鳜弹状病毒N蛋白与鳜c-Myc互作调控谷氨酰胺代谢的研究

为了研究鳜弹状病毒(Siniperca chuatsi rhabdovirus, SCRV)如何调控鳜c-Myc(Sc-c-Myc)进而调控谷氨酰胺代谢的分子机制,本研究通过免疫共沉淀联合蛋白质谱寻找可能与Sc-c-Myc互作的病毒蛋白,初步分析确定为核衣壳蛋白(N蛋白);Co-IP结果显示SCRV的N蛋白与Sc-c-Myc存在相互作用。通过PCR扩增获得了带有Flag标签序列的SCRV-N基因的ORF片段,并构建了SCRV-N蛋白过表达载体pcDNA-N-Flag;将pcDNA-N-Flag质粒转染CPB细胞,荧光观察发现Sc-c-Myc与SCRV-N在细胞质内存在共定位现象;并且通过qRT-PCR和Western blotting检测转染pcDNA-N-Flag的CPB细胞中Sc-c-Myc及谷氨酰胺代谢通路关键酶(GLS1,GDH,IDH2)的表达变化,发现Sc-c-Myc、GLS1的转录水平和蛋白水平均显著上调。以上结果表明SCRV的N蛋白与Sc-c-Myc互作促进Sc-c-Myc的表达,进而调控宿主细胞谷氨酰胺代谢途径,为SCRV防控提供了新的靶点。     

基于CPB细胞扩增体系的鳜弹状病毒增殖条件的优化

为获知鳜弹状病毒(Siniperca chuatsi rhabdovirus,SCRV)QY株在体外培养细胞中最适增殖条件,以鳜脑细胞系(Chinese perch brain cell line,CPB)为增殖体系,采用实时荧光定量PCR(quantitative real-time PCR,q RT-PCR)技术所测病毒拷贝数为判断指标,比较同步接毒和异步接毒2种接种方式以及病毒接种量、血清浓度等培养条件对病毒增殖的影响,确定SCRV-QY株在CPB细胞中的最适增殖条件。结果显示,单位体积病毒增殖量方面,异步接毒法优于同步接毒法,以感染复数(multiplicity of infection,MOI)为10将病毒接种长满单层的CPB细胞中,28°C吸附1.5 h,用含2%胎牛血清的L15培养液于28°C培养时,病毒增殖量最高,为5.59×10~(10)拷贝/mL;而从单位成本所获病毒量考虑,同步接毒法筛选出的4种增殖条件单位成本所获的病毒产量优于异步接毒法,其中当MOI=0.03、胎牛血清终浓度为6%时,同步接种对数生长中期的CPB细胞,28°C恒温培养70 h后收获病毒液,单位成本所获病毒量最高,为4.88×10~(13)个拷贝/元。综上所述,本研究以病毒增殖量和培养基成本作为考量,优化SCRV-QY株的增殖条件,可为SCRV疫苗低成本、规模化生产提供理论依据。     

鳜弹状病毒糖蛋白在杆状病毒表达系统中的表达与鉴定

近年来,鳜弹状病毒引起的病害频发,该病毒传播快,致病性强,是引起鳜病害的主要病原之一。鳜弹状病毒基因组编码5个结构蛋白,其中糖蛋白G是病毒表面的主要抗原,为实现该抗原蛋白的大量表达,利用分子克隆技术将鳜弹状病毒糖蛋白G片段插入杆状病毒穿梭载体pFastBac1中转化至大肠杆菌DH5α感受态细胞后,筛选阳性克隆获得重组转移载体pFastBac-G,再将其转化至大肠杆菌DH10Bac感受态细胞,经蓝白斑与抗性筛选后获取重组杆粒rBacmid-G,随后利用脂质体转染法将重组杆粒转染Sf9昆虫细胞制备重组杆状病毒。将获取到的P3代重组病毒感染Sf9昆虫细胞进行重组蛋白的SDS-PAGE检测,成功表达后利用镍离子亲和层析柱纯化目的蛋白,随后对重组蛋白进行Western blot鉴定,结果显示在大于50 ku处有一条特异性条带,而对照组无条带,表明制备的抗原能与抗体特异性结合。结果表明,鳜弹状病毒糖蛋白G在杆状病毒表达系统成功表达。本试验结果可为鳜弹状病毒糖蛋白G疫苗的研发和大规模生产提供技术支撑。     

不同溶解氧水平对鳜呼吸代谢酶及其基因表达量的影响

鱼类面对低氧胁迫具有不同的感受性与适应调节机理.为探究不同溶解氧含量下鳜呼吸代谢水平,分别在溶解氧质量浓度为5、4、3、2、1 mg/L时测定鳜心脏、脑、肝脏和肌肉组织中丙酮酸脱氢酶(PDH)、乳酸脱氢酶(LDH)活性和乳酸含量及各组织中相关基因(LDH-A、PDH-E1α)的表达量,并计算每个阶段鳜耗氧率.试验结果显...     

体重和温度对大眼鳜静止代谢的影响

采用流水式呼吸仪,测定了97尾大眼鳜(Siniperca kneri Garman)在不同温度条件下的静止代谢率(M),以检验温度与体重对该种鱼代谢水平的影响。结果表明,在15、20、25及30℃条件下大眼鳜的特定体重代谢率(M')分别为(74.06±6.24)、(135.24±18.27)、(186.19±29.03)、(196.78±34.31)mgO2/(kg.h)。在各温度条件下,大眼鳜的静止代谢率均与体重呈显著的双对数直线相关(P<0.01)。协方差分析表明,各回归方程的斜率(体重指数b)之间差异显著(P<0.01),且b值随温度的升高呈下降的趋势,提示体重和温度对大眼鳜静止代谢的影响存在交互作用。特定体重代谢率与体重(W,kg)和温度(T,℃)的复相关关系为M'=3.79T0.902W(0.345-0.202ln T)或者M'=3.79W0.345T(0.902-0.202ln W)(P<0.001),其中,-0.202为体重和温度影响特定体重代谢率的交互作用指数。通过与其它底栖肉食性鱼类的比较发现,大眼鳜具有较高的特定体重代谢率,这可能与其喜溶氧丰富的流水环境、捕食行为耗能较高等特殊的生态特征有关。     

池塘溶氧昼夜变化对鳜葡萄糖代谢和血糖含量的影响

为探究池塘溶氧昼夜变化对鳜(Siniperca chuatsi)葡萄糖代谢和血糖含量的影响,本研究分别在17:00 (T1)、23:00 (T2)、次日07:00 (T3)、11:00 (T4)、17:00 (T5)共5个连续时间点采样并测定分析,比较了鳜池塘常氧组(>5 mg/L)与昼夜溶氧变化组葡萄糖代谢相关指标的时序差异。结果显示,1 d中,池塘溶氧经历常氧与低氧的周期性变化,T3时,池塘溶氧最低,平均为0.93 mg/L;T5时,池塘溶氧最高,平均为10.58 mg/L。T3和T4时,血浆中葡萄糖显著降低,乳酸含量显著升高;肝脏中肝糖原含量下降,乳酸含量显著升高,ATP含量显著降低;肝脏有氧代谢柠檬酸合酶(CS)活性显著降低,无氧代谢酶乳酸脱氢酶(LDH)活性显著升高;糖酵解酶中己糖激酶(HK)、磷酸果糖激酶(PFK)、丙酮酸激酶(PK)活性显著升高,糖原分解酶糖原磷酸化酶(GP)活性无显著变化;hk、pfk、pk、ldh-a和gp基因表达量显著上升,cs表达量显著下降,葡萄糖转运蛋白glut2表达量无显著变化。研究表明,鳜养殖池塘中,溶氧存在昼夜周期性交替变化,溶氧水平...     

碳酸盐碱胁迫下尼罗罗非鱼氨代谢两种途径时序研究

为了解罗非鱼在碱水环境适应过程中的氨代谢机制,将尼罗罗非鱼(Oreochromis niloticus)放在(2、4、6 g/L)碳酸盐(Na HCO3)碱水环境中进行急性胁迫。检测碱胁迫72 h内的血氨浓度,肝、肾、鳃组织及水体、尿液、血液尿素浓度变化,肝、脑、鳃谷氨酰胺(Gln)浓度,肝、脑谷氨酰胺合成酶(GS)活性,肝氨甲酰磷酸合成酶(CPS)活性,不同组织中GS、CPS、谷氨酰胺酶(GLS)的基因表达变化。结果显示:急性胁迫下尼罗罗非鱼血氨浓度上升,于12 h到达峰值。随着血氨升高,各组织中的尿素浓度0~6 h快速升高,CPS活性0~2 h快速升高,基因相对表达量0~24 h升高,表明尿素代谢途径0-6 h内启动。肝谷氨酰胺浓度0~6 h快速升高到达峰值,肝GS活性0~6 h和12~24 h快速升高,组织中GS、GLS基因相对表达量在0~24 h升高,表明谷氨酰胺代谢途径0~6 h内启动。结果表明,在碱胁迫条件下,尼罗罗非鱼在胁迫早期同时启动尿素代谢途径与谷氨酰胺代谢途径共同参与调节血氨浓度。     

鳜SIRT3蛋白在传染性脾肾坏死病毒感染中的作用

沉默交配型信息调节因子2同源蛋白3 (silent mating type information regulation 2 homolog 3,SIRT3)是一种去乙酰化酶,可调节线粒体氧化代谢.为探讨鳜(Siniperca chuatsi) SIRT3在传染性脾肾坏死病毒(infectious spleen and...     

桑叶水提物对鳜生长、脂质代谢、抗氧化能力和肝脏肠道健康的影响

本研究旨在探究饲料中添加不同浓度桑叶水提物(MLWE)对鳜(Siniperca chuatsi)生长性能、脂质代谢、抗氧化性能、肝脏和肠道组织形态的影响。选择经过驯化后的鳜(42.35±0.07) g 540尾,随机分成4组,每组3个重复,每个重复45尾鱼,在基础配合饲料中分别添加0%(对照组)、0.1%、0.2%和0.5%的MLWE,实验周期为72 d。结果显示:1)与对照组相比,饲料中添加0.1%～0.5%的MLWE不影响鳜的增重率、特定生长率、饲料系数、成活率、脏体指数、肝体指数、肠体指数和肥满度(P>0.05); 2)与对照组相比,饲料中添加0.1%的MLWE能够显著降低鳜血清中总胆固醇(T-CHO)和低密度脂蛋白胆固醇(LDL-C)的含量(P<0.05),添加0.2%的MLWE能够显著降低鳜血清中T-CHO、LDL-C和高密度脂蛋白胆固醇(HDL-C)的含量(P<0.05),添加0.5%的MLWE能够显著降低鳜血清中LDL-C和HDL-C的含量(P<0.05); 3)与对照组相比,在饲料中添加0.1%和0.2%的MLWE能够显著提高鳜血清超氧化物歧化酶...     

Weaning Chinese perch Siniperca chuatsi (Basilewsky) onto artificial diets based upon its specific sensory modality in feeding

Chinese perch are one of the most valuable food fish in China, but the sole source of feed for intensive culture is live prey fish. Our previous studies on systematic sensory physiology revealed that this species have a mechanism for this peculiar feeding habit. In the present study, a specific training procedure was designed, and both experimental (initial body weight 171.0 g; 120 days) and commercial (initial body weight 52.4 g; 240 days) net-cage cultures were conducted to investigate the training success, growth performance and survival of the trained yearlings fed with nonlive or Oregon-type moist diet. The training successes of minced prey fish and the Oregon moist diet were 100 and 89.9%, respectively, in experimental culture, and 92.2 and 83.5% in commercial culture. In an experimental trial, the fish fed minced prey fish or the Oregon moist diet attained final body weights of 472.7 g or 344.7 g, although the specific growth rates of these groups were significantly lower than that of the fish fed live prey fish (final body weight 560.0 g). Mortality was not significantly related to dietary treatment. In commercial culture, the final body weights were as follows: 750 g on live prey fish, 705 g on minced prey fish and 651 g on the Oregon moist diet. Feed costs to produce 1 kg fish were estimated to be US$6.59 for live prey fish, US$1.76 for minced prey fish and US$2.07 for the Oregon moist diet. The results of the present study confirmed that sensory modality and associative learning appear to be critical factors in determining food discrimination of Chinese perch, indicating that both minced trash fish and Oregon-type moist diet can be substituted for live prey fish in intensive commercial production.     

Nucleotide promotes feed intake and protein utilization via regulating the gene expression of feeding and nitrogen metabolism in juvenile Chinese perch (Siniperca chuatsi)

Nucleotide (NT) could enhance growth, feeding and immunity in higher vertebrate and fish. Chinese perch (Siniperca chuatsi) refuse artificial diet in natural water, whereas the NT might promote its feeding of domestication diet. The present study was designed to investigate the effect of dietary NT on growth, feed intake, feed utilization, body composition, serum biochemistry and nitrogen metabolism in juvenile Siniperca chuatsi. 0, 0.5, 1.0, 1.5, 2.0 and 4.0 g NT kg^?1 diet were supplemented in each diet, respectively. Triplicate groups of Chinese perch (36.21 ± 0.98 g) were fed twice a day to apparent satiation for 8 weeks. The results showed that fish fed with 1.5 g NT kg^?1 diet had the highest weight gain (WG), specific growth rate, feed efficiency, feed intake, protein efficiency ratio and protein retention efficiency. The crude protein content in whole body was highest in fish fed the diet containing 1.5 g NT kg^?1 diet. Dietary NT supplementation significantly increased the neuropeptide Y gene expression and significantly decreased the agouti‐related protein and pro‐opiomelanocortin gene expressions in brain. NT supplementation decreased urea nitrogen content, aspartate aminotransferase (AST) and alanine aminotransferase activities in serum. The hepatic AST activity was increased first and then decreased, and the highest activity was observed in fish fed with 1 g NT kg^?1 diet. Inversely, the activity of glutamate dehydrogenase (GDH) in liver and adenosine 5′‐monophosphate deaminase in muscle decreased first and then increased, and the lowest activity was observed in fish fed with 1.5 g NT kg^?1 diet. Similarly, the gene expression levels of GDH and arginase in liver were lowest in fish fed with 1.5 g NT kg^?1 diet. Based on the broken‐line regression analysis of WG in the present experimental condition, 1.44 g NT kg^?1 diet was the optimum supplementation level in juvenile Siniperca chuatsi.     

Impact of environmental and innate factors on the food habit of Chinese perch Siniperca chuatsi (Basilewsky) (Percichthyidae)

Laboratory and field investigations were conducted to study the food habit of Chinese perch Siniperca chuatsi (Basilewsky) from first feeding through adult stage. Only fish larvae were consumed by Chinese perch larvae (2–21 days from hatching), and the presence of zooplankton did not have any significant effect on their survival rate. The ability of Chinese perch to feed on zooplankton is clearly limited by some innate factor. Instead of gill rakers, Chinese perch larvae have well‐developed sharp teeth at the first feeding stage, and are well adapted to the piscivorous feeding habit unique to the larvae of Chinese perch, e.g. they bite and ingest the tails of other fish larvae. At the first feeding stage (2 days from hatching), daily rations were both very low, either in light or complete darkness. Although early‐staged Chinese perch larvae (7–17 days from hatching) could feed in complete darkness, their daily rations were always significantly higher in light than in complete darkness. Late‐staged Chinese perch larvae (21 days from hatching) were able to feed in complete darkness as well as in light, similar to the case of Chinese perch yearlings. Chinese perch yearlings (total length, 14–16 cm) consumed prey fish only and refused shrimp when visual cues were available (in light), but they consumed both prey when visual cues were not available (in complete darkness), suggesting that prey consumption by Chinese perch yearlings is affected by their sensory modality in predation. Both prey were found in the stomachs of similar‐sized Chinese perch (total length, 14–32 cm) from their natural habitat, suggesting that shrimp are consumed by Chinese perch at night. Prey selection of Chinese perch with a length >38 cm, which consumed only fish in the field, appears to be based upon prey size instead of prey type. These results suggest that although environmental factors (e.g. light intensity) affect prey detection by Chinese perch, this fish is anatomically and behaviourally predisposed to prey on live fish from first feeding. This makes it a difficult fish to cultivate using conventional feeds.     

鳜UCP1,2基因结构、序列分析及组织表达

从分子水平上探讨了鳜解偶联蛋白1、2 (UCP1, 2)基因结构、组织表达水平及与产热、脂肪代谢等生理机能的关系。通过与脊椎动物UCP1、UCP2基因序列进行比对,设计简并引物与特异引物进行PCR和RACE扩增、测序、拼接序列,获得UCP1、UCP2的基因组序列和内含子/外显子结构。基因组步行法克隆鳜肝脏UCP1、UCP2 基因5′侧翼序列。应用半定量RT-PCR的方法,以β-肌动蛋白作为外参照,在其指数期增长的范围内得到鳜不同组织UCP1、UCP2的相对表达水平。结果表明,UCP1基因组全序列为3 146 bp, 5′侧翼调控区为1 333 bp,含有5个内含子和6个外显子,开放阅读框(ORF)长942 bp,编码一个大小为313个氨基酸的蛋白质。UCP2基因组全序列为2 890 bp, 5′侧翼调控区为1 800 bp,含有7个内含子和8个外显子,ORF长939 bp,编码一个大小为312个氨基酸的蛋白质。UCP1、UCP2间隔外显子的内含子皆符合“GT-AG”规则,内含子的数目与哺乳动物一致。系统进化分析表明,鳜UCP1、UCP2氨基酸序列分别与鱼类UCP1、UCP2氨基酸序列聚为一支,且与UCP3、UCP4、UCP5分支区分明显。鳜UCP1、UCP2基因不同组织表达水平的高低可能与鳜本身的生态习性及各器官在产热、脂质代谢中的作用相关,但明确的分子机制尚待进一步研究。     

鳜脑神经元的原代培养与鉴定

目前鱼类神经细胞模型极少,物种间的差异性使鳜在细胞水平上的研究具有极大的局限性,为建立一种简单易行的鳜脑神经元细胞模型进一步研究鳜神经系统,本实验取3月龄鳜,联合胶原酶消化法和机械吹打法分离出鳜脑细胞,用含20%FBS的L15培养液制成细胞悬液接种在细胞培养瓶内,于28°C无CO₂培养箱中培养,3 d后更换培养基,待细胞贴壁长满瓶底后进行传代培养;采用Neu-N和β-tubulin免疫荧光细胞化学技术鉴定鳜脑细胞神经元纯度。结果显示,鳜脑细胞分离培养2 d后贴壁状态较好,随着时间延长,贴壁细胞增多,细胞突起相互连接,培养5 d后神经元胞体丰满,细胞数量明显增多,突起相互间形成了明显的神经网络。经Neu-N和β-tubulin免疫荧光细胞化学技术鉴定鳜脑神经元细胞纯度可达95%以上。研究表明,该鳜脑细胞的分离培养方法简单易行,且可获得高纯度的鳜脑神经元细胞。     

Diff-Quik染色法在中华鳖血细胞染色中的应用

采用Diff-Quik染色法对中华鳖的血涂片进行染色,用DP-71显微成像系统采集血片细胞显微图像,经Image-proExpress5.1测量和分析中华鳖各类血细胞大小、比例。结果表明：①Diff-Quik染色后中华鳖各类血细胞核质染色差别明显,核质界面明显,各类白细胞的颗粒明显、易分辨。②中华鳖红细胞、嗜中性粒细胞、嗜酸性粒细胞、嗜碱性粒细胞大小分别为17.86μm×12.43μm、16.26μm×13.67μm、12.28μm×10.98μm、14.63μm×12.83μm。嗜中性粒细胞、嗜酸性粒细胞、单核细胞和淋巴细胞占白细胞百分比分别为54.7、13.72、6.22和24.72,嗜碱性粒细胞仅为0.65。③比较Diff-Quik法、Giemsa法、Wright-Giemsa法对染色效果的影响,3种染色方法在细胞大小及白细胞分类比例上无明显差异。从核质界面、细胞颗粒特征性染色等来看,Diff-Quik法和Giemsa法优于Wright-Giemsa法。Diff-Quik法染色快捷、核质和颗粒染色清晰度更高,是一种适合大量标本快速染色的染色法,可适用于中华鳖的血细胞染色。     

Catechol-O-methyltransferase in the brain of the male African catfish,Clarias gariepinus; distribution and significance for the metabolism of catecholestrogens and dopamine

Catechol-O-methyltransferase, involved in the methylation of catechol substrates, was localized in the brain of the male African catfish,Clarias gariepinus, by means of a radiometric assay using [Methyl-³H]S-adenosylmethionine as methyldonor and catecholestrone as substrate. Fore- and midbrain were divided into eighteen, 500 μm thick, transverse sections. With a hollow needle (diameter 1 mm), specific areas of the brain were punched out and assayed. The catechol-O-methyltransferase activity was calculated from the amount of radioactive methoxyestrone formed from catecholestrone and expressed in pmol.mg⁻¹ tissue.h⁻¹. The enzyme could be demonstrated throughout the brain. Although the enzyme activity did not differ very much between the various brain regions (max. 15.4; min. 7.5 pmol), there were some areas in the brain with a more than average activity,i.e., the lateral telencephalon (10.3 pmol), the nucleus preopticus (13.1 pmol), nucleus lateralis tuberis (11.0 pmol) and nucleus recessus posterioris (12.0 pmol) of the diencephalon, the tectum opticum (15.4 pmol) and torus semicircularis (13.6 pmol) of the mesencephalon, and the caudal cerebellum of the metencephalon (10.8 pmol). The lowest activity was detected in the caudal metencephalon (7.5 pmol). The presence of the enzyme catechol-O-methyltransferase in the brain of the African catfish and the observation that both catecholestrogens and dopamine can be methylated by this enzyme suggest that catecholestrogens can influence the methylation (inactivation) of dopamine. Incubations of forebrain homogenates with dopamine and catecholestrone or catecholestradiol confirmed that both catecholestrogens can inhibit the methylation of dopamine. Lineweaver-burk plots with various concentrations of the catecholestrogens indicated that the inhibition is competitive. Dixon plots from the inhibition studies gave inhibition constants of 1.4 and 0.6 μM for catecholestrone and catecholestradiol, respectively, indicating that catecholestradiol is a two times stronger inhibitor than catecholestrone. The significance of the inhibition of the methylation of dopamine by the catecholestrogens in the brain is discussed in the light of the negative feedback of gonadal steroids on the central regulation of reproductive processes.