尼罗罗非鱼耐盐碱选育二代耐受性、生长性能及碱胁迫下氨代谢基因表达变化

发布时间：2018-06-15 00:39

本文选题：尼罗罗非鱼 + 选育二代　；参考：《上海海洋大学》2016年硕士论文

【摘要】：近年来,我国可利用的淡水资源日益紧缺,同时存在大量的盐碱水资源未被利用,这些盐碱水域由于盐度高、碱度大、离子比例复杂等多重原因,使得能生存的水生生物种类比较稀少。尼罗罗非鱼(Oreochromis niloticus)是我国淡水养殖主要对象之一,不仅生长速度快,对盐、碱环境耐受能力也较强,经驯化后可以在一定范围的盐碱水中生存生长,可作为耐盐碱养殖品种培育的重要候选对象之一。本论文以尼罗罗非鱼耐盐碱选育二代为材料,评估了幼苗急性盐碱耐受性能,慢性盐碱耐受性能及盐碱水中生长性能;比较在不同碳酸盐碱度(2、4、6 g/L NaHCO3)胁迫后不同时间(0、2、4、6、12、24、72 h)尼罗罗非鱼血氨浓度变化,并用荧光定量PCR方法检测氨代谢途径相关基因GS、GLS2、CA-5A、Rhcgl2和CPS1基因在不同组织胁迫过程中的表达变化及GS、CA酶活性变化,主要结果如下:(1)以尼罗罗非鱼盐碱驯化选育二代幼鱼为试验材料,通过急性致死试验比较它们在不同盐度组(18 g/L、20 g/L、22 g/L、24 g/L、26g/L)中的半致死盐度,碳酸氢钠(NaHCO3)碱度组(2 g/L、4 g/L、6g/L、8 g/L、10 g/L、12 g/L)中的半致死碱度,以及盐碱混合组(盐度10g/l、15g/l、20g/l,碱度2g/l、4g/l、6g/l)中的半致死盐碱度。试验结果表明,尼罗罗非鱼选育二代幼鱼96h的半致死盐度为21.809g/l,半致死碱度为8.514g/l,半致死盐碱度:10g/l盐度下,碱度为5.925g/l;15g/l盐度下,碱度为4.583g/l;20g/l盐度下,碱度为1.897g/l。选育二代幼鱼的96h半致死盐碱度与选育一代差异不显著(p0.05),但都显著高于基础群体的96h半致死盐碱度(p0.05)。(2)以尼罗罗非鱼耐盐碱选育二代为材料,通过慢性致死试验及在不同盐碱环境(s0a0、s10a0、s10a2、s10a4、s10a6)中生长对比试验,评估选育二代的盐碱耐受性能和生长性能。慢性致死试验结果表明,选育二代与对照组(非选育群体)的半致死盐度分别为57.37±1.56g/l、48.71±1.58g/l,半致死碱度分别为19.09±0.76g/l、16.32±0.45g/l。60d的生长对比试验结果表明,随着盐碱浓度增加,选育二代与对照组日均增重率都表现为下降趋势;在s0a0组中,选育二代日均增重率与对照组间差异不显著(p0.05),在s10a0、s10a2、s10a4、s10a6组中,选育二代日均增重率均显著大于对照组(p0.05)。本实验证明,选育后代的盐碱耐受性能和盐碱环境下的生长性能得到了提高。(3)为了理解尼罗罗非鱼在碳酸盐碱环境适应过程中氨代谢调节途径,本研究选取了5个氨代谢相关基因:谷氨酰胺合成酶(glutaminesynthetase,gs)、碳酸酐酶5a(carbonicanhydrase5a,ca-5a)、谷氨酰胺酶(glutaminase2,gls2)、氨甲酰磷酸合成酶(carbamylphosphate synthetase 1,CPS1)、氨转运蛋白(Ammonium transporter Rh type C-2 like,Rhcgl2),研究了急性碳酸盐碱度胁迫条件下,尼罗罗非鱼血氨浓度变化、氨代谢相关基因表达水平及其酶活性变化。结果表明,随碳酸盐碱胁迫浓度升高,尼罗罗非鱼血氨浓度上升,随时间推移呈先上升、后下降的变化趋势,在胁迫后12 h达到峰值。氨代谢相关基因在不同碱度下、不同组织中均有不同程度的上调表达,随着胁迫时间推移呈先上升、后下降的变化趋势,胁迫后12-24 h各基因表达水平显著升高,随后逐渐恢复到稳定水平;氨代谢相关基因具有一定的组织表达差异:Rhcgl2主要在鳃中表达,CA-5A、GS、CPS1主要在肝脏中表达,GLS2主要在肾脏和鳃中表达。碳酸酐酶和谷氨酰胺合成酶活性随胁迫碱度的升高而上升,碳酸酐酶、谷氨酰胺合成酶活性变化分别在鳃、肝脏中最为显著。研究结果表明,碳酸盐碱度胁迫会引起尼罗罗非鱼血氨水平升高,随着时间推移血氨水平下降,推测鳃、肝脏、肾脏中不同氨代谢基因共同参与调节氨代谢,在鳃中通过直接排氨,在肝脏中通过合成谷氨酰胺、尿素途径,共同调节降低血氨水平。
[Abstract]:In recent years, the available freshwater resources in China are increasingly scarce, and a large number of salt and alkali water resources have not been used. These saline waters are relatively rare because of high salinity, large alkalinity and complex ion ratio. The Nile Luo Fei fish (Oreochromis niloticus) is the main freshwater aquaculture in China. One of them, not only has fast growth rate, but also has strong tolerance to salt and alkali environment. After domestication, it can survive and grow in a certain range of saline and alkaline water. It can be used as one of the important candidates for cultivation of saline alkali breeding varieties. In this paper, the salt tolerance of Nile tilapia was selected as the two generation material to evaluate the acute salt tolerance performance of the seedlings, and the chronic salt tolerance of the seedlings was evaluated. Salt and alkali tolerance performance and growth performance in saline alkali water; compare the change of blood ammonia concentration in Nile tilapia at different time (0,2,4,6,12,24,72 h) after different carbonation alkalinity (2,4,6 g/L NaHCO3) stress, and use the fluorescence quantitative PCR method to detect the gene of ammonia metabolism pathway related genes GS, GLS2, CA-5A, Rhcgl2 and CPS1 in different tissue stress processes The changes and changes in the activity of GS and CA enzymes are as follows: (1) the semi lethal salinity of the two generation of young Nile tilapia in the acclimatization of Nile tilapia, by the acute lethal test, was compared in the different salinity groups (18 g/L, 20 g/L, 22 g/L, 24 g/L, 26g/L), and the sodium bicarbonate (NaHCO3) alkalinity group (2 g/L, 4 g/L, 6g/L, 8 g/L, 10 g/L, 12,) The semi lethal alkalinity of the semi lethal alkalinity and the salt alkali mixture group (salinity 10g/l, 15g/l, 20g/l, alkalinity 2g/l, 4g/l, 6g/l). The results showed that the semi lethal salinity of the two generation of Nile tilapia was 21.809g/l, the semi lethal alkalinity was 8.514g/l, and the half death salt alkalinity: 10g/l salinity, alkalinity was 5.925g/l; under 15g/l salinity, alkali. The degree of degree was 4.583g/l; under the salinity of 20g/l, the difference of the 96h semi lethal salinity of the two generation of two generation young fish with alkalinity was not significant (P0.05), but both were significantly higher than the 96h semi lethal alkalinity (P0.05) of the base population. (2) two generations of Nile tilapia were selected as the material, through the chronic lethal test and in the different saline environment (s0a0, S10). The growth contrast test in A0, s10a2, s10a4, s10a6) was used to evaluate the salt tolerance performance and growth performance of the two generations. The results of chronic lethal test showed that the semi lethal salinity of the two generation and the control group was 57.37 + 1.56g/l, 48.71 + 1.58g/l, and the semi dead alkalinity was 19.09 + 0.76g/l, 16.32 + 0.45g/l.60d, respectively. The test results showed that with the increase of salt and alkali concentration, the daily average weight gain of the two generation and the control group showed a downward trend. In the s0a0 group, the average daily weight gain of the two generation was not significant (P0.05). In the group of s10a0, s10a2, s10a4 and s10a6, the daily average weight gain of the two generation was significantly greater than that of the control group (P0.05). The experiment proved that the selection of the daily average weight gain rate of the two generation was significantly greater than that of the control group. The salt tolerance performance of the offspring and the growth performance in the saline alkali environment were improved. (3) in order to understand the regulation of ammonia metabolism in the adaptation process of Nile tilapia in the carbonation and alkali environment, 5 ammonia metabolism related genes (glutaminesynthetase, GS), carbonic anhydrase 5A (carbonicanhydrase5a, ca-5a) were selected. Glutaminase2 (gls2), carbamylphosphate synthetase 1, CPS1, ammonia transporter (Ammonium transporter Rh type C-2 like, Rhcgl2). The change of blood ammonia concentration in Nile tilapia, the expression level of ammonia metabolism related genes and the changes of enzyme activity under the condition of acute carbonation stress were studied. The results showed that the blood ammonia concentration of Nile tilapia increased with the increase of the concentration of salt and alkali stress. It increased first and then decreased with time, and reached the peak at 12 h after stress. At different alkalinity, the ammonia metabolism related genes were up and down in different degrees under different alkalinity, and increased first and then decreased with the time of stress. The expression level of 12-24 h after stress increased significantly after stress, and then gradually recovered to the level of stability; ammonia metabolism related genes had certain differences in tissue expression: Rhcgl2 was mainly expressed in gills, CA-5A, GS, CPS1 were mainly expressed in the liver, and GLS2 was mainly expressed in the kidneys and gills. The activity of carbonic anhydrase and glutamine synthetase was associated with the activity of carbonic anhydrase and glutamine synthetase. The changes in the activity of carbonic anhydrase and glutamine synthetase were the most significant in the gills and the liver. The results showed that the level of blood ammonia in Nile tilapia could be caused by the stress of carbonate alkalinity, and the levels of blood ammonia decreased with time, and the gene of different ammonia metabolism in the gills, liver and kidneys was involved in the regulation. Ammonia metabolism, through direct ammonia excretion in the gill, can regulate the reduction of blood ammonia level through the synthesis of glutamine and urea pathway in the liver.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S917.4

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