【摘要】：凡納濱對蝦(Litopenaeus vannamei)又稱南美白對蝦,具有較強的逆境適應(yīng)性、較快的生長速度及較高的營養(yǎng)價值。自1988年引入中國,經(jīng)過近30年的培育和發(fā)展,凡納濱對蝦已成為我國對蝦三大養(yǎng)殖品種之一。近年來隨著高密度、集約化養(yǎng)殖及人類生產(chǎn)、生活污水排放,養(yǎng)殖水體中污染物急劇增加,導(dǎo)致水質(zhì)不斷惡化,給凡納濱對蝦養(yǎng)殖業(yè)帶來了嚴(yán)重的經(jīng)濟損失。作為危害對蝦健康養(yǎng)殖最重要的環(huán)境因子一氨氮,它可以通過生理代謝途徑進(jìn)入凡納濱對蝦組織液中,并引起血氨中毒,進(jìn)一步破壞其免疫系統(tǒng)、擾亂其滲透壓平衡,嚴(yán)重影響其生長發(fā)育性能。不同發(fā)育時期的凡納濱對蝦應(yīng)答氨氮脅迫的敏感性不同;另外,不同家系相同規(guī)格的凡納濱對蝦應(yīng)答氨氮脅迫的敏感性也存在顯著差異,但目前有關(guān)凡納濱對蝦應(yīng)答氨氮脅迫的相關(guān)機制尚不清楚。因此,該論文首先研究了凡納濱對蝦不同發(fā)育時期應(yīng)答氨氮脅迫的敏感性;然后選取對環(huán)境抗逆性較差的家系(A3281,A)與抗逆性較強的家系(B3271,B)開展比較研究,側(cè)重探究了氨氮代謝相關(guān)酶類(谷氨酸脫氫酶(GDHase)、谷氨酰胺合成酶(GSase)與谷氨酰胺轉(zhuǎn)胺酶(TGase))活性及其對應(yīng)基因應(yīng)答氨氮脅迫的時空變化規(guī)律,為揭示凡納濱對蝦應(yīng)答氨氮脅迫的敏感性及其分子機制打下一定的基礎(chǔ),同時,為選育耐氨氮新品系提供科學(xué)依據(jù)。主要研究結(jié)果如下:1、凡納濱對蝦早期生長階段,蚤狀幼體Ⅱ期(Z2)對氨氮脅迫最為敏感。凡納濱對蝦不同生長發(fā)育時期對氨氮的敏感性不同,成體比幼體具有更高的氨氮耐受性;從無節(jié)幼體至仔蝦Ⅴ期(P5)發(fā)育階段中的凡納濱對蝦,其Z2階段對氨氮脅迫最為敏感,可作為檢測氨氮脅迫的關(guān)鍵點,其半數(shù)致死濃度LC50為17.811 mg/L。2、三種氨氮代謝相關(guān)酶應(yīng)答氨氮脅迫的時空變化規(guī)律。(1)兩個家系不同組織GDHase活性變化規(guī)律的比較分析不同濃度的氨氮脅迫(3.4-24.6 mg/L)顯著影響凡納濱對蝦各組織GDHase的活性:其中,兩個家系肌肉GDHase活性隨氨氮濃度的升高而表現(xiàn)為被顯著抑制(p0.05);脅迫的前5天兩家系肝胰腺組織GDHase活性隨氨氮濃度的升高均被誘導(dǎo),而脅迫后期(5-10天)呈現(xiàn)顯著抑制(p0.05)。另外,肌肉與肝胰腺組織中GDHase活性差異性不顯著(p0.05)。(2)兩個家系不同組織GSase活性變化規(guī)律的比較分析兩個家系凡納濱對蝦肌肉組織中的GSase活性隨氨氮濃度升高均呈現(xiàn)被誘導(dǎo)的趨勢;而肝胰腺組織中GSase活性變化,家系間存在顯著差異:其中,家系A(chǔ)隨氨氮濃度的升高而呈現(xiàn)被抑制的趨勢;而家系B于脅迫初期呈現(xiàn)上調(diào)趨勢,后期呈現(xiàn)被抑制的狀態(tài)(T5天)。(3)兩個家系不同組織TGase活性變化規(guī)律的比較分析氨氮脅迫前期(T5天),顯著抑制兩家系肌肉與肝胰腺組織的TGase活性,隨氨氮濃度的升高TGase酶活性逐漸降低;脅迫后期(T5天)TGase酶活性呈現(xiàn)顯著的家系差異性:隨氨氮濃度的升高家系B兩個組織中的TGase活性顯著被抑制,而家系A(chǔ)則被顯著誘導(dǎo)(p0.05)。綜合兩組織中三種氨氮代謝酶活性的變化規(guī)律,發(fā)現(xiàn)應(yīng)對不同程度的氨氮脅迫,兩家系凡納濱對蝦的肌肉組織均可以通過激活氨氮轉(zhuǎn)化類酶活性(GSase),同時抑制氨氮生產(chǎn)類酶(TGase)的活性來維持組織中氨氮含量的平衡。3、三種氨氮代謝相關(guān)酶的整合生物標(biāo)志物響應(yīng)(IBR)指數(shù)分析通過對各時間點三種酶在不同濃度氨氮脅迫下的IBR分析表明:氨氮脅迫對兩家系凡納濱對蝦的肌肉組織的GSase與TGase活性影響最大,且其影響力隨氨氮濃度的增加而變大;家系B應(yīng)對脅迫反應(yīng)時間(T5天)明顯早于家系A(chǔ)(5T10天)。綜合分析相關(guān)代謝酶的催化功能,可以推斷出抗性較強的家系B肌肉組織可率先通過氨氮代謝酶的催化作用調(diào)控機體氨氮代謝,尤其是加速催化glutamate與NH4+合成glutamine這一代謝途徑。IBR的劑量-脅迫效應(yīng)顯示:隨環(huán)境中氨氮濃度升高兩家系的肝胰腺組織受到的脅迫效應(yīng)均呈現(xiàn)出上升趨勢,而肌肉組織呈現(xiàn)出下降趨勢。說明隨著環(huán)境中氨氮濃度升高,肝胰腺組織在氨氮脅迫中發(fā)揮更大的作用,肝胰腺是氨氮脅迫的重要靶組織。4、三種氨氮代謝酶對應(yīng)基因應(yīng)答氨氮脅迫的時空變化規(guī)律(1)不同家系肌肉組織中氨氮代謝酶基因表達(dá)規(guī)律的比較分析各濃度氨氮均可顯著影響兩家系肌肉組織GDH-β與GS基因的表達(dá)(p0.05):整體而言,兩基因隨氨氮脅迫時間的延長與脅迫濃度的增加均呈現(xiàn)顯著上調(diào)趨勢;且家系B中兩基因的表達(dá)量顯著高于家系A(chǔ)(p0.05),表明B家系調(diào)動氨氮轉(zhuǎn)化的能力較強,這從基因水平揭示了不同家系應(yīng)答氨氮耐受能力差異的原因。脅迫后期,兩家系肌肉組織中TG基因表達(dá)呈現(xiàn)顯著差異:隨著氨氮濃度的升高家系A(chǔ)肌肉中TG基因表現(xiàn)為顯著下調(diào),而家系B肌肉則表現(xiàn)為顯著上調(diào),兩家系間差異顯著(p0.05)。(2)不同家系肝胰腺組織中氨氮代謝酶基因表達(dá)規(guī)律的比較分析脅迫早期,兩家系肝胰腺組織中GDH-β、GS與TG基因的表達(dá)呈現(xiàn)顯著差異(p0.05):隨著氨氮濃度升高,家系A(chǔ)肝胰腺中這三個基因均被抑制;而家系B則被顯著誘導(dǎo)。脅迫后期(5T10天),兩家系肝胰腺中GDH-β與GS基因表達(dá)量隨氨氮程度增加而上調(diào);另外,TG基因表達(dá)呈現(xiàn)出相同的下調(diào)趨勢。表明隨環(huán)境中氨氮濃度的升高,抗性較強的B家系肝胰腺組織可通過激活氨氮代謝基因,同時抑制氨氮合成相關(guān)基因表達(dá)來提高對氨氮脅迫的耐受性。該研究不僅為豐富凡納濱對蝦不同家系應(yīng)答氨氮脅迫的基礎(chǔ)生物學(xué)知識,同時為揭示凡納濱對蝦應(yīng)答氨氮脅迫的分子機制及其耐氨氮新品系的培育打下了一定的基礎(chǔ)。
[Abstract]:Litopenaeus vannamei, also known as Penaeus vannamei, has a strong adaptability to adversity, a faster growth rate and higher nutritional value. Since its introduction to China in 1988, after nearly 30 years of cultivation and development, Penaeus vannamei has become one of the three major shrimp breeds in China. As the most important environmental factor, ammonia nitrogen, which is harmful to shrimp healthy culture, can enter the tissue fluid of Penaeus vannamei through physiological metabolic pathway and cause blood ammonia. The sensitivity of Penaeus vannamei to ammonia-nitrogen stress was different at different developmental stages. In addition, the sensitivity of Penaeus vannamei to ammonia-nitrogen stress was also significantly different in different families with the same specifications. Therefore, the sensitivity of Penaeus vannamei to ammonia-nitrogen stress at different developmental stages was studied firstly, and then a comparative study was conducted between a family with poor environmental stress resistance (A3281, A) and a family with strong environmental stress resistance (B3271, B), with emphasis on the enzymes related to ammonia-nitrogen metabolism (glutamate). The activities of dehydrogenase (GDHase), glutamine synthase (GSase) and transglutaminase (TGase) and the temporal and spatial variations of their corresponding genes in response to ammonia-nitrogen stress lay a foundation for revealing the sensitivity of Penaeus vannamei to ammonia-nitrogen stress and its molecular mechanism, and provide scientific basis for breeding new strains resistant to ammonia-nitrogen. The results were as follows: 1. Phase II (Z2) of flea-like larvae was the most sensitive to ammonia-nitrogen stress in the early growth stage of Penaeus vannamei. The LC50 was 17.811 mg/L.2, and three enzymes related to ammonia-nitrogen metabolism responded to ammonia-nitrogen stress. (1) Comparative analysis of GDHase activity in different tissues of two families under different concentrations of ammonia-nitrogen stress (3.4-24.6 mg/L) significantly affected Fan. GDHase activity in shrimp tissues: GDHase activity in muscle of two families was significantly inhibited with the increase of ammonia concentration (p0.05); GDHase activity in liver and pancreas tissues of two families was induced with the increase of ammonia concentration in the first five days of stress, but it was significantly inhibited in the later period (5-10 days) of stress (p0.05). There was no significant difference in the activity of GDHase in tissues (p0.05). (2) The activity of GSase in muscle tissue of Penaeus vannamei from two families was induced with the increase of ammonia concentration, but there was significant difference in the activity of GSase in hepatopancreas between families. With the increase of ammonia nitrogen concentration, TGase activity of muscle and hepatopancreas was inhibited significantly in the early stage of ammonia nitrogen stress (T5 days). TGase activity decreased gradually with the increase of ammonia concentration; TGase activity showed significant family differences at the late stage of stress (T5 days). TGase activity in two tissues of family B was significantly inhibited with the increase of ammonia concentration, while that in family A was significantly induced (p0.05). Ammonia-nitrogen stress, the muscle tissue of two families of Penaeus vannamei can maintain ammonia-nitrogen balance by activating ammonia-nitrogen converting enzyme activity (GSase) and inhibiting the activity of ammonia-nitrogen production enzyme (TGase). 3. The integrated biomarker response (IBR) index of three enzymes related to ammonia-nitrogen metabolism was analyzed at three time points. IBR analysis showed that ammonia-nitrogen stress had the greatest effect on the activities of GSase and TGase in the muscle tissues of Penaeus vannamei, and the influence increased with the increase of ammonia-nitrogen concentration. The response time of family B to ammonia-nitrogen stress (T5 days) was significantly earlier than that of family A (5T10 days). It can be inferred that the B muscle tissue of the resistant family could take the lead in regulating the metabolism of ammonia nitrogen through the catalysis of ammonia-nitrogen metabolizing enzymes, especially accelerating the metabolic pathway of glutamate and NH4+ synthesis of glutamine. The results showed that hepatopancreas played a more important role in ammonia-nitrogen stress with the increase of ammonia-nitrogen concentration in the environment. Hepatopancreas was an important target tissue of ammonia-nitrogen stress. 4. The temporal and spatial variation of three ammonia-nitrogen metabolizing enzymes in response to ammonia-nitrogen stress (1) Muscles of different families Comparison of the expression patterns of ammonia-nitrogen metabolizing enzymes genes in meat tissues The expression of GDH-beta and GS genes in muscle tissues of the two families were significantly affected by ammonia-nitrogen concentrations (p0.05). Overall, the expression levels of the two genes were significantly increased with the prolongation of ammonia-nitrogen stress time and the increase of ammonia-nitrogen concentration. Family A (p0.05) showed that family B had a strong ability to mobilize ammonia-nitrogen transformation, which revealed the reasons for the difference of ammonia-nitrogen tolerance in different families. At the later stage of stress, the expression of TG gene in muscle tissue of the two families was significantly different: with the increase of ammonia-nitrogen concentration, TG gene in muscle of family A was significantly down-regulated, while that in muscle of family B was significantly down-regulated. The expression of GDH-beta, GS and TG genes in the hepatopancreas of the two families showed significant difference (p0.05). With the increase of ammonia concentration, the expression of the three genes in the hepatopancreas of the family A were significantly different. The expression of GDH-beta and GS genes in hepatopancreas of both families increased with the increase of ammonia nitrogen in the late stage of stress (5T10 days). In addition, the expression of TG gene showed the same downward trend. It indicated that hepatopancreas of B families with strong resistance could activate ammonia nitrogen metabolic group by increasing ammonia nitrogen concentration in the environment. This study not only enriched the basic biological knowledge of different families of Penaeus vannamei in response to ammonia nitrogen stress, but also laid a foundation for revealing the molecular mechanism of Penaeus vannamei in response to ammonia nitrogen stress and the cultivation of new strains of Penaeus vannamei resistant to ammonia nitrogen stress.
【學(xué)位授予單位】：海南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S917.4

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