草鱼miR-462通过靶向cx32.2、slc9a3.1和tbk1调控嗜水气单胞菌感染诱导的免疫应答

为探究miR-462在嗜水气单胞菌感染草鱼肾脏细胞(Ctenopharyngodon idella kideny,CIK)后的调控机制,实验利用荧光定量技术检测了CIK细胞感染嗜水气单胞菌后miR-462表达水平的变化;运用RNAhybrid软件预测miR-462的靶基因,利用双荧光素酶报告基因系统进行确定;此外还分析了miR-462对靶基因下游基因的调控作用。结果显示,在CIK细胞感染嗜水气单胞菌的过程中,miR-462的表达发生显著变化;cx32.2、slc9a3.1和tbk1的表达先降低后升高,与miR-462的表达模式呈负相关。双荧光素酶报告系统显示,miR-462可靶向cx32.2、slc9a3.1和tbk1的3′非编码区抑制其表达,过表达miR-462可以显著抑制cx32.2、slc9a3.1和tbk1的表达。转染miR-462模拟物后,下游slc4a4a、tnfrsf5、cxcl9和cxcl11基因的表达受到抑制。研究表明,miR-462参与调控嗜水气单胞菌感染后草鱼CIK细胞中的免疫应答。cx32.2、slc9a3.1和tbk1被鉴定为miR-462的靶基因。miR-462可通过靶向slc9a3.1和tbk1影响下游基因的功能。     

低氧胁迫下斑马鱼鳃microRNAs差异分析

为研究microRNAs (miRNAs)应对低氧胁迫的生物学功能,对低氧胁迫和常氧条件下斑马鱼(Danio rerio)鳃组织进行高通量miRNAs测序,分析了低氧胁迫与常氧条件下斑马鱼鳃中miRNAs的表达差异。结果表明,低氧胁迫和常氧条件下,斑马鱼鳃中共有15个miRNAs呈显著差异表达,其中13个miRNAs在低氧胁迫斑马鱼鳃中的表达量显著上调,2个miRNAs表达量显著下调。对miRNAs测序和斑马鱼鳃转录组进行关联分析,针对前期筛选获得的低氧胁迫与常氧条件下显著差异表达的28个热休克蛋白基因进行靶基因预测,结果显示,低氧胁迫下显著低表达的miR-455-3p同时靶向提高热休克蛋白基因hspa14和dnajb6b的表达,以增强对低氧的适应能力。另外,低氧胁迫下显著高表达的miR-194a和miR-155可以分别靶向5个热休克蛋白基因(hspa12a, dnajc5aa, hspb7, hsp70.3, dnajb2)和4个热休克蛋白基因(hspa12a,hspg2, hspa13, dnajb2)来调控斑马鱼对低氧环境的适应。     

蚕豆水提取物及维生素C和E对草鱼肌肉质构、营养成分以及氧化应激的影响

为探究高浓度蚕豆水提取物、维生素C和E (V_C/V_E)提升草鱼肌肉品质(质构特性等)的效果,实验以普通配合饲料为对照,30%蚕豆水提取物、30%蚕豆水提取物+V_C/V_E(400 mg/kg V_C+200 mg/kg V_E)、蚕豆配合饲料为处理组,分别饲喂(250±20) g草鱼120 d。在40、80和120 d 3个时间点测定草鱼的肌肉和肠道中活性氧(ROS)含量及ROS消除和生成相关酶活性、肌肉细胞中线粒体膜通透性转换孔和线粒体膜电位水平的变化。在120 d时分析草鱼的生长特性和肌肉及肠道的显微结构。结果显示,与蚕豆组相比,30%蚕豆水提取物和30%蚕豆水提取物+V_C/V_E两组草鱼的生长增加、饲料系数和腹腔脂肪水平显著下降,但蚕豆水提取物+V_C/V_E组草鱼肌肉质构特性高于30%蚕豆水提取物组。30%蚕豆水提取物和30%蚕豆水提取物+V_C/V     

一种(鱼师)鱼诺卡氏菌酪氨酸蛋白磷酸酶基因的克隆及功能初步研究

這鱼诺卡氏菌是鱼类诺卡氏菌病的主要病原,可导致鱼类慢性系统性肉芽肿疾病.這鱼诺卡氏菌全基因组序列分析发现了一个酪氨酸蛋白磷酸酶(protein tyrosine phosphtase,PTP)基因,生物信息学分析显示该基因很可能编码一个靶向定位于宿主细胞线粒体的分泌蛋白.本实验对這鱼诺卡氏菌PTP进行了基因克隆、分泌蛋白鉴定、亚细胞定位、过表达和线粒体膜电位检测,结果显示,在這鱼诺卡氏菌胞外产物中质谱鉴定到了PTP肽段,证实其为分泌蛋白.亚细胞定位研究观察到PTP-GFP融合蛋白均匀地分布在FHM细胞中,与线粒体分布不重合,说明這鱼诺卡氏菌PTP蛋白并未靶向定位于线粒体.亚细胞定位和过表达研究都显示PTP蛋白在FHM细胞中表达后,细胞核出现固缩浓染、凋亡小体等明显的细胞凋亡特征.通过线粒体膜电位检测表明,在pcDNA-PTP转染后48 h,线粒体跨膜电位被明显破坏,说明這鱼诺卡氏菌PTP很可能是一种可诱导细胞凋亡的细菌蛋白.通过对這鱼诺卡氏菌PTP开展基因克隆和功能初步研究,为进一步揭示该基因的功能和深入了解這鱼诺卡氏菌的分子致病机理奠定了基础.     

JC-1标记的流式细胞术测定虾类血细胞的线粒体膜电位

以新鲜虾血细胞为阴性对照,α-羰基氰化氯苯腙处理的虾血细胞为阳性对照,建立以JC-1为荧光标记的测定虾类血细胞线粒体膜电位的流式细胞术方法.将凡纳滨对虾离体血细胞经不同浓度Cd2+ (10-9～10-3 mol/L)处理6h后,应用该方法测定血细胞线粒体膜电位的变化.结果显示10-4 mol/L和10-3 mol/L Cd2+导致血细胞线粒体膜电位显著下降,表明该浓度的Cd2+会破坏虾血细胞线粒体的功能.本研究结果也显示,应用JC-1标记的流式细胞术测定方法适用于虾类血细胞线粒体膜电位的研究.     

硫辛酸对草鱼脂肪细胞脂质含量及脂代谢相关基因表达的影响

为了探究硫辛酸对草鱼(Ctenopharyngodon idellus)脂肪细胞脂质代谢的影响,分离草鱼腹腔脂肪组织,消化得到脂肪细胞进行体外培养,分别用0、50和200μmol/L硫辛酸孵育细胞6 h,收取细胞和培养基样品后检测细胞甘油三酯(TG)含量、甘油和非酯化脂肪酸(NEFA)释放量及脂代谢相关基因表达。结果显示:50和200μmol/L硫辛酸处理显著降低草鱼脂肪细胞TG含量,200μmol/L硫辛酸显著增加细胞甘油和NEFA的释放量。200μmol/L硫辛酸组脂肪细胞脂肪分解相关基因(HSL、PPARα、CPT1、CD36)mRNA表达显著高于对照组,且ACC和PPARγ的mRNA表达水平显著上升,ATGL、MGL、SREBP-1c、FAS、DGAT等脂代谢基因表达状况无显著变化。综上所述,硫辛酸可能通过诱导草鱼脂肪细胞HSL和CPT1的基因表达促进脂质水解和β-氧化,从而降低脂肪细胞脂质含量。     

一种鰑鱼诺卡氏菌酪氨酸蛋白磷酸酶基因的克隆及功能初步研究

這鱼诺卡氏菌是鱼类诺卡氏菌病的主要病原,可导致鱼类慢性系统性肉芽肿疾病。這鱼诺卡氏菌全基因组序列分析发现了一个酪氨酸蛋白磷酸酶(protein tyrosine phosphtase,PTP)基因,生物信息学分析显示该基因很可能编码一个靶向定位于宿主细胞线粒体的分泌蛋白。本实验对這鱼诺卡氏菌PTP进行了基因克隆、分泌蛋白鉴定、亚细胞定位、过表达和线粒体膜电位检测,结果显示,在這鱼诺卡氏菌胞外产物中质谱鉴定到了PTP肽段,证实其为分泌蛋白。亚细胞定位研究观察到PTP-GFP融合蛋白均匀地分布在FHM细胞中,与线粒体分布不重合,说明這鱼诺卡氏菌PTP蛋白并未靶向定位于线粒体。亚细胞定位和过表达研究都显示PTP蛋白在FHM细胞中表达后,细胞核出现固缩浓染、凋亡小体等明显的细胞凋亡特征。通过线粒体膜电位检测表明,在pcDNA-PTP转染后48 h,线粒体跨膜电位被明显破坏,说明這鱼诺卡氏菌PTP很可能是一种可诱导细胞凋亡的细菌蛋白。通过对這鱼诺卡氏菌PTP开展基因克隆和功能初步研究,为进一步揭示该基因的功能和深入了解這鱼诺卡氏菌的分子致病机理奠定了基础。     

草鱼miR-23a在嗜水气单胞菌感染过程中的调控机制

为探索嗜水气单胞菌感染后草鱼肾脏细胞(Ctenopharyngodon idella kidney,CIK)中miR-23a的调控机制,实验通过实时荧光定量PCR(qPCR)测定了嗜水气单胞菌感染CIK细胞后miR-23a的表达量变化,使用RNAhybrid软件预测miR-23a的靶基因并用双萤光素酶报告基因检测系统进...     

草鱼瘦身养殖过程中营养与风味品质变化规律研究

为探究瘦身养殖时间(1～24个月)对鱼肉营养成分、滋味及气味的影响,以草鱼(Ctenopharyngodon idellus)为研究对象,采用常规生化方法、气相色谱-质谱联用仪及氨基酸自动分析仪检测鱼肉粗蛋白、水分、脂肪酸、氨基酸等成分,并评价营养价值,再结合电子鼻进一步对鱼肉的风味成分进行分析。结果显示,随瘦身养殖时间延长,鱼肉粗脂肪含量显著下降,灰分含量显著增加,粗蛋白和水分含量相对保持稳定;鱼肉的鲜味氨基酸含量显著增加,苦味氨基酸含量下降;单不饱和脂肪酸含量显著上升,多不饱和脂肪酸含量下降;气味特征研究表明,经8个月瘦身养殖的草鱼气味特征发生显著变化,主要腥味物质己醛和1-辛烯-3-醇含量在前8个月养殖过程中显著降低并逐渐趋于稳定,进一步延长养殖时间,气味不再发生显著变化。综上,通过适当时间的瘦身养殖处理可有效提升草鱼鱼肉的营养和风味品质;瘦身养殖8个月后,草鱼体重继续降低但鱼肉品质不再发生显著变化。     

Involvement of the miR-462/731 cluster in hypoxia response in <Emphasis Type="Italic">Megalobrama amblycephala</Emphasis>

MicroRNAs (miRNAs) are non-coding small RNAs showing both evolutionarily conserved and unique features and are involved in nearly all biological processes. In the present study, the role played by miR-462/731 cluster miRNAs in hypoxia response in Megalobrama amblycephala, an important freshwater fish, was investigated. The M. amblycephala miR-462/731 cluster locus and their 5′ flanking sequences were sequenced and analyzed. In M. amblycephala and other teleost fish species, the mature sequences of miR-462 and miR-731 were identical and hypoxia-responsive elements (HREs) were identified upstream of the miR-462/731 loci. The two miRNAs were significantly induced in the liver, spleen, gill, muscle, and brain after hypoxia treatment. The expression of both miRNAs was also upregulated in cells that received treatment which mimicked hypoxia. Furthermore, reporter assay revealed that M. amblycephala HREs can be activated by hypoxia. Taken together, the 462/731 cluster may play a role in the regulation of the hypoxia response in M. amblycephala.     

Screening of new Paenibacillus polymyxa S3 and its disease resistance of grass carp (Ctenopharyngodon idellus)

A new strain of Paenibacillus polymyxa S3 with antagonistic effects on 11 major fish pathogens (especially Aeromonas hydrophila), but had no toxicity to grass carp, was screened from the sediment of fishponds. In vivo colonization studies showed that strain S3 could be colonized and distributed in the gill and abdomen of the grass carp. Bioassay results showed that the weight growth rate of grass carp in the strain S3 oral group (16.01%) and strain S3 immersion group (16.44%) was significantly higher than those of the control group (8.61%). At the same time, the activities of ACP, AKP, CAT and GSH-Px in the serum of grass carp in oral and immersion groups were significantly higher than those of the control group. In addition, the treatment with strain S3 could significantly upregulate the expression of the antioxidant-related genes and immune-related genes Keap1, Nrf2, C3, LZM, IgM, TLR-4 and MyD-88 in grass carp tissues. The challenge test showed that strain S3 treatment significantly increased the survival rate of grass carp infected with Aeromonas hydrophila. Whole genome sequencing analysis showed that strain S3 had 16 active metabolite gene clusters, indicating that it had abundant gene resources, which provided important support for its development for fish microecological preparations. In summary, a new strain of Paenibacillus polymyxa S3 with antibacterial activity against a variety of fish pathogens was screened in this study and its probiotic function was evaluated, proving its potential value in fisheries.     

草鱼SREBP-1基因的克隆及糖对其在肝脏中表达的影响

固醇调节元件结合蛋白(SREBPs)是调控糖脂代谢相关基因表达的关键核转录因子。为获知草鱼SREBP-1基因的序列及其在肝脏中的表达规律,本实验采用同源克隆和RACE方法获得了草鱼SREBP-1基因的部分cDNA序列,并通过生物信息学方法对该基因及所编码蛋白的结构特征进行了分析;采用实时荧光定量PCR技术,对SREBP-1基因在8种不同组织的表达规律及低糖(糖含量24%)和高糖(糖含量42%)投喂条件下肝脏中的表达水平进行了研究。结果显示,所克隆到的草鱼SREBP-1基因cDNA长4 760 bp,其中包括开放读码框3 426bp,编码1 141个氨基酸;草鱼SREBP-1具有一个典型的碱性螺旋-环-螺旋亮氨酸拉链结构(bHLH-zip);氨基酸序列比对结果显示,草鱼SREBP-1与其他鱼类的同源性在76%~88%之间,与斑马鱼的进化关系最近;草鱼SREBP-1基因在脑中的表达量最高,肝脏和肠次之,在肾脏、脾脏、肌肉、脂肪和性腺中均有少量表达;与对照组相比,SREBP-1基因在高糖诱导下表达量显著提高(P0.05),低糖诱导下没有显著性差异。研究表明,在高糖负荷条件下,草鱼肝脏中SREBP-1可能会促进糖的利用和转化,从而参与糖代谢调节过程,为丰富鱼类糖代谢调控机理提供研究资料,并有望为提高鱼类对饲料糖的利用效率提供理论依据。     

Effects of dissolved oxygen and dietary lysine levels on growth performance,feed conversion ratio and body composition of grass carp,Ctenopharyngodon idella

In order to investigate the effects of lysine and dissolved oxygen on grass carp, the grass carp were fed with 13, 15 and 17 g kg ⁻¹ lysine diet at about 6 mg L⁻¹ (high dissolved oxygen, HO group) or 3.5 mg L⁻¹ (low dissolved oxygen, LO group) dissolved oxygen level, for 8 weeks. The fish were fed to apparent satiation by hand. The results showed that apparent digestibility of protein, energy and dry matter were decreased significantly when grass carp were fed at 3.5 mg L⁻¹ dissolved oxygen, and feed intake (FI) was also inhibited by low dissolved oxygen (P < 0.05). Weight gain, protein retention, protein efficiency, feed conversion ratio and amino acid retention of fish at 6 mg L⁻¹ dissolved oxygen level were significantly improved at 3.5 mg L⁻¹ dissolved oxygen level (P < 0.05). Weight gain, protein and amino acid retention, and feed efficiency of grass carp at the two dissolved oxygen levels were significantly improved by lysine supplementation (P < 0.05). The dietary lysine level and dissolved oxygen of water had an interaction effect on feed conversion ratios (P < 0.05). Grass carp fed at low dissolved oxygen level showed lower liver protein and fat contents. Plasma aspartate aminotransferase (AST) activity of grass carp fed at 3.5 mg L⁻¹ dissolved oxygen level was significantly increased compared to 6 mg L⁻¹ dissolved oxygen level (P < 0.05). Our results show that low dissolved oxygen level of water is harmful to the liver of grass carp.     

Changes in mitochondrial fatty acid composition following temperature acclimation of carp and their possible effects on FoF1-ATPase activity

Carp undergo temperature acclimation of respiratory function by altering mitochondrial ATP synthase (F_oF₁-ATPase) both quantitatively and qualitatively (Itoi et al. 2003). To address such acclimation temperature-dependent changes of F_oF₁-ATPase activity, we investigated in this study the correlation between the fatty acid composition and F_oF₁-ATPase activity in fast muscle of thermally acclimated carp. The quantities of saturated fatty acids of mitochondria from carp acclimated to 10 °C were significantly lower than those of carp acclimated to 30 °C. While mono- and poly-unsaturated fatty acids tended to increase with cold acclimation of carp, the molar concentration of 16:0 aldehyde in mitochondria from the 10 °C-acclimated carp were less than those from the 30 °C-acclimated fish. The specific activities of F_oF₁-ATPase in the 10 °C- and 30 °C-acclimated fish mitochondria were calculated to be 167±22 and 56±10 nmol/min ⋅ mg mitochondrial protein, respectively, the difference being significant at P<0.005. Taken together, the increase in F_oF₁-ATPase activity in fast muscle mitochondria of carp after cold temperature acclimation may be closely related to the increase of unsaturated fatty acids in mitochondria. Abbreviations: BSA - bovine serum albumin; DHA - docosahexaenoic acid; EGTA - ethyleneglycol bis(2-aminoethylether)tetraacetic acid; EPA - eicosapentaenoic acid; F_oF₁-ATPase - mitochondrial ATP synthase; α-F₁-ATPase - F_oF₁-ATPase α-subunit; β-F₁-ATPase - F_oF₁-ATPase β-subunit; HEPES - 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid; SDS - sodium dodecyl sulfate; SDS-PAGE - SDS-polyacrylamide gel electrophoresis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ammonia nitrogen excretion in Mandarin Fish (Siniperca chuatsi) and Grass Carp (Ctenopharyngodon idellus) fed practical diets: the effects of water temperature

To understand the actual production of fish culture about the utilization of dietary protein and excreta impact on the environment between mandarin fish (Siniperca chuatsi) and grass carp (Ctenopharyngodon idellus), the study to investigate the effect of temperatures (19 ± 0.5°C, 24 ± 0.5°C and 29 ± 0.5°C) on ammonia‐N excretion in mandarin fish and grass carp under fed and fasted states was conducted. These two species were fed a practical diet containing 325.2 g kg^?1 crude protein at 3% body weight per day. The ammonia‐N excretion rate was significantly increased when temperature increased from 19 to 29°C, and a linear relationship between ammonia‐N excretion rate and temperature. The maximum ammonia‐N excretion levels of mandarin fish and grass carp were observed at 4–8 h and 2–4 h after feeding, respectively, and the minimum values for both species were observed at 24 h after feeding. Under the feeding condition, mandarin fish had a lower ammonia‐N excretion level compared to grass carp at 24°C and 29°C. The average amount of ammonia‐N excreted by mandarin fish at 24 h is significantly higher than grass carp under fasting conditions, except 19 ± 0.5°C. These results indicated that mandarin fish might make better use of protein at higher temperature than grass carp when fed practical diets in commercial production. These results of this study suggested that mandarin fish had a lower ammonia‐N excretion level compared with grass carp, making a less contribution to environmental loading in an intensive fish culture.     

草鱼肝细胞外泌体的分离鉴定及其对肝细胞miR-122/33和免疫相关基因表达的影响

外泌体是具有磷脂双分子膜结构的纳米级囊泡,能够参与机体多种生理过程。实验探讨了草鱼肝细胞外泌体的分离鉴定方法,并初步研究外泌体对草鱼肝细胞中miRNAs及免疫相关基因表达的影响。实验以草鱼肝细胞L8824为材料,通过超速离心获得外泌体,利用电子显微镜观察外泌体形态,采用纳米颗粒示踪分析(nanoparticle tracking analysis,NTA)技术检测外泌体粒径和数量,同时利用Western blot分析其标志蛋白CD63的表达,最后用正常肝细胞和油酸诱导的脂肪肝细胞源外泌体孵育草鱼肝细胞,通过Real-time qPCR技术检测两种不同来源的外泌体对草鱼肝细胞中miR-122/33及免疫相关基因(TNF-α,NF-κB,IL-1β,IL-6和IL-10)转录水平的影响。结果显示,草鱼肝细胞外泌体为30~150 nm的不均匀囊泡,呈圆形或椭圆形,有完整的膜结构;外泌体标志蛋白CD63呈阳性表达;NTA技术检测显示外泌体囊泡占所有囊泡的50%以上;脂肪肝细胞源外泌体显著提高了肝细胞中miR-122及炎症因子TNF-α、IL-1β和IL-6的mRNA转录水平。研究表明,通过超速离心法可成功分离草鱼肝细胞外泌体,且脂肪肝细胞源外泌体在草鱼肝细胞免疫调节中可能发挥重要作用。     

杜仲对草鱼生长、肌肉品质和胶原蛋白基因表达的影响

为研究杜仲对草鱼生长性能、肌肉品质及胶原蛋白基因COL1A1和COL1A2表达的影响,实验采用初始体质量为(215.0±0.4)g的草鱼120尾,随机分为2处理组(每组3重复,每重复20尾鱼),分别饲喂基础饲料(对照组)和添加2%杜仲的实验饲料(杜仲组),养殖时间为8周。结果显示,与对照组相比,添加2%杜仲对草鱼生长性能无显著影响,但能显著增加肌肉、皮肤和肝脏胶原蛋白水平,增加肌肉总必需氨基酸(TEAA)、总氨基酸(TAA)水平。2%杜仲可显著降低草鱼肌肉的冷冻失水率、离心失水率,但对肌纤维密度和肌纤维直径无显著影响。在胶原蛋白基因表达方面,2%杜仲显著增加了第4周、8周时草鱼的肌肉、皮肤和第8周时的肝脏组织COL1A1、COL1A2基因m RNA表达量。研究表明,饲料中添加2%杜仲可改善大规格草鱼的肌肉品质。