【作者】 明建华；

【导师】 徐跑； 刘文斌；

【作者基本信息】 南京农业大学 ， 水产养殖， 2011， 博士

【摘要】 大黄素(Emodin)是从中草药大黄(Rheum officinale Baill)根茎中提取的主要有效成分,具有抗菌消炎、抗病毒、抗氧化以及清除氧自由基、降血脂、保护肝脏和免疫调节等多种功效。维生素C(Vc)是鱼类必需的营养素,兼有免疫增强和抗氧化的双重功效,但大多数鱼类缺乏合成Vc所需的L-古洛糖酸内酯氧化酶(L-gulonolactone oxidase, GLO),必须从食物中摄取Vc。为了研究大黄素和高剂量Vc对鱼类生长、非特异性免疫以及抗应激的影响,选用我国主要淡水养殖鱼类之一的团头鲂(Megalobrama amblycephala, Yih)作为研究对象,首先分离、克隆出团头鲂两种热休克蛋白70 cDNA的全长序列,并对其基因结构和表达特征进行了分析。然后,在此基础上构建了团头鲂两种HSP70s的原核表达系统,并优化了表达条件,对表达产物进行了纯化和鉴定。同时,选取1200尾健康的团头鲂,体质量(133.44±2.11)g,随机分成4组,其中1组为对照组,投喂基础日粮(Vc含量50.3mg/kg),另外3组为试验组,投喂饲料是在基础日粮中分别添加60 mg/kg大黄素、700 mg/kg Vc、60 mg/kg大黄素+700 mg/kg Vc,每组设3个重复。连续投喂60 d后结束饲养试验,取鱼进行拥挤胁迫、高温应激和感染嗜水气单胞菌的应激试验。1团头鲂两种热休克蛋白70基因克隆与序列分析采用RT-PCR和cDNA末端快速扩增法(rapid amplification of cDNA ends, RACE),分别从室温和34℃热激团头鲂的肝脏中克隆到两种热休克蛋白70基因,分别命名为Ma-HSC70和Ma-HSP70基因。它们的cDNA全长分别为2336和2224 bp[不包括poly(A)],分别含有1950和1932 bp的开放阅读框,编码649和643个氨基酸,分子量约为71.24和70.52 kD,理论等电点为5.25和5.30。Ma-HSC70基因在编码区含有7段内含子,所有内含子均遵守GT/AG法则,为组成型HSC70,而Ma-HSP70基因在编码区不含内含子,为诱导型HSP70。氨基酸序列分析表明,Ma-HSC70和Ma-HSP70均含有HSP70家族的3个签名序列,两个部分重叠的双向核定位信号序列(nuclear localization signal sequence, NLS)以及胞质的特征基序EEVD等。同源性分析表明,Ma-HSC70与异育银鲫等脊椎动物HSC70的氨基酸序列相似性高达93.0%以上,而Ma-HSP70与鲫鱼等脊椎动物HSP70氨基酸序列的相似性高达85.0%以上,它们之间氨基酸序列相似性为86.5%。生物信息学分析显示,团头鲂两种HSP70s基因所编码的蛋白以亲水性区域为主,均具有丰富的B细胞抗原位点,无信号肽,为非典型的分泌型蛋白,无明显的跨膜区域；同时还含有众多的蛋白激酶C磷酸化位点、N-肉豆蔻酰化位点、酪蛋白激酶Ⅱ磷酸化位点和N-糖基化位点,推测这些位点在蛋白质折叠,转运,细胞内定位以及细胞信号转导与调控中发挥重要作用。这两种蛋白的二级结构均以a-螺旋和无规卷曲为主,空间结构包括N端ATPase功能域和C端多肽结合功能域。2大黄素和维生素C对团头鲂生长、生理生化指标以及两种HSP70s mRNA表达的影响饲养试验结束后,测定团头鲂的生长性能,并采样检测鱼的肌肉成分、血液和肝脏生化指标以及肝脏两种HSP70s mRNA的表达水平。结果表明,与对照组相比,大黄素、Vc组显著提高了鱼体增重率和特定生长率,血清中总蛋白(TP)、溶菌酶(LSZ)和碱性磷酸酶(AKP)的水平,肝脏中超氧化物歧化酶(SOD)的活性和诱导型HSP70 mRNA的基础表达水平,降低了饵料系数、死亡率、血清中皮质醇(COR)、甘油三酯(TG)以及肝脏丙二醛(MDA)的含量(P<0.05)；配伍组虽然血清中TP、LSZ的含量以及肝脏HSP70 mRNA的水平显著升高,肝脏MDA的含量也显著降低(P<0.05),但均未表现出协同增效作用。此外,与对照组相比,大黄素组还显著提高了团头鲂肝脏过氧化氢酶(CAT)的活性(P<0.05),而Vc和配伍组CAT的活性与对照组的差异不显著(P>0.05)；其它一些指标如血清中葡萄糖(GLu)、谷丙转氨酶(GPT)、谷草转氨酶(GOT)、胆固醇(CHOL)、肝脏组成型HSC70 mRNA的表达水平、鱼体形体指标以及肌肉组成成分等各组之间差异均不显著(P>0.05)。3大黄素和维生素C对团头鲂在拥挤胁迫下的生化指标及两种HSP70s mRNA表达的影响饲养试验结束后,从各池中取25尾规格基本一致的团头鲂,进行连续48 h的拥挤胁迫(100g/L)试验,分别于0h、12h、24h、48h取样分析团头鲂血液和肝脏的生化指标以及肝脏两种HSP70s mRNA的表达水平,并统计各组鱼的累积死亡率。结果表明,在拥挤胁迫后,与对照组相比,大黄素、Vc组不同程度地提高了团头鲂血清中TP和AKP的水平,肝脏中SOD和CAT的活性以及HSC70和HSP70 mRNAs的表达水平,降低了血清中COR、GLU、GPT、GOT、TG以及肝脏MDA的水平,而LSZ的活性表现为先下降后升高；配伍组中,这些指标虽然有类似以上的变化趋势,但大多差异不显著(P>0.05),且同样未表现出协同增效作用。此外,各组鱼血清CHOL的水平在拥挤胁迫前后的差异均不显著(P>0.05)。统计表明,大黄素和Vc组鱼的累积死亡率在拥挤胁迫24h、48h均显著低于对照组(P<0.05),而配伍组与对照组的差异均不显著(P>0.05)。4大黄素和维生素C对团头鲂在高温应激下的生化指标及两种HSP70s mRNA表达的影响饲养试验结束后,从各池中取25尾规格基本一致的鱼进行高温应激(34℃)试验。取样检测团头鲂在高温应激前后血液和肝脏的生化指标以及肝脏两种HSP70s mRNA的表达水平,并统计各组鱼在高温应激下的累积死亡率。结果表明,在高温应激后,与对照组相比,大黄素、Vc组不同程度地提高了团头鲂血清TP、LSZ和AKP的水平,肝脏SOD和CAT的活性以及HSC70和HSP70 mRNAs的表达水平,降低了血清COR、GLU、GPT、GOT、TG以及肝脏MDA的水平；配伍组中,这些指标虽然也有类似以上的变化趋势,但大多差异不显著(P>0.05),且未表现出协同增效作用。此外,各组鱼血清胆固醇的含量在高温应激前后的差异均不显著(P>0.05)。统计表明,在高温应激下,Vc组鱼的累积死亡率均显著低于对照组,大黄素组鱼的累积死亡率除6h外,也显著低于对照组(P<0.05),而配伍组与对照组的差异均不显著(P>0.05)。5大黄素和维生素C对团头鲂感染嗜水气单胞菌后的生化指标及两种HSP70s mRNA表达的影响饲养试验结束后,从各池中取15尾团头鲂进行腹腔注射感染嗜水气单胞菌试验,并在攻毒前后采样检测团头鲂血液和肝脏的生化指标以及肝脏两种HSP70s mRNA的表达水平；另从各池中取10尾鱼进行同样的攻毒试验,并统计攻毒后鱼的累积死亡率。结果表明,经攻毒后,与对照组相比,大黄素、Vc组不同程度地提高了团头鲂血清TP.LSZ和AKP的水平,肝脏SOD和CAT的活性以及HSC70和HSP70 mRNAs的表达水平,降低了血清COR、GLU、GPT、GOT、TG以及肝脏MDA的水平；配伍组中,这些指标虽然有类似上述的变化趋势,但大多与对照组的差异不显著(P>0.05),且未表现出协同增效作用。此外,各组鱼血清胆固醇的含量在攻毒前后的差异均不显著(P>0.05)。统计也表明,经攻毒后,大黄素和Vc组鱼的累积死亡率在24 h、48 h均显著低于对照组(P<0.05),而配伍组鱼的累积死亡率与对照组的差异均不显著(P>0.05)。6团头鲂两种热休克蛋白70的原核表达、纯化及鉴定采用PCR方法扩增团头鲂组成型HSC70和诱导型HSP70基因完整的编码区片段,并分别克隆到原核表达载体pET-22b(+)中,经双酶切和DNA测序鉴定后,将这两种重组表达质粒分别转化大肠杆菌BL21 (DE3),用1 mmol/L IPTG在不同温度及时间下进行诱导表达,并对表达产物进行SDS-PAGE分析,建立最佳诱导表达条件。采用Ni-NTA His Bind Resins亲和层析和DEAE-Sepharose FF阴离子交换柱层析对目的蛋白进行纯化,并进行Western-blotting验证。结果表明,成功构建了团头鲂两种重组表达质粒pET-22b(+)/Ma-HSC70和pET-22b(+)/Ma-HSP70,表达融合蛋白的相对分子量均约为72 kD,并能与兔抗人HSP70多抗进行特异性结合,这两种HSP70s融合蛋白经纯化后的纯度均达到95%以上。在较高温度(37℃)下,有利于融合蛋白的快速大量表达,但易形成包涵体；在较低温度(25℃)下,有利于融合蛋白的可溶性表达,但也会降低融合蛋白的表达速度。综合各种因素,本实验选择融合蛋白Ma-HSC70在25℃和Ma-HSP70在30℃下分别诱导7h作为可溶性表达的最佳条件。总之,本研究首次从团头鲂肝脏中克隆到两种HSP70s基因,并对该基因的结构和表达特征进行了分析,确定Ma-HSC70基因为组成型,而Ma-HSP70基因为诱导型。在此基础上成功构建了团头鲂两种HSP70s的原核表达系统,并优化了表达条件,最后得到了纯度在95%以上的两种热休克蛋白70的融合蛋白。这些为进一步研究这两种蛋白的结构和功能以及相应抗体的制备打下了基础。饲养和应激试验则表明,在基础日粮中添加大黄素60mg/kg或Vc 700 mg/kg可促进团头鲂的生长,降低饵料系数,提高鱼体的非特异性免疫力、抗氧化和抗应激能力,减少应激死亡率。二者配伍则效果不佳,其相互作用的机理还有待于进一步的研究。更多还原

【Abstract】 Emodin is an active anthraquinone constituent extracted from the rhizome of rhubarb Rheum officinale Baill. It has many functions such as anti-bacterial and anti-inflammatory, antivirus, antioxidation and scavenging free radicals, reducing blood lipid, protecting the liver, and regulating immunity. Vitamin C (Vc), also known as L-ascorbic acid, is an essential nutrient for normal physiological functions in fish. Vc is an important immunostimulant and antioxidant. but most teleosts are unable to synthesize ascorbic acid due to lack of L-gulonolactone oxidase (EC 1.1.3.8) responsible for the de novo synthesis of Vc. Therefore, exogenous Vc is required in fish diet. In order to study the effects of dietary emodin, high-dose Vc and their combination on growth, non-specific immunity and anti-stress of fish, Wuchang bream (Megalobrama amblycephala, Yih), one of the major freshwater aquaculture fish, was selected as the research subject. Firstly, two full-length cDNA sequences of two HSP70s genes were isolated and cloned from the liver of Wuchang bream, and their gene structure and expression characteristics were analyzed. Then, two HSP70s prokaryotic expression systems were constructed on the basis of above two HSP70s genes, and the expression conditions were optimized. The expressed products were purified and identified. At the same time,1200 healthy fish with initial body weight of (133.44±2.11) g were selected and randomly divided into four groups:a control group fed with basal diet (containing 50.3 mg/kg Vc) and three treated groups fed with basal diets supplemented with 60 mg/kg emodin,700 mg/kg Vc, and the combination of 60 mg/kg emodin+700 mg/kg Vc, respectively. Each diet was tested in triplicate. After continuous feeding for 60 days, the rearing experiment was over. Then, the experiments of crowding stress, heat stress and infected with Aeromonas hydrophila were carried out.1 Molecular cloning and sequence analysis of two HSP70 genes in Wuchang breamTwo full-length cDNA sequences of two HSP70s genes were isolated from the liver of Wuchang bream using RT-PCR and rapid amplification of cDNA ends (RACE). They were named Ma-HSC70 and Ma-HSP70 genes, respectively. The cDNAs were 2336 and 2224 bp in length [not including poly(A)] and contained 1950 and 1932 bp open reading frames (ORFs), respectively. The ORFs encoded proteins of 649 and 643 amino acids with predicted molecular weights of 71.24 and 70.52 kD, and theoretical isoelectric points of 5.25 and 5.30, respectively. Genomic DNA structure analysis revealed that Ma-HSC70 gene contained seven introns with all introns conforming to the GT/AG rule, belonging to the constitutive HSC70 whereas Ma-HSP70 gene did not contain any intron in the coding region, belonging to the inducible HSP70. Amino acid sequence analysis indicated that both Ma-HSC70 and Ma-HSP70 contained three signature sequences of HSP70 family, two partial overlapping bipartite nuclear localization signal sequences (NLS) and cytoplasmic characteristic motif (EEVD). Homology analysis revealed that Ma-HSC70 shared more than 93.0% identity with the known HSC70s of other vertebrates while Ma-HSP70 shared more than 85.0% identity with the known HSP70s of other vertebrates, and Ma-HSC70 and Ma-HSP70 shared 86.5% identity. Bioinformatics analysis indicated that the proteins encoded by Ma-HSC70 and Ma-HSP70 genes were hydrophilic, rich in B cells antigenic sites, without any signal peptide or transmembrane region. The two proteins also contained many protein kinase C phosphorylation sites, N-myristoylation sites, casein kinaseⅡphosphorylation sites, and N-glycosylation sites, predicting that they could play essential roles in protein folding, translocation, intracellular localization, signal transduction and regulation. The predominant secondary structures of the two proteins wereα-helix and random coil, and their spatial structures included N-terminal ATPase domain and C-terminal polypeptide-binding domain.2 Effects of emodin and vitamin C on growth, physiological and biochemical parameters, and two HSP70s mRNA expression of Wuchang breamAt the end of the rearing experiment, the growth performance, muscle composition, biochemical parameters of serum and liver, and the mRNA levels of liver two HSP70s genes were investigated. The results showed that compared with the control, the weight gain and specific growth rate, the levels of total protein (TP), lysozyme (LSZ) and alkaline phosphatase (AKP) in serum, the activity of superoxide dismutase (SOD) and basal expression level of inducible HSP70 mRNA in liver significantly increased (P<0.05) while feed coefficient, mortality, the contents of serum cortisol (COR) and triglycerides (TG), and liver malondialdehyde (MDA) decreased (P<0.05) in emodin and Vc groups. Although serum TP, LSZ levels, and the mRNA level of liver HSP70 gene increased significantly (P <0.05), and liver MDA content also significantly decreased (P<0.05) in combination group, the synergism didn’t appear. In addition, liver catalase (CAT) activity was also significantly improved in emodin group compared with the control (P<0.05) while the CAT activities in Vc and combination groups were not significantly different with the control (P> 0.05). The other indices, such as serum glucose (GLU), glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), cholesterol (CHOL), the mRNA level of liver constitutive HSC70, the fish body indices, and muscle composition were not significantly different among all groups (P> 0.05).3 Effects of emodin and vitamin C on biochemical parameters and two HSP70s mRNA expression of Wuchang bream under crowding stressAt the end of the rearing experiment,25 fish per tank were exposed to crowding stress (100 g/L) for 48 h, the biochemical parameters of blood and liver, mRNA levels of liver two HSP70s genes, and cumulative mortalities of fish in each group were investigated at 0 h,12 h,24 h and 48 h, respectively. The results showed that, after crowding stress, compared with the control, serum TP and AKP levels, liver SOD and CAT activities, and the expression levels of liver HSC70 and HSP70 mRNAs increased in emodin and Vc groups in varying degrees, and serum COR, GLU, GPT, GOT, TG and liver MDA levels decreased to some extent, while the LSZ activity decreased at first and then increased significantly (P<0.05). In combination group, although these parameters had similar changing trend as above ones, most of them were not significant differences with the control(P>0.05), and did not show any synergism either. In addition, serum CHOL levels among all groups before and after crowding stress were not significantly different (P> 0.05). The statistics showed that, the cumulative mortalities of the emodin and Vc groups were significantly lower than that of the control at 24 h and 48 h (P<0.05), but there was no significant difference between the combination and control groups (P> 0.05).4 Effects of emodin and vitamin C on biochemical parameters and two HSP70s mRNA expression of Wuchang bream under high temperature stress At the end of the rearing experiment,25 fish per tank were exposed to high temperature stress of 34℃. The biochemical parameters of blood and liver, and expression levels of liver two HSP70s mRNA before and after heat stress were determined and the cumulative mortality of each group under heat stress was counted. The results showed that, after heat stress, compared with the control, serum TP, LSZ, and AKP levels, liver SOD, CAT activities, and expression levels of HSC70 and HSP70 mRNAs increased in emodin and Vc groups in varying degrees and serum COR, GLU, GPT, GOT, TG and liver MDA levels decreased to some extent. Although these parameters had similar changing trend as above ones in combination group, it did not show any synergism either. In addition, serum cholesterol levels among all groups before and after heat stress were not significantly different (P>0.05). Statistics showed that under heat stress, the cumulative mortalities of emodin and Vc groups, except at 6 h in emodin group, were significantly lower than that of the control (P<0.05) while the difference between the combination and control groups was not significant (P>0.05).5 Effects of emodin and vitamin C on biochemical parameters and two HSP70s mRNA expression of Wuchang bream infected with Aeromonas hydrophilaAt the end of the rearing experiment,15 fish per tank were intraperitoneally infected with A. hydrophila (BSK-10), and the biochemical parameters of serum and liver, the mRNA expression levels of liver two HSP70s genes before and after infection were determined. Another ten fish taken from each tank were challenged with A. hydrophila as the same method as above, the cumulative mortality of each group was counted at different time point of post-infection. The results showed that after infection of fish, compared with the control, serum TP, LSZ and AKP levels, liver SOD, CAT activities, and the expression levels of HSC70 and HSP70 mRNA increased in emodin and Vc groups in varying degrees, while serum COR, GLU, GPT, GOT, TG and liver MDA levels decreased to some extent. In combination group, although these parameters had similar changing trend as above ones, most of them were not significant differences with the control(P>0.05), and did not show any synergism either. In addition, serum cholesterol levels among all groups before and after infection were not significantly different (P>0.05). Statistics also showed that after infected with A. hydrophila, the cumulative mortalities of emodin and Vc groups were significantly lower than that of the control in 24 h and 48 h (P<0.05), but the difference of the cumulative mortality between the combination and control groups was not significant (P>0.05).6 Prokaryotic expression, purification and identification of two HSP70s in Wuchang breamThe fragments of constitutive HSC70 and inducible HSP70 ORFs of Wuchang bream were amplified by PCR, and then cloned into prokaryotic expression vectors of pET-22b(+), respectively. The two recombinant plasmids, determined by double-endonuclease digestion and sequencing, were transformed into competent E. coli BL21 (DE3), respectively. The two recombinant strains were induced by 1 mmol/L IPTG at different temperatures and times, establishing optimal conditions for inducible expression. The expression products were purified by Ni-NTA His Bind Resins affinity chromatography and DEAE-Sepharose FF anion-exchange chromatography, and detected by SDS-PAGE and Western-blotting, respectively. The results show that two recombinant expression plasmids of pET-22b(+)/Ma-HSC70 and pET-22b(+)/Ma-HSP70, each expressing a fusion protein of about 72 kD identified by rabbit source anti-HSP70 polyclonal antibody, were successfully constructed, and the purity of two purified fusion proteins was more than 95%. A higher temperature (37℃) is conducive to the rapid expression of fusion protein, but it is easy to form inclusion body. A lower temperature (25℃) is in favor of the soluble expression of fusion protein, but it could also reduce the expression rate of fusion protein. All things considered, the optimal conditions for soluble expression of the two fusion proteins of Ma-HSC70 and Ma-HSP70 are induced for 7 h at 25℃and 30℃, respectively.In conclusion, two full-length HSP70 cDNAs were cloned and characterized from Wuchang bream liver for the first time. There are seven introns in the coding region of Ma-HSC70 gene while there is no intron in the Ma-HSP70 gene, suggesting constitutive and inducible expression, respectively. Through optimizing the expression conditions, two purified fusion proteins of Ma-HSC70 and Ma-HSP70, which purities were more than 95%, have been obtained by prokaryotic expression systems of two HSP70s genes of Wuchang bream. Thus it laid a foundation for further study on molecular structure and function of the two proteins and associated antibodies. The rearing and stress experiments indicate that, the basal diet supplemented with either 60 mg/kg emodin or 700 mg/kg Vc could promote the growth of Wuchang bream, lower FCR, increase non-specific immunity, antioxidant capacity, and stress resistance, reduce stress mortality. However, the combination of emodin and high-dose Vc showed no better effects. The mechanism of their interaction needs to be further studied.更多还原