)、中华绒螯蟹(Eriocheir sinensis)、拟穴青蟹(Scylla paramamosain)肌肉GDH序列片段；通过多序列比对,发现其氨基酸序列中的若干高度保守区域,其中GGVTVS片段在所有已获得的真核生物GDH序列中表现出高度同源性；在脊椎动物和无脊椎动物类群中又分别存在一个高度保守区,综合已有文献,根据结构保守区的差异,GDH在真核生物中可分为GDH-1和GDH-2两大类,本研究所获得的5种GDH序列属于GDH-2。2.凡纳滨对虾谷氨酸脱氢酶的生物信息学分析对获得的凡纳滨对虾GDH-B全长序列进行进一步的生物信息学分析。发现凡纳滨对虾的GDH-B二级结构中存在20个α-螺旋和14个β-折叠结构,而利用Swiss-MODEL对其进行三维结构模拟则发现14个α-螺旋和10个β-折叠结构,两个氨基酸保守区域则分别存在于两个β-折叠集中区域；同时发现在其170-183aa处存在一个多种氨基酸脱氢酶的活性位点(Glu/Leu/Phe/Val dehydrogenases active site) VpfGGAKaGlkiNP,利用Bioedit软件对谷氨酸脱氢酶的氨基酸组成进行分析表明,该蛋白富含甘氨酸、丙氨酸和谷氨酸等与渗透调节相关的氨基酸类。结果提示GDH-B的结构与其功能密切相关。3.急性盐度胁迫下谷氨酸脱氢酶表达变化研究利用实时荧光定量PCR技术研究了急性盐度胁迫下凡纳滨对虾肌肉和鳃中GDH的表达变化情况。发现无论面对低盐胁迫还是高盐胁迫,GDH-A在肌肉和鳃中均是胁迫后24 hr其表达量最高,在高盐胁迫下,同一时刻GDH-A在鳃中的表达量始终高于其在肌肉中的表达量,在24 hr时差异最为显著。GDH-B的变化则稍有差异,在低盐胁迫时,其在肌肉和鳃中的表达量在48 hr最高,在高盐胁迫时,肌肉中的GDH-B表达量在48 hr最高,但变化不显著,而鳃中该基因的表达量在96 hr达到最高,且显著高于其他时刻的表达量。结果提示,在应对盐度急性胁迫时,GDH-A响应时间要早于GDH-B,其主要靶器官为鳃,可能与急性盐度胁迫下的渗透调节关系更为密切,GDH-B响应时间稍晚,可能在更为复杂的代谢过程中发挥作用。4.长期盐度胁迫下谷氨酸脱氢酶表达变化研究利用实时荧光定量PCR技术研究了在长期盐度胁迫下凡纳滨对虾肌肉和鳃中GDH的表达变化情况。发现在低盐度(5‰)下,GDH-A在肌肉和鳃中的表达量要显著低于中盐度(20‰)及高盐度组(32‰),各盐度下的组织表达差异并不显著；而对于GDH-B,高盐度组对虾肌肉中的表达量显著高于其他两组,鳃中表达差异并不显著。由于肌肉是氨基酸的主要积累场所,推测GDH-B在谷氨酸合成方面起主要作用,而GDH-A在面对长期盐度胁迫时,其在渗透调节方面的作用已不明显。更多还原

【Abstract】 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.更多还原