凡纳滨对虾不同家系氨氮代谢相关酶及相应基因应答氨氮胁迫的比较研究

发布时间：2018-09-07 19:28

【摘要】：凡纳滨对虾(Litopenaeus vannamei)又称南美白对虾,具有较强的逆境适应性、较快的生长速度及较高的营养价值。自1988年引入中国,经过近30年的培育和发展,凡纳滨对虾已成为我国对虾三大养殖品种之一。近年来随着高密度、集约化养殖及人类生产、生活污水排放,养殖水体中污染物急剧增加,导致水质不断恶化,给凡纳滨对虾养殖业带来了严重的经济损失。作为危害对虾健康养殖最重要的环境因子一氨氮,它可以通过生理代谢途径进入凡纳滨对虾组织液中,并引起血氨中毒,进一步破坏其免疫系统、扰乱其渗透压平衡,严重影响其生长发育性能。不同发育时期的凡纳滨对虾应答氨氮胁迫的敏感性不同;另外,不同家系相同规格的凡纳滨对虾应答氨氮胁迫的敏感性也存在显著差异,但目前有关凡纳滨对虾应答氨氮胁迫的相关机制尚不清楚。因此,该论文首先研究了凡纳滨对虾不同发育时期应答氨氮胁迫的敏感性;然后选取对环境抗逆性较差的家系(A3281,A)与抗逆性较强的家系(B3271,B)开展比较研究,侧重探究了氨氮代谢相关酶类(谷氨酸脱氢酶(GDHase)、谷氨酰胺合成酶(GSase)与谷氨酰胺转胺酶(TGase))活性及其对应基因应答氨氮胁迫的时空变化规律,为揭示凡纳滨对虾应答氨氮胁迫的敏感性及其分子机制打下一定的基础,同时,为选育耐氨氮新品系提供科学依据。主要研究结果如下:1、凡纳滨对虾早期生长阶段,蚤状幼体Ⅱ期(Z2)对氨氮胁迫最为敏感。凡纳滨对虾不同生长发育时期对氨氮的敏感性不同,成体比幼体具有更高的氨氮耐受性;从无节幼体至仔虾Ⅴ期(P5)发育阶段中的凡纳滨对虾,其Z2阶段对氨氮胁迫最为敏感,可作为检测氨氮胁迫的关键点,其半数致死浓度LC50为17.811 mg/L。2、三种氨氮代谢相关酶应答氨氮胁迫的时空变化规律。(1)两个家系不同组织GDHase活性变化规律的比较分析不同浓度的氨氮胁迫(3.4-24.6 mg/L)显著影响凡纳滨对虾各组织GDHase的活性:其中,两个家系肌肉GDHase活性随氨氮浓度的升高而表现为被显著抑制(p0.05);胁迫的前5天两家系肝胰腺组织GDHase活性随氨氮浓度的升高均被诱导,而胁迫后期(5-10天)呈现显著抑制(p0.05)。另外,肌肉与肝胰腺组织中GDHase活性差异性不显著(p0.05)。(2)两个家系不同组织GSase活性变化规律的比较分析两个家系凡纳滨对虾肌肉组织中的GSase活性随氨氮浓度升高均呈现被诱导的趋势;而肝胰腺组织中GSase活性变化,家系间存在显著差异:其中,家系A随氨氮浓度的升高而呈现被抑制的趋势;而家系B于胁迫初期呈现上调趋势,后期呈现被抑制的状态(T5天)。(3)两个家系不同组织TGase活性变化规律的比较分析氨氮胁迫前期(T5天),显著抑制两家系肌肉与肝胰腺组织的TGase活性,随氨氮浓度的升高TGase酶活性逐渐降低;胁迫后期(T5天)TGase酶活性呈现显著的家系差异性:随氨氮浓度的升高家系B两个组织中的TGase活性显著被抑制,而家系A则被显著诱导(p0.05)。综合两组织中三种氨氮代谢酶活性的变化规律,发现应对不同程度的氨氮胁迫,两家系凡纳滨对虾的肌肉组织均可以通过激活氨氮转化类酶活性(GSase),同时抑制氨氮生产类酶(TGase)的活性来维持组织中氨氮含量的平衡。3、三种氨氮代谢相关酶的整合生物标志物响应(IBR)指数分析通过对各时间点三种酶在不同浓度氨氮胁迫下的IBR分析表明:氨氮胁迫对两家系凡纳滨对虾的肌肉组织的GSase与TGase活性影响最大,且其影响力随氨氮浓度的增加而变大;家系B应对胁迫反应时间(T5天)明显早于家系A(5T10天)。综合分析相关代谢酶的催化功能,可以推断出抗性较强的家系B肌肉组织可率先通过氨氮代谢酶的催化作用调控机体氨氮代谢,尤其是加速催化glutamate与NH4+合成glutamine这一代谢途径。IBR的剂量-胁迫效应显示:随环境中氨氮浓度升高两家系的肝胰腺组织受到的胁迫效应均呈现出上升趋势,而肌肉组织呈现出下降趋势。说明随着环境中氨氮浓度升高,肝胰腺组织在氨氮胁迫中发挥更大的作用,肝胰腺是氨氮胁迫的重要靶组织。4、三种氨氮代谢酶对应基因应答氨氮胁迫的时空变化规律(1)不同家系肌肉组织中氨氮代谢酶基因表达规律的比较分析各浓度氨氮均可显著影响两家系肌肉组织GDH-β与GS基因的表达(p0.05):整体而言,两基因随氨氮胁迫时间的延长与胁迫浓度的增加均呈现显著上调趋势;且家系B中两基因的表达量显著高于家系A(p0.05),表明B家系调动氨氮转化的能力较强,这从基因水平揭示了不同家系应答氨氮耐受能力差异的原因。胁迫后期,两家系肌肉组织中TG基因表达呈现显著差异:随着氨氮浓度的升高家系A肌肉中TG基因表现为显著下调,而家系B肌肉则表现为显著上调,两家系间差异显著(p0.05)。(2)不同家系肝胰腺组织中氨氮代谢酶基因表达规律的比较分析胁迫早期,两家系肝胰腺组织中GDH-β、GS与TG基因的表达呈现显著差异(p0.05):随着氨氮浓度升高,家系A肝胰腺中这三个基因均被抑制;而家系B则被显著诱导。胁迫后期(5T10天),两家系肝胰腺中GDH-β与GS基因表达量随氨氮程度增加而上调;另外,TG基因表达呈现出相同的下调趋势。表明随环境中氨氮浓度的升高,抗性较强的B家系肝胰腺组织可通过激活氨氮代谢基因,同时抑制氨氮合成相关基因表达来提高对氨氮胁迫的耐受性。该研究不仅为丰富凡纳滨对虾不同家系应答氨氮胁迫的基础生物学知识,同时为揭示凡纳滨对虾应答氨氮胁迫的分子机制及其耐氨氮新品系的培育打下了一定的基础。
[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.
【学位授予单位】：海南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

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