实验海洋生物学重点实验室知识库

环境胁迫可能引起广盐性对虾的应激反应，我们首先评估了凡纳滨对虾在逐渐降低pH（6.65-8.20）和逐渐升高pH（8.20-9.81）对比正常pH（8.14-8.31）条件下饲养28天的死亡率、生长性能、渗透调节基因的表达、消化酶活性和对副溶血性弧菌的抵抗力。结果，在逐渐升高pH条件下，虾累计死亡率不断增加，第28天达到39.9%，增重率和增长率不断下降。然而，在逐渐降低pH条件下，虾累计死亡率在7~28天稳定于6.67%，增重率和增长率先下降后恢复正常。此结果表明，虾对逐渐降低pH环境表现出适度耐受性。同时，在逐渐降低pH条件下，虾的渗透调节基因Na⁺/K⁺-ATPase、CAc、CAg转录不断增加或后来恢复正常，淀粉酶、脂肪酶和胰蛋白酶活性先下降后恢复正常或不断增加，副溶血弧菌浸浴的死亡率不断减少。因此，虾对逐渐降低pH环境的主要适应机制可能是其高渗透调节能力，使机体达到一个新的平衡稳态，消化酶活性增强满足能量消耗。同时，此环境还会抑制虾养殖中的疾病爆发。然后，对比分析虾肝胰腺和中肠的氧化应激、抗氧化反应、氧化损伤。结果，在短期（≤7天）逐渐降低和逐渐升高pH条件下，虾增强肝胰腺和中肠的抗氧化酶基因MnSOD、GPx和应激蛋白基因Hsp70转录作为早期适应机制，以清除过量的活性氧（ROS）。同时，肝胰腺因为较早的产生活性氧、抗氧化反应和氧化损伤，所以比中肠对pH变化及其氧化应激更敏感。而长期（≥14天）逐渐升高pH条件下加重的氧化损伤引起了虾肝胰腺和中肠丧失了抗氧化调节能力，导致虾连续死亡。相比，长期（≥14天）逐渐降低pH条件下肝胰腺和中肠的抗氧化酶基因GPx、GST和应激蛋白基因Hsp70的转录增强可能是虾的抗氧化适应机制，防止ROS进一步干扰，并减弱氧化损伤，达到新的免疫稳态，有助于存活率的稳定。因此，在逐渐升高pH条件下，可以通过保护肝胰腺来控制虾的死亡和生长抑制。

然后评估了凡纳滨对虾在循环重/中度低氧（0.8-3.5 mg/L）对比常氧（6.4-7.5 mg/L）条件下饲养28天的死亡率、生长性能、渗透调节基因的表达、消化酶活性和对副溶血性弧菌的抵抗力。结果，在循环重/中度低氧条件下，虾的累积死亡率不断增加，增重率和增长率不断下降，渗透调节基因Na⁺/K⁺-ATPase、CAc、CAg转录先增加后恢复正常或下降，淀粉酶、脂肪酶、胰蛋白酶活性不断下降，副溶血弧菌浸浴的死亡率不断增加。因此，循环重/中度低氧降低凡纳滨对虾的生存和生长性能，主要原因是渗透调节机制被破坏，使机体失去了平衡稳态，消化酶活性降低。同时，此环境可能导致虾养殖中的疾病爆发。然后，对比分析了肝胰腺和中肠的低氧诱导因子1a（HIF-1a）的表达，抗氧化反应和氧化损伤。结果显示，短期（≤7天）循环重/中度低氧条件下，虾增强肝胰腺和中肠HIF-1a、抗氧化酶基因MnSOD、Gpx、GST、MT和应激蛋白基因Hsp70转录作为虾早期的适应机制，以耐受低氧并清除过量的活性氧。同时，肝胰腺因为较早的HIF-1a转录和氧化损伤，所以比中肠对低氧及其氧化应激更敏感。然而，长期（≥14天）循环重/中度低氧加重的氧化损伤引起了虾肝胰腺和中肠丧失了抗氧化调节能力，导致虾连续死亡。特别是肝胰腺比中肠更早丧失了抗氧化调节能力。因此，循环重/中度低氧条件下，可以通过保护肝胰腺来控制虾的死亡和生长抑制。

最后研究了谷胱甘肽（GSH）在循环重/中度低氧条件下对虾养殖的影响，评估了常氧、循环重/中度低氧、循环重/中度低氧并补充75 mg/kg GSH和循环重/中度低氧并补充150 mg/kg GSH条件下凡纳滨对虾的生存、生长性能、及胰胰腺氧化指标和转录表达。结果，补充75 mg/kg GSH能显著提高虾在循环重/中度低氧条件下肝胰腺GSH含量和肝胰腺指数，并保持组织结构完整性，提高存活率和生长性能，特别是其生长性能与常氧虾基本相同。然而，补充150 mg/kg GSH却没有意义。分析肝胰腺氧化指标及转录组，发现补充75 mg/kg、150 mg/kg GSH显著增强虾在循环重/中度低氧条件下肝胰腺HIF-1信号通路、GSH代谢通路、抗氧化酶基因（SOD、CAT、GPX、POD、GST）和应激蛋白基因HSP转录，降低ROS和丙二醛（MDA）含量，并且虾补充75 mg/kg比150 mg/kg GSH具有更高水平的低氧适应和抗氧化防御系统，更低的ROS和MDA含量和更完整的组织结构，特别是ROS和MDA含量和组织结构与常氧虾基本相同。补充75 mg/kg GSH，而非150 mg/kg GSH，能显著增强虾在循环重/中度低氧条件下肝胰腺的免疫识别基因LRR、TIR和体液免疫基因Serpin转录。补充75 mg/kg、150 mg/kg GSH能显著增强虾在循环重/中度低氧条件下肝胰腺的细胞免疫基因Rab、Ran、integrin转录，并且虾补充75 mg/kg比150 mg/kg GSH具有更好的增强效果。因此，补充75 mg/kg GSH在循环重/中度低氧条件下增强了肝胰腺抗氧化防御系统和先天免疫反应，维持组织结构完整性，缓解虾死亡和生长抑制。

Environment stress could cause a stress response in euryhaline penaeids. Fistly, the mortality, growth performance, osmoregulation gene expression, digestive enzyme activity, and resistance against Vibrio parahemolyticus of white shrimp Litopenaeus vannamei reared under conditions of gradual changes to a low-pH (6.65–8.20) and gradual changes to a high-pH (8.20–9.81) versus a normal pH environment (8.14–8.31) during a 28-d experiment were evaluated. Consequently, under gradual-high pH, the cumulative mortality rate (CMR) rose with time until 39.9% on days 28, the weight gain percentage (WGP) and length gain percentage (LGP) decreased continuously. However, under gradual-low pH, the CMR of shrimp stabilized at 6.67% during 7-28 d; the WGP and LGP decreased first and then returned to normal. These results indicated that L. vannamei displayed a moderate tolerance to gradual-low pH. Under gradual-low pH, the osmoregulation gene Na⁺/K⁺-ATPase, CAc, and CAg transcripts of shrimp increased continuously or then back to normal, the amylase, lipase, and trypsin activities decreased first and then returned to normal or increased, the MR with V. parahaemolyticus immersion decreased continuously. Thus, the major adaptation mechanism of shrimp to gradual-low pH might be its high osmoregulation ability, which made shrimp achieve a new, balanced steady-state with enhanced digestive enzyme activities to meet energy requirement after long-term exposure. Meanwhile, the gradual-low pH environment would probably inhibit disease outbreak in the shrimp farming. Then, the oxidative stress, antioxidant responses and oxidative damage in the hepatopancreas and midgut of shrimp were investigated. Consequently, shrimp enhanced antioxidant enzyme gene MnSOD, GPx, and stress protein gene Hsp70 transcripts in the hepatopancreas and midgut as early defense mechanism to scavenge excessive ROS during short-term (≤ 7 d) gradual-low and high pH stress. Meanwhile, the hepatopancreas was more sensitive to pH variation and its oxidative stress than midgut because of earlier ROS production increase, antioxidant response and oxidative damage. Then, suppressed antioxidant response in the hepatopancreas and midgut of shrimp suggested a loss of antioxidant regulatory capacity caused by aggravated oxidative damage after long-term (≥ 14 d) gradual-high pH stress, leading to continuous death. However, enhanced antioxidant enzyme GPx, GST, and Hsp70 transcripts in the hepatopancreas and midgut might be long-term (≥ 14 d) antioxidant adaptation mechanism of shrimp to gradual-low pH stress, which could prevent further ROS perturbation and weaken oxidative damage to achieve a new immune homeostasis, contributing to stable survival rate. Therefore, it is vital to protect hepatopancreas for controlling shrimp death under gradual-high pH stress.

Secondly, the survival, growth performance, osmoregulation gene expression, digestive enzyme activity, and resistance against Vibrio parahemolyticus of the L. vannamei reared under conditions of cyclic serious/medium hypoxia (0.8-3.5 mg/L) versus normoxia (6.4-7.5 mg/L) during a 28-d experiment were investigated. Consequently, under cyclic serious/medium hypoxia, the CMR of shrimp increased continuously. The WGP and LGP of shrimp decreased continuously, the osmoregulation gene Na⁺/K⁺-ATPase, CAc, and CAg transcripts in the gill of shrimp increased first and then returned to normal or decreased. The amylase, lipase, and trypsin activities in the hepatopancreas of CSMH shrimp decreased continuously, the MR with V. parahaemolyticus immersion increased continuously. Therefore, cyclic serious/medium hypoxia could reduce survival and growth performance of shrimp during long-term exposure, the major reason was broken osmoregulation mechanism, which made shrimp lose balanced steady-state and reduce digestive enzyme activities. Meanwhile, cyclic serious/medium hypoxia would probably lead to outbreak of infectious diseases in the shrimp farming. Then, the hypoxia inducible factors 1a (HIF-1a), antioxidant responses and oxidative damage in the hepatopancreas and midgut of shrimp were investigated. Results showed enhanced HIF-1a, antioxidant enzyme gene MnSOD, Gpx, GST, MT, and stress protein gene Hsp70 transcript in the hepatopancreas and midgut during short-term cyclic serious/medium hypoxia (≤ 7 d), which suggested early adaptive mechanism of shrimp to scavenge excessive ROS. Meanwhile, HIF-1a transcript and oxidative damage were induced earlier in the hepatopancreas than the midgut. Thus, the hepatopancreas could be more sensitive to hypoxia and its oxidative stress than the midgut. However, long-term (≥ 14 d) cyclic serious/medium hypoxia could disrupt cellular antioxidant mechanism with depressed antioxidant responses, and then aggravate oxidative damage in the hepatopancreas and midgut, particularly the hepatopancreas would lose antioxidant ability earlier than the midgut. Therefore, it is vital to protect hepatopancreas for controlling shrimp death and growth inhibition under cyclic serious/medium hypoxia.

Finally, effect of glutathione (GSH) on shrimp under cyclic serious/medium hypoxia was studied. The survival, growth performance, and oxidative index and transcriptome in the hepatopancreas of L. vannamei reared under conditions of normoxia, cyclic serious/medium hypoxia, cyclic serious/medium hypoxia and supplement of 75 mg/kg GSH, and cyclic serious/medium hypoxia and supplement of 150 mg/kg GSH. Consequently, supplementation of 75 mg/kg GSH significantly improved GSH content and hepatopancreas index, and maintained integrity of histological structure in the hepatopancreas of shrimp under cyclic serious/medium hypoxia, promoting significantly enhanced shrimp survival and growth. Especially, shrimp supplemented with 75 mg/kg GSH under cyclic serious/medium hypoxia even have the basically same growth performance with the shrimp under normoxia. However, supplementation of 150 mg/kg GSH made no sense. Obviously, supplementation of 75 mg/kg GSH could relieve shrimp death and growth inhibition under cyclic serious/medium hypoxia. Then, oxidative indexes and transcriptome in the hepatopancreas were analyzed in this experiment. Supplementation of 75 mg/kg and 150 mg/kg GSH significantly enhanced HIF-1 signal pathway, GSH metabolism pathway, antioxidant enzyme (SOD, CAT, GPx, POD, GST) and stress protein HSP gene transcripts, and reduced ROS and malondialdehyde (MDA) content in the hepatopancreas of shrimp under cyclic serious/medium hypoxia, and shrimp supplemented with 75 mg/kg GSH have a higher basal level antioxidant defense system, lower ROS and MDA content and more integrated histological structure than shrimp supplemented with 150 mg/kg GSH, even the basically same ROS production and MDA content and histological structure with the shrimp under normoxia. Supplementation of 75 mg/kg rather than 150 mg/kg GSH significantly enhanced immune recognition gene LRR, TIR and humoral immunity gene Serpin transcripts in the hepatopancreas of shrimp under cyclic serious/medium hypoxia. Supplementation of 75 mg/kg and 150 mg/kg GSH significantly enhanced cellular immunity gene Rab, Ran and integrin gene transcripts in the hepatopancreas of shrimp under cyclic serious/medium hypoxia, and shrimp supplemented with 75 mg/kg GSH have a higher basal level of cellular immunity level than shrimp supplemented with 150 mg/kg GSH. Therefore, supplementation of 75 mg/kg GSH could relieve shrimp death and growth inhibition by enhancing antioxidant defense system and innate immune response, and maintaining histological structure under cyclic serious/medium hypoxia.

目 录

摘 要 I

ABSTRACT III

第一章 绪论 1

1.1 环境胁迫对水产动物的影响 1

1.1.1 低氧... 1

1.1.2 盐度... 1

1.1.3 pH.. 2

1.1.4氨氮... 3

1.1.5重金属... 3

1.2 环境胁迫下水产动物的新陈代谢策略 4

1.2.1 适度胁迫的代谢反应：补偿... 4

1.2.2 极端胁迫的代谢反应：保留... 6

1.3 环境胁迫下水产动物的氧化应激反应 8

1.3.1 ROS的产生和发展... 9

1.3.2 ROS的消除策略... 10

1.4 GSH的生物学功能及对动物肝脏的应用研究 11

1.4.1 GSH消除ROS. 11

1.4.2 GSH消除内源性毒物... 12

1.4.3 GSH调节细胞生长和死亡... 12

1.4.4 GSH对动物肝脏的应用研究... 13

1.5 本文主要研究内容 14

1.6 本研究的目的和意义 15

第二章 逐渐降低或升高pH对凡纳滨对虾生长、渗透调节、消化酶活性、及弧菌抵抗力的影响 17

2.1 引言 17

2.2 材料与方法 17

2.2.1 实验虾... 17

2.2.2 副溶血性弧菌准备... 18

2.2.3 逐渐降低pH或逐渐升高pH胁迫的实验设计... 18

2.2.4 虾死亡率、生长性能的测定和取样程序... 19

2.2.5 鳃的渗透调节基因表达分析... 19

2.2.6 肝胰腺消化酶活性的测定... 20

2.2.7 虾对副溶血性弧菌的抵抗力... 20

2.2.8 统计分析... 21

2.3 结果 21

2.3.1 不同pH条件下虾的死亡率... 21

2.3.2 不同pH条件下虾的生长性能... 22

2.3.3 不同pH条件下虾的渗透调节基因表达... 22

2.3.4 不同pH条件下虾的消化酶活性... 23

2.3.5 不同pH条件下虾对副溶血弧菌的抵抗力... 24

2.4 讨论 25

2.5 小结 27

第三章 凡纳滨对虾的肝胰腺和肠道对逐渐降低或升高pH的氧化应 激比较研究 29

3.1 引言 29

3.2 材料与方法 30

3.2.1 实验虾... 30

3.2.2 逐渐降低pH或逐渐升高pH胁迫的实验设计... 30

3.2.3 虾取样程序... 30

3.2.4 肝胰腺和中肠的ROS与MDA含量测定... 30

3.2.5 肝胰腺和中肠的基因表达分析... 31

3.2.6 肝胰腺和中肠的DNA ladder分析... 31

3.2.7 肝胰腺和中肠的的组织结构分析... 32

3.2.8 统计分析... 32

3.3 结果 32

3.3.1 不同pH条件下虾肝胰腺和中肠ROS含量... 32

3.3.2 不同pH条件下虾肝胰腺和中肠MnSOD基因表达... 32

3.3.3 不同pH条件下虾肝胰腺和中肠GPx基因表达... 33

3.3.4 不同pH条件下虾肝胰腺和中肠GST基因表达... 34

3.3.5 不同pH条件下虾肝胰腺和中肠Hsp70基因表达... 34

3.3.6 不同pH条件下虾肝胰腺和中肠MDA含量... 35

3.3.7 不同pH条件下虾肝胰腺和中肠的细胞凋亡... 36

3.3.8 不同pH条件下虾肝胰腺和中肠的组织结构... 36

3.4 讨论 37

3.5 小结 39

第四章 循环重/中度低氧胁迫对凡纳滨对虾生长、渗透调节、消化酶活性、及弧菌抵抗力的影响 41

4.1 引言 41

4.2 材料与方法 42

4.2.1 实验虾... 42

4.2.2 副溶血性弧菌准备... 42

4.2.3 循环重/中度低氧胁迫的实验设计... 42

4.2.4 虾死亡率、生长性能的测定和取样程序... 43

4.2.5 鳃的渗透调节基因表达分析... 43

4.2.6 肝胰腺消化酶活性的测定... 43

4.2.7 虾对副溶血性弧菌的抵抗力... 43

4.2.8 统计分析... 44

4.3 结果 44

4.3.1 循环重/中度低氧条件下虾的死亡率... 44

4.3.2 循环重/中度低氧条件下虾的生长性能... 44

4.3.3 循环重/中度低氧条件下虾的渗透调节基因表达... 45

4.3.4 循环重/中度低氧条件下虾的消化酶活性... 46

4.3.5 循环重/中度低氧条件下虾对副溶血弧菌的抵抗力... 46

4.4 讨论 47

4.5 小结 49

第五章 凡纳滨对虾的肝胰腺和肠道对循环重/中度低氧的氧化应激 比较研究 51

5.1 引言 51

5.2 材料与方法 52

5.2.1 实验虾... 52

5.2.2 循环重/中度低氧胁迫的实验设计... 52

5.2.3 虾取样程序... 52

5.2.4 肝胰腺和中肠的基因表达分析... 52

5.2.5 肝胰腺和中肠的DNA ladder分析... 53

5.2.6肝胰腺和中肠的的组织结构分析... 53

5.2.7 统计分析... 53

5.3 结果 54

5.3.1 循环重/中度低氧条件下虾肝胰腺和中肠HIF-1a基因表达... 54

5.3.2 循环重/中度低氧条件下虾肝胰腺和中肠MnSOD，GPx，GST和 MT基因表达... 54

5.3.3 循环重/中度低氧条件下虾肝胰腺和中肠Hsp70基因表达... 56

5.3.4 循环重/中度低氧条件下虾肝胰腺和中肠DNA ladder分析... 56

5.3.5 循环重/中度低氧条件下虾肝胰腺和中肠组织结构分析... 57

5.4 讨论 58

5.5 小结 60

第六章 谷胱甘肽缓解循环重/中度低氧胁迫凡纳滨对虾造成损伤的 作用机理 61

6.1 引言 61

6.2 材料与方法 62

6.2.1 实验虾... 62

6.2.2 补充GSH及循环重/中度低氧胁迫的实验设计... 62

6.2.3 虾死亡率、生长性能的测定和取样程序... 63

6.2.4 肝胰腺氧化指标测定... 64

6.2.5 RNA分离和cDNA文库构建... 64

6.2.6 转录组测序，基因组装与注释... 64

6.2.7 肝胰腺的组织结构分析... 65

6.2.8 统计分析... 65

6.3 结果 65

6.3.1 虾的生存和生长性能... 65

6.3.2 肝胰腺氧化指标分析... 65

6.3.3 转录组测序和组装... 66

6.3.4 功能注释和分类分析... 67

6.3.5 差异表达基因分析... 69

6.3.6 差异表达基因可能涉及的肝胰腺功能... 71

6.3.7 肝胰腺组织结构分析... 76

6.4 讨论 76

第七章 结论与创新点 81

7.1 主要结论 81

7.2 创新点 82

参考文献 83

致 谢 107

作者简历及攻读博士学位期间发表的学术论文与研究成果 108