罗氏沼虾Rab11基因cDNA克隆及其组织表达分析

为了发掘罗氏沼虾(Macrobrachium rosenbergii)免疫相关基因, 研究Rab蛋白(Ras-related proteins inbrain)在罗氏沼虾免疫应答中发挥的作用, 研究采用RACE-PCR技术克隆了罗氏沼虾Rab11基因全长cDNA序列, 记为MrRab11。全长1381 bp, 包括226 bp的5'UTR, 511 bp的3'UTR和645 bp的开放阅读框, 编码214个氨基酸, 含有一个Rab结构域。氨基酸序列比对显示, 罗氏沼虾与冈比亚按蚊(Anopheles gambiae)、蚤状溞(Daphniapulex)、叶蝉(Homalodisca vitripennis) Rab11一致性分别为82%、83%和82%。软件预测, MrRab11编码的蛋白分子量约为23.75 kD, 等电点约为5.34。实时荧光定量表达分析表明, MrRab11基因在罗氏沼虾各组织中都有表达, 肝胰腺中的表达量最高, 其次是肌肉和肠道。在阴沟肠杆菌(Enterobacter cloacae)感染12h后, 罗氏沼虾肝胰腺中MrRab11的表达量上升, 显著高于对照组(P0.05), 推测这是MrRab11对阴沟肠杆菌的应激表达,MrRab11在肝胰腺中参与了罗氏沼虾免疫应答过程。     

日本沼虾含硒谷胱甘肽过氧化物酶全长克隆及表达分析

为探讨日本沼虾(Macrobrachium nipponense)的免疫机制及含硒谷胱甘肽过氧化物酶(Se-GPx)在甲壳动物解毒和免疫应激反应中的作用,本文采用RACE法克隆了日本沼虾Se-GPx cDNA全长,其cDNA全长为908 bp,5′-UTR的长度为91 bp,3′-UTR长为256 bp,包括1个保守的硒代半胱氨酸插入序列(SECIS)和1个polyA尾,开放阅读框长度为561 bp,编码由186个氨基酸组成的多肽,其中,第39个氨基酸为TGA编码的硒代半胱氨酸。同源性和相似性分析显示日本沼虾的Se-GPx在甲壳动物中与罗氏沼虾相似度最高,在脊椎动物中与GPx1和GPx2家族的相似度比较高。日本沼虾Se-GPx基因在血细胞、肝胰腺、肌肉、卵巢、表皮以及大颚器官中都有表达,尤其在血细胞中表达量相对较高。用嗜水气单胞菌(Aeromonas hydrophila)刺激后3h和6h,血细胞Se-GPx基因表达量显著升高。结果表明,日本沼虾Se-GPx作为一种重要的抗氧化酶,在维护组织正常功能方面及免疫应激反应中发挥重要作用。     

饲料中铜水平对凡纳滨对虾免疫相关基因表达和抗菌能力的影响

在饲料中添加0、30和50 mg Cu/kg饲料的蛋氨酸铜,投喂凡纳滨对虾(Litopenaeus vannamei)7、14和21d,检测对虾体组织铜蓄积、免疫相关基因(Toll受体mRNA和溶菌酶mRNA)表达水平和免疫抗菌能力的变化。结果表明:凡纳滨对虾肝胰腺铜含量随着饲料蛋氨酸铜添加量增加及投喂时间延长而显著增加(P0.05);对虾肌肉的铜含量显著低于肝胰腺的铜含量。饲料中铜水平对凡纳滨对虾肌肉、血淋巴及肝胰腺中溶菌酶活性无显著影响(P0.05)。对虾组织SOD活性因饲料中铜水平和投喂时间变化显著,添加30 mgCu/kg组对虾肌肉、血淋巴和肝胰腺中SOD活性在第21天时显著高于其他两组(P0.05)。饲料中铜水平对凡纳滨对虾鳃组织中溶菌酶mRNA表达水平无显著影响,但显著影响鳃组织Toll受体mRNA表达水平(P0.05)。第7天时凡纳滨对虾Toll受体mRNA表达水平随着饲料铜水平升高而显著升高(P0.05);第14和第21天时,Toll受体mRNA表达水平在摄食添加30 mg Cu/kg组最高。人工急性感染溶藻弧菌(Vibrioalginolyticus)实验表明,第7天时,摄食添加50 mg Cu/kg组凡纳滨对虾全致死时间和半致死时间长于未添加铜组和添加30 mgCu/kg组,但在第14天,摄食添加30 mg Cu/kg组的全致死时间和半致死时间最长。研究表明饲料铜添加水平不但影响组织中铜的蓄积,还影响凡纳滨对虾SOD活性和Toll受体mRNA表达水平,从而影响机体的抗弧菌能力。     

两种沼虾溶菌酶基因ORF的克隆和罗氏沼虾溶菌酶基因的组织表达(英文)

分别提取罗氏沼虾和日本沼虾血细胞总RNA,RT-PCR扩增获得特异性cDNA片段,纯化后克隆到T载体上。序列测定表明所克隆的两种沼虾溶菌酶基因的开放阅读框(ORF)为477bp,共编码158个氨基酸,包括溶菌酶成熟肽140个氨基酸残基和信号肽18个氨基酸残基。同源性分析表明,罗氏沼虾和日本沼虾溶菌酶基因的碱基序列及推测氨基酸序列高度同源,分别为99.4%和98.1%。两种沼虾溶菌酶基因的碱基序列和推测氨基酸序列与Gen-Bank上其他对虾溶菌酶的同源性达83.0%和80.0%以上。两种沼虾溶菌酶都具有c-型溶菌酶典型的两个酶活性位点(Glu51)和(Asp68),以及8个保守结构氨基酸残基Cys,且在101、106和107位上缺少Asp,因而推测本实验所克隆的两种沼虾溶菌酶基因属c-型溶菌酶基因的非钙结合亚型。以PCR法制备罗氏沼虾溶菌酶基因的生物素标记探针,斑点杂交检测感染弧菌后溶菌酶基因mRNA在各组织中的转录水平,结果表明受感染6h后在眼、肌肉、鳃、肝胰腺、肠管中的表达量均有升高,其中在肝胰腺中的表达量最高,约为对照组的560%。在不同感染时间里,肝胰腺中该基因表达量有较大的变化:感染后3h表达量最低,24h后表达量升至最高,大约为对照组的430%,48h时的表达量又有所下降,但仍明显高于对照组(约为330%)。受弧菌感染后罗氏沼虾溶菌酶基因转录的上调证明溶菌酶基因在非特异性免疫中的直接作用,同时表明肝胰腺可能在沼虾的免疫防御过程起重要作用。       

罗氏沼虾蜕皮激素受体EcR基因的克隆及表达分析

为获得罗氏沼虾蜕皮激素受体(ecdysone receptor, EcR)基因编码序列,并检测mRNA表达模式。本实验拟利用克隆测序技术,以肝胰腺为实验素材,扩增EcR基因的编码区;借助生物信息学手段对所得序列进行分析;采用实时荧光定量PCR技术检测EcR基因在肌肉和肝胰腺等8个组织中的m RNA表达量。结果表明,罗氏沼虾EcR基因的编码区长度为1 716 bp,共编码571个氨基酸。物种间同源性比较分析发现,罗氏沼虾EcR基因序列与褐虾、黑背陆地蟹、日本沼虾、美洲龙螯虾、大西洋砂招潮蟹、蓝蟹、三疣梭子蟹、中华绒螯蟹和拟穴青蟹的同源性分别为90.7%、84.1%、83%、81%、81%、80.6%、79.8%、77.2%和75.9%。组织表达谱结果显示,EcR基因在所检8种组织中广泛表达,且不同性别相同组织间和相同性别不同组织间EcR的表达均存在不同程度的差异性。其中EcR基因在雄虾的鳃组织中表达量最高,在肌肉组织表达量最低;在雌虾的卵巢组织中高表达,在腹节神经组织中低表达。此外,EcR基因在雌虾的性腺、肠和肝胰腺组织中的表达量极显著或显著高于雄虾(p0.01或p0.05),在鳃组织中极显著低于雄虾(p0.01)。本研究成功克隆了罗氏沼虾的EcR基因,检测到EcR基因在各组织中广泛表达,且在不同性别相同组织和相同性别不同组织中存在显著差异。本实验为进一步研究EcR基因与生长发育调控作用提供了理论依据。     

虾夷扇贝铜锌超氧化物歧化酶基因的克隆与转录表达特性分析

利用RACE和克隆等方法得到了虾夷扇贝铜锌超氧化物歧化酶(Cu/Zn-SOD)基因全长cDNA序列,该序列全长1 064 bp,5′和3′非编码区(UTR)分别为61 bp和541 bp,开放阅读框(ORF)462 bp,编码153个氨基酸和一个终止密码子.分析发现,此氨基酸序列含有形成二硫键的两个半胱氨酸残基以及4个铜结合位点、4个锌结合位点,与已知的Cu/Zn-SOD结构相似,与其它物种同源性较高.进行虾夷扇贝组织分布及发育过程中不同时期的实时荧光定量监测,发现鳃中Cu/Zn-SOD基因mRNA相对表达量最高,肾次之,血淋巴中最低;不同发育阶段Cu/Zn-SOD表达量变化明显,其中受精卵和早期D形幼体两个时期表达量相对较高.     

饲料中添加合成虾青素对中华绒螯蟹成体雌蟹性腺发育、色泽和抗氧化能力的影响

采用在基础饲料中分别添加0、130和260 mg/kg的合成虾青素配制成3种粗蛋白和粗脂肪含量分别为42%和16%的等氮等脂的中华绒螯蟹(Eriocheir sinensis)育肥饲料(分别记为饲料1、2和3), 以中华绒螯蟹商业育肥饲料作为饲料4, 分别投喂4组雌蟹(每个饲料组3个重复水槽, 每个水槽中12只蟹), 进行为期60d的室内养殖实验, 以探讨添加合成虾青素对中华绒螯蟹成体雌蟹性腺发育、色泽及抗氧化能力的影响。结果显示: (1)实验进行到30d和60d, 在饲料中添加虾青素对雌体肝胰腺指数(HSI)和性腺指数(GSI)均无显著影响(P>0.05)。(2)性腺、肝胰腺和头胸甲中的总类胡萝卜素含量和红度值(a值)均以饲料3组最高, 性腺亮度值(L值)和黄度值(b值)以饲料1组最高(P<0.05); 饲料2组头胸甲的b值最高, 饲料1组最低(P<0.05)。(3) 饲料1组中华绒螯蟹血清过氧化物酶(POD)活性显著高于其他组, 饲料4组的过氧化氢酶(CAT)、谷胱甘肽还原酶(GR)、乳酸脱氢酶(LDH)、总抗氧化能力(T-AOC)、丙二醛(MDA)和乳酸(LD)含量最高(P<0.05); 肝胰腺中的SOD、T-AOC、GSH-Px和GR活性均以饲料1组最高, 饲料2组最低(P<0.05)。(4) 饲料2组血清酸性磷酸酶(ACP)活性和血蓝蛋白(Hc)含量显著高于其他组, 其余各组间差异不显著。血清中的碱性磷酸酶(ALP)和γ-谷氨酰转移酶(γ-GT)活性和一氧化氮(NO)含量均以饲料4组最高(P<0.05), 肝胰腺中的ACP、ALP和γ-GT活性以饲料1组最高。综上, 在育肥饲料中添加合成虾青素对成体雌蟹性腺发育无显著影响, 但可显著提高头胸甲、肝胰腺和卵巢中的类胡萝卜素总量、色泽和抗氧化能力, 建议雌蟹育肥饲料中合成虾青素的含量为90 mg/kg左右。     

饲料中黄曲霉毒素B1对克氏原螯虾幼虾生长、饲料利用和肝胰腺组织结构的影响

以含不同浓度黄曲霉毒素B₁(AFB₁)(0、10、100和1000μg/kg饲料)的4种饲料饲喂初始均重为(0.382±0.005) g的克氏原螯虾(Procambarus clarkii)幼虾42d,探讨AFB₁对克氏原螯虾幼虾生长性能、饲料效率和肝胰腺组织结构的影响。结果显示, 100和1000μg/kg毒素组幼虾的存活率、摄食率、终末体重、特定生长率和饲料效率均显著低于对照组, 10μg/kg毒素组与对照组无显著差异。10μg/kg毒素组幼虾肝胰腺碱性磷酸酶(AKP)、谷丙转氨酶(ALT)、谷草转氨酶(AST)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)、谷胱甘肽转移酶(GST)活性和丙二醛(MDA)含量均与对照组无显著差异,超氧化物歧化酶(SOD)活性显著低于对照组。饲料AFB₁含量≥100μg/kg时显著影响了克氏原螯虾幼虾上述肝胰腺酶的活性。10μg/kg毒素组肝胰腺组织结构发生轻微变化, 100和1000μg/kg毒素组幼虾的肝胰腺表现出严重病变, R细胞数量减少而B细胞...     

饲料中添加白术提取物对日本沼虾生长、健康和抗病力的影响

文章研究了饲料中添加不同水平的白术提取物(Extraction of Atractylodes macrocephala Koidz, EAMK)对日本沼虾(Macrobrachium nipponense)生产性能、器官指数、血液生化指标、抗氧化能力、消化系统健康和抗病力的影响。实验以初始体重为(0.21±0.05) g的日本沼虾为研究对象,设置6个饲料处理,白术提取物添加量为0、100、200、400、600和1200 mg/kg(Control、A100、A200、A400、A600和A1200)。每个饲料处理设6个重复,每个养殖缸放入60尾虾,实验周期为75d,每天记录采食量。养殖实验期间监测水体温度为(20±3)℃, pH为7.8—8.2,亚硝酸盐为0.16—0.2 mg/L,氨氮为0.02—0.1 mg/L,溶氧>6.0 mg/L。在实验结束后,分析日本沼虾生长性能、血清生化指标、肠道消化酶和免疫指标及肝胰腺抗氧化能力,观察日本沼虾的组织结构,用嗜水气单胞菌(Aeromonas hydrophila)进行攻毒并记录存活率。结果表明:饲料中添加200 mg/kg的白术提...     

Copper transporter 1, metallothionein and glutathione reductase genes are differentially expressed in tissues of sea bream (Sparus aurata) after exposure to dietary or waterborne copper

The high affinity copper transporter 1 (Ctr1), metallothionein (MT) and glutathione reductase (GR) are essential for copper uptake, sequestration and defense respectively. Following rearing on a normal commercial diet (12.6+/-0.2 mg kg(-1) Cu), sea bream were fed an experimental control diet lacking mineral mix (7.7+/-0.3 mg kg(-1) Cu), an experimental diet enhanced with Cu (135+/-4 mg kg(-1) Cu) or an experimental diet (7.7+/-0.3 mg kg(-1) Cu) whilst exposed to Cu in water (0.294+/-0.013 mg L(-1)). Fish were sampled at 0, 15 and 30 days after exposures. Fish fed the Cu-enhanced experimental diet showed lower levels of expression of Ctr1 in the intestine and liver compared to fish fed control experimental diets, whilst Ctr1 expression in the gill and kidney was unaffected by excess dietary Cu exposure. Waterborne-Cu exposure increased Ctr1 mRNA levels in the intestine and the kidney compared to experimental controls. Excess dietary Cu exposure had no effect on levels of metallothionein (MT) mRNA, and the only effect of dietary excess Cu on glutathione reductase (GR) mRNA was a decrease in the intestine. Both MT mRNA and GR were increased in the liver and gill after waterborne-Cu exposure, compared to levels in fish fed experimental control low Cu diets. Thus, Ctr1, MT and GR mRNA expression in response to excess Cu is dependent on the route of exposure. Furthermore, the tissue expression profile of sea bream Ctr1 is consistent with the known physiology of copper exposure in fish and indicates a role both in essential copper uptake and in avoidance of excess dietary and waterborne copper influx.     

Copper absorption in young men fed adequate and low zinc diets

Copper absorption was measured at two levels of dietary zinc in six healthy young men who were confined to a metabolic unit for a 75 d study of zinc utilization. A diet of conventional foods was fed, providing either 16.5 or 5.5 mg zinc and 1.3 mg copper daily. Copper absorption was determined by feeding⁶⁵Cu, a stable isotope of copper, once during the 16.5 mg Zn diet and near the beginning and end of the 5.5 mg Zn diet. Apparent copper absorption averaged 48.1% when the 16.5 mg Zn diet was fed. This was significantly higher than the averages of 37.2 and 38.5% when the 5.5 mg Zn diet was fed. Absorption also differed significantly among subjects. Fecal copper did not differ between diets or among subjects. All subjects were in positive copper balance at both levels of dietary zinc. These results suggest that a dietary zinc intake slightly above the Recommended Dietary Allowance of 15 mg/d does not increase fecal copper loss and does not interfere with copper absorption.     

Dietary pharmacological or excess zinc and phytase effects on tissue mineral concentrations, metallothionein, and apparent mineral retention in the newly weaned pig

Feeding pharmacological zinc (Zn) to weaned pigs improves growth, and dietary phytase improves P and Zn availability. Metallothionein (MT) increases in the duodenum, kidney, and liver of pigs fed 1000 mg Zn/kg with phytase or 2000 mg Zn/kg with or without phytase when fed for 14 d postweaning. The goal of this study was to determine the effects of feeding pharmacological Zn and phytase on tissue minerals, MT, mineral excretion, and apparent retention. Twenty-four newly weaned pigs (20 d; 7.2 kg) were individually fed twice daily, a basal diet supplemented with 0, 1000, or 4000 mg Zn/kg as Zn oxide, without or with phytase (500 phytase units [FTU]/kg) for 14 d, followed by a basal diet (100 mg Zn/kg) without phytase for 7 d. Pigs fed 4000 mg Zn/kg without phytase had higher (p=0.01) plasma, hepatic, renal Zn, renal Cu, and hepatic, renal, and jejunal MT than pigs fed the basal diet or 1000 mg Zn/kg. Duodenal MT was higher (p=0.0001) in pigs fed 1000 and 4000 mg Zn/kg than in pigs fed the basal diet. In pigs fed 1000 and 4000 mg Zn/kg, Zn loading occurred during the first 11 d of supplementation; by d 14, excess Zn was being excreted in the feces.     

Effect of zinc supplementation on metallothionein,copper, and zinc concentration in various tissues of copper-loaded rats

The present study was designed to investigate the effects of Zn administration on metallothionein concentrations in the liver, kidney, and intestine of copper-loaded rats. Male CD rats were fed a diet containing 12 mg Cu and 67 mg Zn/kg body wt. They were divided into either acute or chronic experimental protocols. Rats undergoing acute experiments received daily ip injections of either Cu (3 mg/kg body wt) or Zn (10 mg/kg body wt) for 3 d. Chronic experiments were carried out on rats receiving Cu ip injections on d 1, 2, 3, 10, 17, and 24, Cu injections plus a Zn-supplemented diet containing 5 g Zn/kg solid diet, or a Zn-supplemented diet alone. Rats injected Zn or Cu had increased MT concentrations in liver and kidney. Zn produced the most important effects and the liver was the most responsive organ. Rats fed a Zn-supplemented diet had significantly higher MT concentrations in liver and intestine with respect to controls. Increased MT synthesis in the liver may contribute to copper detoxification; the hypothesis of copper entrapment in enterocytes cannot be confirmed.     

Effects of dietary zinc on gene expression of antioxidant enzymes and heat shock proteins in hepatopancreas of abalone Haliotis discus hannai

The expression patterns of different genes encoding antioxidant enzymes and heat shock proteins were investigated, in present study, by real-time quantitative PCR in the hepatopancreas of abalone Haliotis discus hannai fed with different levels of dietary zinc (6.69, 33.8, 710.6 and 3462.5 mg/kg) for 20 weeks. The antioxidant enzymes include Cu/Zn-superoxide dismutase (Cu/Zn-SOD), Mn-superoxide dismutase (Mn-SOD), catalase (CAT), mu-glutathione-s-transferase (mu-GST) and thioredoxin peroxidase (TPx). The results showed that the mRNA expression of these antioxidant enzymes increased and reached the maximum at the dietary zinc level of 33.8 mg/kg, and then dropped progressively. Expression levels of the heat shock proteins (HSP26, HSP70 and HSP90) firstly increased at 33.8 mg/kg dietary Zn level, and reached to the maximum at 710.6 mg/kg, then dropped at 3462.5 mg/kg (p<0.05). Excessive dietary Zn (710.6 and 3462.5 mg/kg) significantly increases the Zn content and significantly decreases the total antioxidant capacity (T-AOC) in hepatopancreas (p<0.05). These findings showed that dietary Zn (33.8 mg/kg) could highly trigger the expression levels of antioxidant enzymes and heat shock proteins, but excessive dietary Zn (710.6 and 3462.5 mg/kg) induces a high oxidative stress in abalone.     

Regional distribution of metallothionein and zinc in the mouse gut

Gut Zn homeostatic responses to low, replete, and excess dietary Zn (10, 150, and 400 mg Zn/kg, respectively) were compared in mice with (MT+/+) and without (MT?/?) metallothionein (MT) expression. MT concentrations decreased progressively from stomach (12.9 nmol Cd bound/g) to colon (4.6 nmol Cd bound/g). Small intestinal MT was increased in mice fed the 400-mg Zn/kg diet (+130%, duodenum; +56%, jejunum; +29%, terminal ileum), but not in the stomach, cecum and colon. Zn concentrations were much higher in the distal gut at increasing Zn intakes in MT+/+ mice but to a lesser extent in MT?/? mice. On the 10-mg Zn/kg diet, MT?/? mice had 45% more Zn in the jejunum/ileum than MT+/+ mice. In fasted (20 h) mice, Zn concentrations in all gut regions were similar to those of MT+/+ mice fed the 10-mg Zn/kg diet, irrespective of prior Zn intake or genotype. Liver MT quadrupled in mice fasted after the 10-mg Zn/kg diet but only doubled after the 400-mg Zn/kg diet, a trend also present in gut MT. Glucagon administration stimulated gut as well as liver MT, implicating it as a major component of the MT response to fasting. MT?/? mice had five times more variation than MT+/+ mice in plasma Zn over all dietary groups. Together, these findings demonstrate that without MT, there is little modification of regional gut Zn concentrations in response to extremes of dietary Zn and poorer regulation of Zn homeostasis.     

皱纹盘鲍伴侣蛋白CCTζ的分子克隆及其在不同锌含量日粮处理下的表达分析

伴侣蛋白CCT在蛋白折叠、抗胁迫以及调节细胞生长等生理过程中担负重要作用。研究利用同源克隆和RACE技术克隆了皱纹盘鲍伴侣蛋白CCTζ(HdhCCTζ)cDNA全长序列,并对CCTζ基因的序列特征进行了分析。HdhCCTζcDNA序列长1960 bp,开放阅读框为1599 bp,编码532个氨基酸,生物学软件推测其编码蛋白相对分子量为58.46 kD,等电点为6.53。BLAST分析结果表明HdhCCTζ与哺乳动物、鸟和鱼类等物种的CCT具有较高的同源性。生物信息学分析结果显示,HdhCCTζ蛋白序列中包含3个典型的CCT信号基序(35RTNLGPKGTLKML47,56LTKDGNVLLHEMQIQHP72和84QDDITGDGT92)以及1个腺苷三磷酸结合基序(89GDGTTS94)。此外,在HdhCCTζ蛋白序列中还包含3个糖基化位点(23NISA26,128NKSL131and234NVSL237)。实时荧光定量PCR检测结果显示,HdhCCTζ在皱纹盘鲍组织中为组成型表达,但主要集中在性腺、血淋巴和肝胰腺中表达。使用不同锌含量(6.69 mg/kg、33.85 mg/kg、710.63 mg/kg和3462.5 mg/kg)的日粮来饲喂幼鲍20周后,取其肝脏和血细胞总RNA,利用实时荧光定量PCR技术进行HdhCCTζmRNA的表达水平分析。分析结果显示,随着日粮中锌含量的增加,HdhCCTζmRNA在血淋巴和肝胰腺中的表达水平呈现上调的趋势。相比于锌适量组(33.85 mg/kg),过量的锌(3462.5 mg/kg)却能够下调HdhCCTζmRNA的表达水平。上述结果表明,HdhCCTζ的表达受到日粮中锌添加量的影响,而高表达量的HdhCCTζ可能在提高皱纹盘鲍机体抗胁迫过程中发挥重要作用。