热应激对大正三色锦鲤非特异性免疫指标及HSP70基因表达的影响

为探讨锦鲤的抗逆机理,在实验室条件下研究了持续热应激对大正三色锦鲤非特异性免疫指标及HSP70基因相对表达量的影响。分别于应激前、应激后2、6、10、14、18、22、26 h进行取样测定呼吸爆发、补体蛋白3(C3)、超氧化物歧化酶(SOD)、丙二醛(MDA)以及热休克蛋白70(HSP70)基因的相对表达量。结果发现,热应激后呼吸爆发降低,在应激后2、10、18～26h降低显著(P<0.05);热应激2 h后血清中C3含量略有上升,应激6～26 h过程中与应激前相比均下降,应激14～22 h显著地下降(P<0.05);热应激下血清中SOD有上升的趋势,但差异不显著(P>0.05);热应激下,血清中MDA浓度升高,应激10～18 h上升显著(P<0.05);应激后2、26 h HSP70基因的相对表达量与应激前相比显著性地上升了5.93倍、2倍(P<0.05),应激后6～22 h HSP70基因的相对表达量与应激前水平无显著性差异(P>0.05)。结果表明,热应激影响锦鲤非特性免疫指标,降低锦鲤非特异性免疫力;HSP70的表达受热应激调节,热应激下诱导合成的HSP70对锦鲤起到一定的应激保护作用。     

克氏原螯虾对高温应激的生理学响应

本实验旨在研究高温对克氏原螯虾(Procambarus clarkia)血液生化指标、抗氧化能力以及热休克蛋白70基因表达的影响。实验分别采用30℃和35℃水温进行高温应激试验,应激前(0 h)和在应激后3、6、12、24和48 h分别采集样本,测定血淋巴液中LDH、GLU、TCHO、TP和TG含量,测定肝胰腺T-AOC、CAT、MDA以及HSP70基因的表达变化。结果显示:在高温应激的初级阶段(6 h),克氏原螯虾血淋巴中的TCHO和LD含量急剧上升,而GLU含量持续上升到24 h;TG含量在应激的初级阶段变化不明显,但在6 h后急速上升,在12 h达到最高后开始回落;TP的含量在初级阶段出现下降,然后在12 h恢复至应激前水平。肝胰腺中的MDA含量在应激后持续上升,在24 h后达到最高值,随后下降至应激前水平;T-AOC在应激后3 h上升至最高水平,随后降低;CAT的含量分别在应激后6 h和12 h上升至最高值然后回落。HSP70基因在应激前未检测到有表达,高温应激后3 h即可检测到少量表达,35℃组在6 h出现大量表达,并持续到12 h,随后24 h降低至无法检出;而30℃组在应激6 h后降低至无法检出。结果表明,30℃和35℃高温能引起克氏原螯虾机体产生应激反应,并对机体的生理机能产生影响。     

罗非鱼HSP70(tHSP70)蛋白的生物信息学分析、真核表达及质谱鉴定

运用生物信息学技术分析罗非鱼热休克蛋白70(tilapia heat shock protein,tHSP70)的理化特性、糖基化位点、跨膜区域、蛋白的细胞定位及信号肽与虹鳟等其他动物HSP70的相似性,以人工合成cDNA为模板,通过聚合酶链式反应扩增其完整CDS,并将此DNA片段与真核表达载体pGAPZa-A连接,构建pGAPZa-HSP70表达质粒,在毕赤酵母GS115中表达tHSP70蛋白,对表达上清液进行SDS-PAGE及Western-blotting分析后,采用Ni²⁺IDA层析脱盐纯化目的蛋白,并对所表达的蛋白进行糖基化PAS染色鉴定。对表达的蛋白进行SDS-PAGE后,将所获得的非预定大小(约100 ku)条带进行电离飞行时间质谱鉴定,并确定其蛋白种类。结果表明:tHSP70所含氨基酸数量为640,分子量为70 274.5 u,等电点为5.49。在哺乳动物网织红细胞(体外)的半衰期为30 h,在酵母内半衰期大于20 h,而在大肠杆菌内的半衰期也大于10 h,不稳定指数为36.64,脂肪族指数为85.58。可能分别含有6个N-和O-糖基化位点,所克隆tHSP70基因与目的基因完整编码区序列完全一致,所构建的pGAPZa-HSP70质粒能在GS115中成功表达,诱导表达上清液经SDS-PAGE及Western-blotting分析后发现,除70 ku目的蛋白外,还出现一条100 ku条带。经糖基化PAS染色证明,此100 ku蛋白可能是HSP70糖基化的结果,且能被人的兔抗HSP70抗体识别,质谱鉴定证明该条带即是tHSP70蛋白。本研究所表达tHSP70蛋白为后续罗非鱼细胞学及抗原提呈等免疫学研究奠定了基础。     

Physiological responses and HSP70 mRNA expression in GIFT tilapia juveniles,Oreochromis niloticus under short‐term crowding

Physiological responses and HSP70 mRNA expression to short‐term crowding were tested in juveniles genetically improved farmed tilapia (Oreochromis niloticus) (70.43 ± 4.43 g). Fish were kept at control group (5 g L^?1) and stress groups (low density 10 g L^?1, medium density 40 g L^?1 and high density 70 and 100 g L^?1) for 48 h. Each density was tested in triplicates. The effects of a short‐term exposure on the physiological responses of fish were determined before stress (0 h) and at 6, 12, 24 or 48 h post‐crowding. There was a significant increase in serum cortisol, total protein, lysozyme and aspartate aminotransferase (AST) activities in all stressed groups at 24 h post‐crowding (P < 0.05). Serum glucose and alanine aminotransferase (ALT) of 100 g L^?1 stressed group were lower than that of the control after 48 h of crowding stress (P < 0.05). The levels of serum triglyceride and cholesterol in crowding stress with high density were significantly decreased compared with the control group at 48 h. The mRNA expression data showed that hepatic HSP70 mRNA levels were markedly elevated at all stressed groups. HSP70 mRNA levels of 70 and 100 g L^?1 stressed groups decreased at 48 h compared with the 24 h post‐crowding. The protective ability of HSP70 was limited.     

pH、氨氮胁迫对中国对虾HSP90基因表达的影响

研究了pH、氨氮胁迫对中国对虾Fenneropenaeus chinensis血细胞、肝胰腺、鳃和肌肉组织HSP90基因时空表达的影响.分别将中国对虾暴露于pH7.0、9.0的水体中148h和不同氨氮浓度的水体中96h,结果表明,pH（7.0,9.0）胁迫条件下中国对虾鳃、肌肉和血细胞HSP90基因表达均上调,肝胰腺HSP90基因表达对两种pH胁迫差异明显：pH7.0胁迫条件下,HSP90基因表达3h达峰值后明显降低;pH 9.0胁迫时,HSP90基因表达水平逐渐升高,整个胁迫过程中均显著高于对照组（P＜0.05）,说明中国对虾肝胰腺组织是高pH胁迫的响应器官.6mg/L氨氮浓度组鳃和肌肉组织HSP90基因表达水平24h达最高值,分别为对照组的5.46和1.55倍;各胁迫组肝胰腺和血细胞HSP90基因表达水平分别于6h和48h达到最高值,为对照组的1.33～2.08倍和2.20～5.45倍.肝胰腺组织对氨氮胁迫表现敏感,短时间内（6h）通过上调HSP90基因表达水平保护细胞;鳃组织HSP90基因表达波动范围最大,说明鳃组织需要更高表达量的HSP90保护细胞.当氨氮浓度持续48～96h维持在2～6mg/L,各组织HSP90基因表达水平显著降低.     

Effect of heat stress and recovery on viability,oxidative damage,and heat shock protein expression in hepatic cells of grass carp (Ctenopharyngodon idellus)

In this study, we investigated the effects of hyperthermia and recovery on cell viability, lactate dehydrogenase (LDH) activity, superoxide dismutase (SOD) activity, malondialdehyde (MDA), total antioxidant capacity (T-AOC), and heat shock protein (HSP60, 70, and 90) mRNA expression in the hepatic cells of the grass carp, Ctenopharyngodon idellus. Triplicate groups of cultured cells were exposed to 30, 32, or 34 °C for 0.5 h and then immediately incubated at 27 °C in 5 % CO₂ for 6, 12, 24, or 48 h. Hyperthermia stress greatly reduced cell viability and increased LDH release. Cell damage declined after recovery. Hyperthermia stress increased the lipid peroxide levels and reduced the antioxidant capacity (e.g., reduced SOD and T-AOC) of the cells. However, oxidative damage declined as the recovery period increased, and the levels of MDA, SOD, and T-AOC were restored. After cells were exposed to 32 °C, the expression of HSP60 after recovery for 1, 2, and 4 h (P < 0.05), the expression of HSP70 after recovery for 0.5 and 1 h (P < 0.01), and the expression of HSP90 throughout recovery were significantly higher (P < 0.01) than the prestress levels. During the recovery period, the variations in HSP gene expression reflected the transition period from a state of cellular growth to one of the cellular repairs. In conclusion, hyperthermia depresses cell viability, induces oxidative damage, and increases HSP expression, which plays an important role during hyperthermic stress in grass carp hepatic cells.     

EDCs‐induced glucocorticoid receptor related genes expression of the river pufferfish,Takifugu obscurus

Endocrine disrupting chemicals (EDCs) are of substantial concern when contaminating waters. We studied gene expression of larvae from river pufferfish, Takifugu obscurus exposed to EDCs. We cloned the full‐ or partial‐length cDNA sequence of genes related to the cortisol pathway, such as the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), heat shock protein (HSP) 70 and 90, phosphoenolpyruvate carboxykinase (PEPCK) and the FK506‐binding protein 4 (FKBP52). The tissue and time‐dependent expression of mRNA was studied by real‐time PCR in T. obscurus exposed to two representative EDCs (bisphenol‐A and 4‐tert‐octylphenol). HSP70, HSP90, MR and FKBP52 mRNA expression level was much higher than GR and PEPCK expression. GR mRNA expression level was significantly upregulated after 48 h in both EDC‐treated fish groups compared with the control. Both groups also showed down‐regulation patterns after 48 h. The initial expression of PEPCK in both groups was down‐regulated after 24 h. In the bisphenol‐A treated group, the expression of FKBP52 showed substantially high up‐regulation from 6 to 48 h after exposure, and then strikingly reduced its expression level at 72 h. Our results provide insights into the EDCs metabolizing system of T. obscurus and offers baseline information for further research related to the possible use as a bio‐indicator of this commercially important aquaculture fish species.     

氨氮胁迫对凡纳滨对虾肠道免疫相关指标的影响

为研究急性氨氮胁迫对凡纳滨对虾(Litopenaeus vannamei)肠道免疫功能的影响,将对虾暴露于氨氮浓度为20 mg·L^-1的海水中72 h,测定了不同时间点肠道中抗病原感染指标如酸性磷酸酶(ACP)、碱性磷酸酶(ALP)、溶菌酶(Lys)、酚氧化酶原(proPO)以及抗氧化功能指标如总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)、热休克蛋白70(HSP70)、热休克蛋白90(HSP90)等变化。结果显示,与对照组相比,氨氮胁迫后:1)ACP和ALP活性均于6 h显著升高(P<0.05),随后于48~72 h显著低于对照组(P<0.05);Lys活性于24 h显著升高至最大值(P<0.05),随后于72 h显著低于对照组(P<0.05)。2)T-AOC和SOD活性均于6 h和12 h显著高于对照组(P<0.05),随后于48 h和72 h显著降低(P<0.05)。3)HSP70基因表达水平于24 h显著升高至最大值,随后虽有降低,但仍显著高于对照组(P<0.05);HSP90和proPO基因表达水平均于12 h显著升高至最大值(P<0.05),随后于72 h降低至对照组水平(P>0.05)。研究表明,急性氨氮胁迫对凡纳滨对虾肠道免疫功能相关指标影响显著,对其肠道免疫防御系统有明显的损伤作用。