本文選題：尼羅羅非魚 + 選育二代　； 參考：《上海海洋大學(xué)》2016年碩士論文

【摘要】：近年來,我國可利用的淡水資源日益緊缺,同時存在大量的鹽堿水資源未被利用,這些鹽堿水域由于鹽度高、堿度大、離子比例復(fù)雜等多重原因,使得能生存的水生生物種類比較稀少。尼羅羅非魚(Oreochromis niloticus)是我國淡水養(yǎng)殖主要對象之一,不僅生長速度快,對鹽、堿環(huán)境耐受能力也較強,經(jīng)馴化后可以在一定范圍的鹽堿水中生存生長,可作為耐鹽堿養(yǎng)殖品種培育的重要候選對象之一。本論文以尼羅羅非魚耐鹽堿選育二代為材料,評估了幼苗急性鹽堿耐受性能,慢性鹽堿耐受性能及鹽堿水中生長性能;比較在不同碳酸鹽堿度(2、4、6 g/L NaHCO3)脅迫后不同時間(0、2、4、6、12、24、72 h)尼羅羅非魚血氨濃度變化,并用熒光定量PCR方法檢測氨代謝途徑相關(guān)基因GS、GLS2、CA-5A、Rhcgl2和CPS1基因在不同組織脅迫過程中的表達變化及GS、CA酶活性變化,主要結(jié)果如下:(1)以尼羅羅非魚鹽堿馴化選育二代幼魚為試驗材料,通過急性致死試驗比較它們在不同鹽度組(18 g/L、20 g/L、22 g/L、24 g/L、26g/L)中的半致死鹽度,碳酸氫鈉(NaHCO3)堿度組(2 g/L、4 g/L、6g/L、8 g/L、10 g/L、12 g/L)中的半致死堿度,以及鹽堿混合組(鹽度10g/l、15g/l、20g/l,堿度2g/l、4g/l、6g/l)中的半致死鹽堿度。試驗結(jié)果表明,尼羅羅非魚選育二代幼魚96h的半致死鹽度為21.809g/l,半致死堿度為8.514g/l,半致死鹽堿度:10g/l鹽度下,堿度為5.925g/l;15g/l鹽度下,堿度為4.583g/l;20g/l鹽度下,堿度為1.897g/l。選育二代幼魚的96h半致死鹽堿度與選育一代差異不顯著(p0.05),但都顯著高于基礎(chǔ)群體的96h半致死鹽堿度(p0.05)。(2)以尼羅羅非魚耐鹽堿選育二代為材料,通過慢性致死試驗及在不同鹽堿環(huán)境(s0a0、s10a0、s10a2、s10a4、s10a6)中生長對比試驗,評估選育二代的鹽堿耐受性能和生長性能。慢性致死試驗結(jié)果表明,選育二代與對照組(非選育群體)的半致死鹽度分別為57.37±1.56g/l、48.71±1.58g/l,半致死堿度分別為19.09±0.76g/l、16.32±0.45g/l。60d的生長對比試驗結(jié)果表明,隨著鹽堿濃度增加,選育二代與對照組日均增重率都表現(xiàn)為下降趨勢;在s0a0組中,選育二代日均增重率與對照組間差異不顯著(p0.05),在s10a0、s10a2、s10a4、s10a6組中,選育二代日均增重率均顯著大于對照組(p0.05)。本實驗證明,選育后代的鹽堿耐受性能和鹽堿環(huán)境下的生長性能得到了提高。(3)為了理解尼羅羅非魚在碳酸鹽堿環(huán)境適應(yīng)過程中氨代謝調(diào)節(jié)途徑,本研究選取了5個氨代謝相關(guān)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)、氨轉(zhuǎn)運蛋白(Ammonium transporter Rh type C-2 like,Rhcgl2),研究了急性碳酸鹽堿度脅迫條件下,尼羅羅非魚血氨濃度變化、氨代謝相關(guān)基因表達水平及其酶活性變化。結(jié)果表明,隨碳酸鹽堿脅迫濃度升高,尼羅羅非魚血氨濃度上升,隨時間推移呈先上升、后下降的變化趨勢,在脅迫后12 h達到峰值。氨代謝相關(guān)基因在不同堿度下、不同組織中均有不同程度的上調(diào)表達,隨著脅迫時間推移呈先上升、后下降的變化趨勢,脅迫后12-24 h各基因表達水平顯著升高,隨后逐漸恢復(fù)到穩(wěn)定水平;氨代謝相關(guān)基因具有一定的組織表達差異:Rhcgl2主要在鰓中表達,CA-5A、GS、CPS1主要在肝臟中表達,GLS2主要在腎臟和鰓中表達。碳酸酐酶和谷氨酰胺合成酶活性隨脅迫堿度的升高而上升,碳酸酐酶、谷氨酰胺合成酶活性變化分別在鰓、肝臟中最為顯著。研究結(jié)果表明,碳酸鹽堿度脅迫會引起尼羅羅非魚血氨水平升高,隨著時間推移血氨水平下降,推測鰓、肝臟、腎臟中不同氨代謝基因共同參與調(diào)節(jié)氨代謝,在鰓中通過直接排氨,在肝臟中通過合成谷氨酰胺、尿素途徑,共同調(diào)節(jié)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.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S917.4

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