【作者】 王芸；

【导师】 李健；

【作者基本信息】 上海海洋大学 ， 水产养殖， 2011， 博士

【摘要】 中国对虾（Fenneropenaeus chinensis）是我国重要的海水养殖品种,具有适应能力强、生长速度快、耐低温、营养丰富等优点。养殖环境恶化、管理不当等因素引起的环境胁迫所诱导的应激反应,不仅对中国对虾的生理状态及抗病力有显著的影响,甚至严重影响了对虾的养殖成活率。良好的水环境是对虾养殖成败的关键,pH、氨氮是衡量养殖水质质量的重要指标,本文通过研究pH、氨氮胁迫对中国对虾细胞凋亡和抗氧化系统影响,揭示了中国对虾应对pH、氨氮胁迫的应激机理,为对虾抗逆选育和健康养殖提供技术支撑。研究内容包括以下四个部分:第一部分,中国对虾细胞凋亡因子Caspase基因cDNA克隆和表达分析。从中国对虾肝胰腺中克隆了Caspase基因,该基因cDNA全长1329b（pGenBank注册号为GU597089）,其中开放式阅读框为972bp,包含109bp的5′非编码区（5′-UTR）,248bp的3′非编码区（3′-UTR）及一个多腺苷酸信号AATAAA。该基因编码323个氨基酸,预测分子量为36.0kDa,pI为6.27。Caspase编码的氨基酸包含Caspase家族保守的活性位点中心（QACRG五肽）和两个结构域（大亚基p20和小亚基p10）,推导的氨基酸不存在信号肽序列。中国对虾Caspase蛋白包含特殊的氨基酸残基His150和Cys198,这两个氨基酸对于催化Caspase酶活性起到至关重要的作用。多序列分析结果表明F. chinensis Caspase与Penaeus monodon Caspase、Fenneropenaeus merguiensis Caspase、Litopenaeus vannamei Caspase-3蛋白质序列同源性分别为83%、82%、76%。应用荧光定量PCR方法分析了中国对虾暴露于pH7.0和pH9.0水环境中,各组织Caspase基因表达的水平。结果显示:pH7.0组中国对虾血淋巴细胞Caspase基因表达水平12h显著升高,48h达最低,96h达最高值,148h显著低于对照组（P<0.05）;pH9.0组中国对虾血淋巴细胞Caspase基因表达水平3h显著升高,24h显著低于对照组（P<0.05）,120h达最大值并显著高于对照组。pH7.0组中国对虾鳃Caspase基因表达水平在48h和120h低于对照组,其余各时间点表达水平均高于对照组,且在148h达最高值;pH9.0组中国对虾鳃Caspase表达水平3h显著低于对照组（P<0.05）,之后逐渐增加,24h达最高值,48h后随着胁迫时间的延长Caspase基因表达水平逐渐降低。pH7.0组中国对虾肝胰腺Caspase基因表达水平均低于对照组,但3h、48h和96h例外;pH9.0组中国对虾肝胰腺Caspase基因表达水平3h显著增加,之后表达水平逐渐增加,120h达最高值,且在24h至148h期间均显著高于对照组（P<0.05）。高、低pH胁迫对中国对虾肌肉Caspase基因表达变化相似,3h略有升高,96h达最高值,148h均显著低于对照组（P<0.05）。高、低pH胁迫中国对虾3h后,其淋巴器官Caspase基因表达水平增加,且随着胁迫时间延长,各组Caspase基因表达水平一直处于较高水平。pH7.0组中国对虾胃Caspase基因表达3-12h显著低于对照组（P<0.05）,之后呈波浪式变化,148h达最高值,与对照组相比差异显著（P<0.05）;pH9.0组中国对虾胃Caspase基因表达3h显著降低,12-72h表达水平逐渐升高,120h达最高值。TUNEL分析结果表明中国对虾暴露于pH7.0和pH9.0的水环境中12h,肝胰腺组织开始出现细胞凋亡现象,并且随着胁迫时间的延长肝胰腺细胞凋亡现象越明显,与TUNEL分析中的阳性对照表现相一致。相反pH8.2（对照组）中国对虾肝胰腺没有发现被深染的细胞核,与TUNEL阴性对照组的表现一致。这说明高、低pH胁迫均能够诱导中国对虾肝胰腺组织的细胞凋亡。第二部分,中国对虾Caspase基因的重组表达、分离纯化和多克隆抗体的制备。克隆中国对虾Caspase基因开放式阅读框序列,构建了该基因原核表达载体pET30a-CAS,通过E. coli稀有密码子分析,将该重组质粒转化入大肠杆菌BL21 （DE） pLysS,成功实现了Caspase基因在E. Coli中的表达。SDS-PAGE分析显示该重组蛋白分子大小约为50 kDa,略大于软件预测的分子量。MALDI-TOF-MS分析验证了该蛋白是中国对虾Caspase蛋白。利用固定金属亲和层析和Co-NTA技术获得的纯化Caspase重组蛋白,免疫新西兰大白兔制备了特异性多克隆抗体,Western blot结果表明中国对虾血淋巴细胞、鳃、肌肉、淋巴器官、心脏、胃和肝胰腺组织中均有Caspase蛋白的表达,但在血清中未检测到特异性的谱带。第三部分,pH胁迫对中国对虾抗氧化系统及HSP90基因表达的影响。将中国对虾暴露于pH8.2（对照组）、7.0、9.0（胁迫组）的水体中148 h,于胁迫后0、3、12、24、48、72、96、120、148h测定鳃、肝胰腺、肌肉和血淋巴总抗氧化活力（T-AOC）、抗超氧阴离子活力、过氧化氢酶（CAT）活力和CAT、过氧化物还原酶（Prx）和HSP90基因表达的情况。结果显示, pH胁迫12h-24h,对虾各组织T-AOC、抗超氧阴离子、CAT酶活力及CAT基因表达均增加,pH胁迫120h-148h上述指标受到抑制。对虾肝胰腺和肌肉Prx基因表达随胁迫时间增加逐渐升高,鳃和血淋巴Prx基因表达随胁迫时间增加呈下降趋势。高pH（9.0）胁迫时,对虾鳃抗氧化系统酶活力及基因表达水平达峰时间较其他3个组织明显缩短;而低pH（7.0）胁迫时,对虾肝胰腺抗氧化系统酶活力比其他组织变化快。高pH（9.0）组中国对虾肝胰腺HSP90基因表达显著高于对照组（P<0.05）,肌肉组织HSP90基因表达除72h外均显著低于对照组（P<0.05）。试验结果表明,pH胁迫3h-24h对中国对虾抗氧化系统酶活力及相关基因表达有一定的诱导效果,但胁迫120h-148h则抑制其抗氧化系统功能,可能会导致中国对虾机体的氧化损伤;中国对虾鳃和肝胰腺分别对高、低pH胁迫较敏感。第四部分,氨氮胁迫对中国对虾抗氧化系统、血淋巴氨氮成分及HSP90基因表达的影响。将中国对虾暴露于不同氨氮浓度（0mg/L、2mg/L、4mg/L、6mg/L、8mg/L）的水体中96h,于胁迫后0、6、24、48、72、96h测定中国对虾血淋巴氨氮、尿素氮浓度和鳃、肝胰腺、肌肉和血淋巴的T-AOC、抗超氧阴离子、Na⁺ K⁺-ATPase、Ca²⁺ Mg²⁺-ATPase活性及CAT、Prx、Caspase、HSP90基因表达水平,结果显示:（1）当水体氨氮浓度从0.220mg/L到13.449mg/L时,中国对虾血淋巴氨氮浓度明显升高,于24h-28h达最低值,72-96h达最高值,但均显著高于对照组（P<0.05）。（2）当水体氨氮浓度从0.220mg/L到9.446mg/L时:中国对虾血淋巴尿素氮含量明显升高且显著高于对照组（P<0.05）,各组尿素氮浓度6h达最高值;中国对虾鳃、肝胰腺和肌肉Na⁺ K⁺-ATPase、Ca²⁺ Mg²⁺-ATPase活性及HSP90基因表达水平明显升高,并且各指标变化基本一致。当水体氨氮浓度从9.446mg/L到13.449mg/L时,血淋巴尿素氮浓度和鳃、肝胰腺、肌肉Na⁺ K⁺-ATPase、Ca²⁺ Mg²⁺-ATPase活性及HSP90基因表达水平明显降低,说明高氨氮浓度抑制血淋巴尿素氮的合成、ATPase活性及HSP90基因表达水平（3）当水体氨氮浓度从0.303mg/L到8.625mg/L时,中国对虾T-AOC、抗超氧阴离子活力及CAT基因表达水平升高,并在胁迫后6h或96h达最高值;Prx基因表达水平均在72-96h达最高值。当水体氨氮浓度大于8.625mg/L时中国对虾的抗氧化系统受到抑制。（4）Caspase基因表达水平均在氨氮胁迫后的72-96h达最高值（实验后期）,该表达变化与pH胁迫后Caspase基因的表达变化相似。推测高浓度氨氮能够诱导中国对虾Caspase基因的表达,进而诱导中国对虾的细胞凋亡。更多还原

【Abstract】 Chinese shrimp, Fenneropenaeus chinensis, is an important mariculture species in China and has many advantages, such as wild adaptability, fast growth, low temperature resistance and rich in nutrition. However, with the constant expansion of shrimp culture, deteriorated pond environment due to management subsequently resulted in the increased incidences of stress-induced physiology reaction and diseases of shrimp, or even seriously affected the survival rate of farming shrimp. pH and ammonia are both important factors to measure water quality. Good water environment is a key to acquire success in shimp culture. In order to reveal the stress mechanism on pH and ammonia stress of Chinese shrimp, we do the study of pH and ammonia stress on apoptosis and antioxidant system of Chinese shrimp. This will give a theoretical foundation for stress-resistance traits breeding and healthy culture. The study consists of two parts:The first part: Molecular clonging and characterization of apoptosis factor Caspase gene from Chinese shrimp Fenneropenaeus chinensis. In the present study, a Caspase gene was cloned from the Chinese shrimp, Fenneropenaeus chinensis （GenBank No. GU597089）. The full-length of Caspase was of 1329 bp with 972 bp ORF, 109 bp of the 5′-untranslated region, 248 bp of the 3′-untranslated region and a poly（A） tail and a putative polyadenylation signal （AATAAA）. The entire open reading frame encoded a deduced protein of 323 aminoacids with the putative initiation methioine codon （ATG）. The calculated molecular mass and pI of Caspase protein was 36.0 kDa and 6.27 respectively. The deduced aminoacid sequence of Caspase contained a potential active site （QACRG pentapeptide） conserved in most Caspases and two profile hits （p20 and p10 domain profile）. No putative signal peptide aminoacids was present in the Caspase sequence. The deduced protein Caspase domain contais two conserved residue sites: a histidine150 residue and a cysteine198 residue which play important to catalysis the activity of Caspase. Sequence conparision showed that the Caspase of F. chinensis shares 83%, 82% and 76% identity with that of Penaeus monodon Caspase, Fenneropenaeus merguiensis Caspase, Litopenaeus vannamei Caspase-3.The expression of Caspase gene in different tissues of Chinese shrimp after exposure to pH 7.0 and 9.0 stress was analyzed by Real-time PCR. The results showed that the number of Caspase transcripts in the pH 7.0 group increased gradually in the haemocytes during the the first 12h, then decreased to their lowest level by 48h. Following this, expression of the transcripts increased again to their highest level at 96h. The levels at the end of the experiment were significantly lower than those in the control group （P<0.05）. Levels of the Caspase transcript in the pH 9.0 group also increased in the haemocytes at 3h, then decreased slightly by 24 h （P<0.05）. The highest levels were observed after 120 h. The Caspase expression in the gill of pH 7.0 was almost higher than in the control and reached to its highest level at 148 h, except at 48 and 120 h. The Caspase transcript levels were significantly lower at 3 h in the pH 9.0 group compared to the control group （P<0.05）, but then gradually increased up to 24 h before decreasing untile 48 h. The Caspase gene expression was lower in the pH 7.0 than in the control group untile the end of the experiment, except at 3 h and 96 h. The transcript levels in the hepatopancreas of the pH 9.0 group increased after 3 h, but then decreased and were slightly lower than the control group at 12 h. The transcript levels peaked at 120 h and were higher than the control group from 24 h to 148 h. The pattern of Caspase transcript expression was similar at the pH 7.0 and 9.0 groups in the muscle tissue. Levels increased slightly 3 h post-stress, and then decreased significantly from 12 h to 48 h. Following this, levels increased again and peaked at 96 h. At the end of experiment, the expression of Caspase transcripts was significantly lower after 120 h and was lower than the control group by 148 h. The number of Caspase transcripts in lymphoid organ of shrimp in the pH 7.0 and 9.0 groups increased by 3 h then decreased to baseline by 12 h. Levels increased up to 24 h. Thereafter, the expressions were almost always higher than in the control group. The expression of Caspase in stomach was also lower after 3 h and 12 h in the pH 7.0 group, but had increased by 24 h. Levels then decreased and were at their lowest by 72 h. The highest expression levels were observed by 148 h. The expression of Caspase decreased significantly by 3 h in the stomach tissue of shrimp in the pH 9.0. Levels then increased groudually between 12 h and 72 h, and decreased again up to 96 h before peaking at 120.TUNEL analysis results showed that apoptosis began to appear in the hepatopancreas cells of Chinese shrimp exposure to pH 7.0 and 9.0 for 12 h. The amount of apoptosis seems positively correlated with length of exposure to the pH stressor. Conversely, Brownish nuclei （TUNEL-positive） representing fragmented DNA were not observed in the control （Ph 8.2） and negative control. These results suggested that both high and low pH stress induced apoptosis in hepatopancreas cells of F. chinensis.The second part: The recombinant expression, purification and polyclonal antibody of Chinese shrimp Caspase. Specific primers were designed according to the opean reading frame （ORF） sequence of Caspase cDNA of Chinese shimp. Prokaryotic expression vector pET30a-CAS was contructed and transformed into the host E. coli BL21 （DE） pLysS. SDS-PAGE analysis results showed that a band of approximately 50 kDa corresponding to the His-tag Caspase fusion protein was observed after IPTG induction. Peptide fragments of the recombinant Caspase matched with the known aminoacid sequence of Caspase based on the comparison of peptide mass fingerprint using the MALDI-TOF-MS analytical system. The recombinant Caspase protein was purified using TALON Resin affinity chromatography. A polyclonal antiserum against Caspase was obtained from rabbits by injection of the purified fusion protein in complete Freund’s adjuvant and incomplete Freund’s adjuvant. To characterize the anti-Caspase serum, total protein extract of hemolymph, haemocytes, gill, muscle, lymphoid organ, heart, stomach and hepatopancreas were subject to western blot analysis. The results showed that Caspase was present in all tissues, whereas no target band was detected from the hemolymph.The third part: Effects of pH stress on antioxidant system and HSP90 gene expression of F. chinensis. The activity of T-AOC, anti-superoxide anion, catalase （CAT） and the gene expression of catalase （CAT）, peroxiredoxin （Prx） , HSP90 were analyzed in the gill, hepatopancreas, muscle and haemocytes of Chinese shrimp, Fenneropenaeus chinensis, after exposure to pH 7.0 and 9.0 stress. The activity of T-AOC, anti-superoxide anion, CAT and the gene expression of CAT in different tissues of Chinese shrimps were increased from 12 h to 24 h after pH stress. However, they were inhibited with the extending periods of pH stress time. The gene expression of Prx in heaptopancreas and muscle was gradually increased, but it was decreased in gill and haemocytes with prolonging pH stress time. The enzyme activities and gene expression of antioxidant system in shrimp gills of pH 9.0 group were reached to its highest level, which were earlier than other tissues in pH 7.0 and 9.0 group. The antioxidant enzyme activities in shrimp hepatopancreas of pH 7.0 group changed faster than other tissues’antioxidant enzyme activities. The gene expression of HSP90 in hepatopancreas and muscle of neutral stress group was significantly lower than the control group （P<0.05）, but it was significantly higher than the control group in hepatopancreas of alkaline pH stress group （P<0.05）. The gene expression of HSP90 in muscle of alkaline pH stress group reached to its highest level at 72h and was significantly lower at other sampled times. The results indicate that the antioxidant enzyme activities and gene expression of Chinese shrimp were induced between 3 h to 24 h after pH stress which is the response to oxidative stress, while they were inhibited with the extending pH stress time. It was showed that long time neutral and alkaline-induced oxidative stress probably caused the antioxidant defense system injury in Chinese shrimp. Gills and hepatopancreas could be as sensitive tissues to resist high and low pH stress respectively. The fouth part: The effect of ammonia stress on antioxidant system, haemocyte nitrogenous contents and HSP90 gene expression in Chinese shrimp. In order to investigate the mechanism of ammonia stress on Chinese shrimp, the concentration of haemocyte ammonia, urea, the activity of T-AOC, anti-superoxide anion, Na⁺ K⁺-ATPase、Ca²⁺ Mg²⁺-ATPase and the gene expression of catalase （CAT）, peroxiredoxin （Prx）, Caspase, HSP90 were analyzed in the gill, hepatopancreas, muscle and haemocytes of Chinese shrimp, Fenneropenaeus chinensis, after exposure to different ammonia concentrations （0mg/L, 2mg/L, 4mg/L, 6mg/L, 8mg/L） stress. The actual ammonia concentration of water was measured at the same time of sampling, and then calculated the concentration of ammonia molecules. The results showed at: （1）When the actual ammonia concentration of water was from 0.220mg/L to 13.449mg/L, the contents of haemocyte ammonia in Chinese shrimp were significantly increased. And the contents of haemocyte ammonia was decreased to its lowest level at 24-48h, reached to its highest level at 72-96, but they were always significantly higher than control group （P<0.05）. （2） When the actual ammonia concentration of water was from 0.220mg/L to 9.446mg/L, the contents of haemocyte urea in Chinese shrimp were increased gradually and significantly higher than control group （P<0.05）. They reached to their highest level at 6h; The activity of Na⁺ K⁺-ATPase, Ca²⁺ Mg²⁺-ATPase and HSP90 gene expression were obviously increased and they were shown the same trend. However, when the actual ammonia concentration of water was from 9.446mg/L to 13.449mg/L, the contents of haemocyte urea and the activity of Na⁺ K⁺-ATPase, Ca²⁺ Mg²⁺-ATPase and HSP90 gene expression were all decreased. This indicated that the high ammonia inhibited the haemocyte synthesis of urea and the activity of ATPase. （3） As the actual ammonia concentration of water was from 0.303mg/L to 8.625mg/L, the activity of T-AOC, anti-superoxide anion and CAT gene expression were increased and reached to their highest level at 6h or 96h; Prx gene expression was reached to its highest level at 72-96h. However, the antioxidant enzyme activities and gene expression of Chinese shrimp were inhibited with the content of ammonia in water larger than 8.625mg/L. （4） Caspase gene expression in ammonia stress showed similar with its in pH stress, it reached to highest level at 72-96h. This indicated that high content of ammonia could induce high expression of Caspase gene, which may cause the apoptosis of Chinese shrimp.更多还原