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为建立实现牡蛎疱疹病毒(OsHV-1)在贝类宿主组织中的精确定位方法,基于原位杂交PCR (ISPCR)技术建立了OsHV-1的间接原位杂交PCR (indirect ISPCR)检测方法,并利用该方法对OsHV-1在魁蚶主要器官的分布情况进行了检测和分析。首先选择适合原位杂交PCR的引物,在载玻片上实现对原位固定靶组织内病毒DNA的稳定、特异扩增,然后与使用相同引物制作的地高辛标记核酸探针进行原位杂交,最后通过免疫酶标技术显示样本内OsHV-1的组织分布。为了达到最佳检测效果,对间接ISPCR的扩增循环数以及组织消化的蛋白酶K浓度进行优化。结果显示,最适PCR扩增循环数为20,蛋白酶K浓度为20μg/mL。利用经优化的间接ISPCR检测方法,对OsHV-1在魁蚶外套膜、鳃、肝胰腺、斧足和闭壳肌5个样本中的组织分布情况和亲嗜性进行检测和分析。病毒阳性信号主要分布于魁蚶外套膜结缔组织中浸润的血细胞和成纤维细胞、肝胰腺和鳃浸润的血细胞、斧足和闭壳肌中坏死肌肉细胞的细胞核中。本研究建立的间接ISPCR检测方法具有灵敏、特异和精确定位的优点,通过组织切片上显示的病毒核酸阳性信号,能判别OsHV-1在不同组织部位的分布特点和受感染的细胞类型;适用于OsHV-1感染的确诊、病毒对不同组织器官的亲嗜性、病毒侵染途径和致病机理等相关研究。 相似文献
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为建立牡蛎疱疹病毒魁蚶株快速、灵敏、准确和操作简便的检测方法,实验根据已测序完成的OsHV-1-SB全基因组序列,选择其保守区域,建立了交叉引物等温扩增检测方法,并对反应温度、dNTPs、Mg2+浓度和反应时间进行了优化.结果显示,最佳温度为63℃,反应时间为60 min,dNTPs浓度为1.4 mmol/L,Mg2+浓度为6 mmol/L.该方法检测灵敏度为30拷贝的质粒DNA,并且特异性较强,与贝类常见病原急性病毒性坏死病毒、鲍鱼疱疹病毒、派琴虫、包纳米虫、马尔泰虫以及对虾白斑综合征病毒和副溶血弧菌均无交叉反应.使用实验建立的CPA检测方法对2012年分别采自韩国庆尚南道、山东长岛、辽宁大连共计22份OsHV-1-SB感染情况未知的魁蚶样品进行了检测.研究表明,实验所建立的OsHV-1-SB的CPA检测方法简单、快速、灵敏且特异性强.由于其检测结果可通过简单离心或者向反应管中加入核酸荧光染料GeneFinderTM进行肉眼观察,所以可以在沿海贝类养殖厂及条件简陋的实验室使用,具有较好的应用前景. 相似文献
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牡蛎疱疹病毒对魁蚶的致病性 总被引：1，自引：1，他引：0
为探明牡蛎疱疹病毒(Os HV-1)对魁蚶的致病性,本研究使用发病魁蚶组织制作病毒悬液进行感染实验。感染实验分为空白组、阴性悬液注射组和病毒悬液注射组,并使用实时定量PCR法对感染后魁蚶体内病毒的时空分布进行检测。实验结果显示,空白组和阴性悬液组魁蚶感染后未检测到病毒粒子,病毒悬液注射组魁蚶经人工感染后,各部位病毒含量均呈先上升再下降随后又上升的趋势,最终达到106拷贝/ng DNA左右。通过电镜观察,在感染魁蚶的鳃、肝胰腺、外套膜中出现染色质边缘化甚至消失,细胞核肿胀、溶解,核仁消失,核膜扩张、不清晰,线粒体肿大,脊崩解,核糖体脱落等一系列细胞病理变化。在其细胞核和细胞质中均能发现大量直径为90~110 nm球形病毒粒子,该病毒粒子具囊膜,囊膜内可见均匀高电子密度的核衣壳,与自然发病魁蚶负染电镜中的病毒粒子形态相同。研究结果表明,Os HV-1可以感染魁蚶并与魁蚶大规模死亡有直接相关关系;魁蚶感染Os HV-1后机体产生应激反应,对Os HV-1有一定抑制作用,但其作用机制还有待进一步实验验证。 相似文献
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通过cDNA末端快速扩增(Rapid amplification of c DNA ends,RACE)技术克隆得到魁蚶(Scapharca broughtonii)C型凝集素(C-type lectin,Sb-Lec1)基因,该基因全长为700 bp,其中,5′-UTR为29 bp,3′-UTR为167 bp,开放阅读框长度为504 bp,编码167个氨基酸,包括长度为23个氨基酸的信号肽序列、129个氨基酸的糖识别结构域(CRD)以及参与二硫键形成的6个半胱氨酸。预测蛋白分子量为19.11 k Da,理论等电点为4.74。多序列比对结果显示,Sb-Lec1基因CRD编码的氨基酸序列与长牡蛎(Crassostrea gigas)、紫贻贝(Mytilus galloprovincialis)和海湾扇贝(Argopecten irradians)C型凝集素的同源性分别为38%~40%、34%~35%和38%~39%,Sb-Lec1基因编码的氨基酸序列与其他物种的凝集素基因具有相似的结构,均含有形成二硫键的4个保守半胱氨酸。系统进化分析结果显示,魁蚶先与贝类聚为一支,再与脊椎动物聚在一起,表明魁蚶Sb-Lec1基因在进化树上的位置与其传统分类所处位置一致。采用荧光定量PCR技术,检测了Sb-Lec1基因在组织中的表达情况,发现其在肝胰腺、血淋巴、鳃、外套膜、闭壳肌、斧足中均有表达,其中,肝胰腺表达量最高。同时,分析了Sb-Lec1基因在鳗弧菌(Vibrio anguillarum)刺激下的mRNA表达量变化情况。结果显示,与对照组相比,菌刺激组Sb-Lec1基因mRNA在各检测组织中的表达量均显著上调(P0.05),随着刺激时间的延长,表达量呈先升高后降低的趋势。本研究表明,魁蚶Sb-Lec1基因在机体免疫防御方面发挥重要功能。 相似文献
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牡蛎疱疹病毒(Ostreid herpesvirus 1, OsHV-1)给世界双壳贝类养殖业造成了严重的经济损失。10余种双壳贝类陆续被认定为易感宿主,仍有其他几种贝类仅有PCR核酸阳性数据,因确诊证据不足导致其易感性未得到充分评估。原位环介导等温核酸扩增(LAMP)检测技术相对传统原位杂交技术具有灵敏度高、方便快捷、可作为病原微生物感染证据的优点。为了在OsHV-1流行病学调查过程中实现病毒感染的快速检测和确诊,根据已报道的OsHV-1特异性LAMP检测引物,设计内引物,优化反应条件,建立了OsHV-1的原位LAMP检测方法。基于该方法对2019年以来采集的长牡蛎(Crassostrea gigas)、福建牡蛎(Crassostrea angulata)、栉孔扇贝(Chlamys farreri)、虾夷扇贝(Mizuhopecten yessoensis)、毛蚶(Scapharca subcrenata)和菲律宾蛤仔(Ruditapes philippinarum)样本进行检测。结果显示,毛蚶样本的OsHV-1原位LAMP检测结果呈阳性;其他几种贝类部分样本的实时定量PCR (qPCR)检测呈阳性,但原位LAMP检测呈阴性。对毛蚶样本的原位LAMP检测结果分析发现,病毒杂交信号主要分布在外套膜和肝胰腺等器官的结缔组织,推测感染的细胞为成纤维细胞和血淋巴细胞;在闭壳肌和斧足肌肉组织的肌细胞细胞核中也发现较多杂交信号。鳃丝内和周边偶现阳性信号,推测来自渗出的血淋巴细胞。基于原位LAMP技术的OsHV-1检测结果显示,毛蚶是OsHV-1的一种易感宿主,毛蚶结缔组织、肌肉组织和血淋巴细胞对该病毒有强亲嗜性。 相似文献
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本研究对编码牡蛎疱疹病毒(Ostreid herpesvirus 1, OsHV-1)囊膜蛋白的orf111基因进行了生物信息学分析、克隆和表达。首先,通过特异性PCR扩增得到orf111基因全长序列,并对其编码囊膜蛋白的理化性质、高级结构、跨膜区和抗原决定簇等进行生物信息学分析。结果显示,orf111编码一种稳定的疏水性蛋白,具有5个跨膜结构域和9个抗原决定簇,同时,氨基酸序列中还包含1个高度保守的精氨酰–甘氨酰–天冬氨酸(Arg-Gly-Asp, RGD)结构域。随后,构建了pET28a-orf111重组质粒,并将其转化到大肠杆菌DH5α感受态细胞中。最后,通过使用异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白表达,表达产物分子量约为32 kDa。本研究应用原核表达得到了含RGD结构域的OsHV-1囊膜蛋白,为进一步制备ORF111蛋白单克隆抗体及开展牡蛎疱疹病毒侵染机制的研究奠定了重要基础,为将来OsHV-1的防控工作提供新的思路。 相似文献
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魁蚶(Scapharca broughtonii)的血淋巴中因含有血红蛋白而不同于其他多数双壳贝类。为明确魁蚶血红蛋白基因的结构特征、组织发生分布、免疫活性等特点,本研究以魁蚶转录组数据库中的部分序列为基础,通过cDNA末端快速扩增(Rapid amplication of cDNA ends, RACE)技术,克隆获得了一种魁蚶血红蛋白基因cDNA全长序列(命名为SbHbⅠ),并研究了不同条件下的mRNA表达。序列和结构分析显示,SbHbⅠ基因cDNA全长为867 bp,其中包括长度为483 bp的开放阅读框(Open reading frame, ORF),编码160个氨基酸,预测蛋白分子量为17.5 kDa,理论等电点为9.68;氨基酸序列具有多个血红素结合位点,序列与所选其他物种的相似度范围为57%~93%,与蚶科相似度较高。实时荧光定量PCR (qRT-PCR)结果显示,在魁蚶成体的斧足、鳃、外套膜、闭壳肌、血淋巴、肝胰腺及受精卵至发育23 d的幼虫体内,均能检测到SbHbⅠ基因;该基因在成体魁蚶血淋巴中表达量最高,与其他5个组织中的表达量差异显著(P<0.05);受精卵至23 d幼虫期的表达,基本呈现为先下降后上升的趋势,且在12 d时急剧上升(P<0.05);在鳗弧菌(Vibrio anguillarum)刺激和低氧胁迫后,血淋巴中SbHbⅠ基因的表达均呈先上升后下降的趋势,表达量最高值均出现在 16 h。本研究丰富了贝类血红蛋白相关研究资料。 相似文献
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魁蚶(Scapharca broughtonii)的血淋巴中因含有血红蛋白而不同于其他多数双壳贝类。为明确魁蚶血红蛋白基因的结构特征、组织发生分布、免疫活性等特点,本研究以魁蚶转录组数据库中的部分序列为基础,通过cDNA末端快速扩增(Rapid amplication of cDNA ends, RACE)技术,克隆获得了一种魁蚶血红蛋白基因cDNA全长序列(命名为SbHbⅠ),并研究了不同条件下的m RNA表达。序列和结构分析显示,SbHbⅠ基因cDNA全长为867 bp,其中包括长度为483 bp的开放阅读框(Open reading frame, ORF),编码160个氨基酸,预测蛋白分子量为17.5 kDa,理论等电点为9.68;氨基酸序列具有多个血红素结合位点,序列与所选其他物种的相似度范围为57%～93%,与蚶科相似度较高。实时荧光定量PCR (qRT-PCR)结果显示,在魁蚶成体的斧足、鳃、外套膜、闭壳肌、血淋巴、肝胰腺及受精卵至发育23 d的幼虫体内,均能检测到SbHbⅠ基因;该基因在成体魁蚶血淋巴中表达量最高,与其他5个组织中的表达量差异显著(P0.05);受精卵至23 d幼虫期的表达,基本呈现为先下降后上升的趋势,且在12d时急剧上升(P0.05);在鳗弧菌(Vibrioanguillarum)刺激和低氧胁迫后,血淋巴中SbHbⅠ基因的表达均呈先上升后下降的趋势,表达量最高值均出现在16 h。本研究丰富了贝类血红蛋白相关研究资料。 相似文献
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An analysis of the food of 274 specimens of Tilapia aurea (Steindachner), which had been collected in Lake Kinneret, showed that this species is mainly a zooplankton consumer.A study of the quantitative dynamics of the food components in the course of a year has proved that the species in question feeds more intensely in spring, this being the time when zooplankton forms are more abundant in the water of the lake. Vegetable detritus, mixed with plankton and benthos forms, served as additional and alternative food.The lack of clearly positive degree of food component selectivity, as well as the low values of the intestine repletion index and of the condition coefficient, account for the slowness in growth of Tilapia aurea in Lake Kinneret.The low values of the growth and feeding indices are due, according to the authors, to the pressure brought to bear by the other local fish species, which are competitors for the same food. 相似文献
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Pepietta Spataru 《Aquaculture (Amsterdam, Netherlands)》1978,14(4):327-338
Analysis of the gut content of 329 specimens of T. zillii (Cichlidae) collected from Lake Kinneret, has shown great variation in the sorts of food.A study of seasonal dynamics has proved the prevalence in the food of Chironomida pupae (Diptera) in winter and spring and of zooplankton forms in summer and autumn. The additional food consisted of the various groups of algae, the most frequently found being Cyanophyta (100%) and Pyrrophyta (64.16%). An extremely voraceous species, it consumes — while searching for its preferred food — anything that comes its way in the water: algae, scraps of macrophytes, autochthonous and allochthonous insects, and forms of benthic origin, such as Nematoda, Ostracoda, Porifera and Chironomida (larvae).The satiation index is high (between 4.08 and 5.63), in contradiction to the low values of the coefficient of condition (between 3.05 and 3.51), and with the slow rate of growth in Lake Kinneret. The main food of Tilapia zillii, consists of arthropod species with a chitin content (which is eliminated unchanged) of more than 50% of the total weight, and this may account for the poor exploitation of the trophic base.This species of fish may be considered as being detrimental to others (i.e. commercially important species) because of its successful competition for food, and not, as is often incorrectly assumed, because it is an aggressive consumer of their spawn and fry. 相似文献
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Moshe Gophen 《Aquaculture (Amsterdam, Netherlands)》1979,16(1):67-71
Daphnia lumholtzi (Sars) was found in Lake Kinneret until the late nineteen-fifties. Lake Kinneret was the northern-most limit of the distribution of this species. It is suggested that fingerlings of grey mullets and Sarotherodon aureus that were introduced into Lake Kinneret caused the extinction of D. lumholtzi. 相似文献
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In this study, the embryonic development of the eggs in the Mastacembelus mastacembelus (Bank & Solender, 1794) was examined. At the same time, possibilities of artificial breeding through artificial insemination were investigated. Artificial insemination was achieved by mixing the eggs of the mature female and sperm of the mature male samples caught with gill nets (22 × 22) in Ataturk Dam Lake in Turkey. To this end, first in a Petri dish (100 × 20), the testes were cut into small pieces with a lancet and the mixture of sperm–testes‐tissue was obtained. The fertilization rate of the eggs was found to be 80%. The diameter of the eggs ranged from 2.015to 1.147 mm. The perivitelline space formed 0.5 h after insemination. The first cleavage occurred at the animal pool 4 h after insemination. The oil droplets had fused to a single droplet 19 h after insemination. The blastoderm became an embryonic shield 30 h after insemination. The blastoderm covered almost half the egg 40 h after insemination and embryonic body was formed. The blastoderm covered almost the whole egg 50 h after insemination. Some somites were discernible 59 h after insemination. The embryonic body reached two‐third of the circumference of the egg 70 h after insemination. The tail bud began to separate from the yolk 77 h after insemination. A newly hatched larva was observed at 85 h after insemination. 相似文献
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Substitution effect of sea tangle (ST) (Laminaria japonica) with tunic of sea squirt (SS) (Halocynthia roretzi) in diet on growth and carcass composition of juvenile abalone (Haliotis discus,Reeve 1846) 
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下载免费PDF全文 B. Jang P.Y. Kim H.S. Kim K.W. Lee H.J. Kim D.G. Choi S.H. Cho B. Min K. Kim H. Han 《Aquaculture Nutrition》2018,24(1):586-593
Substitution effect of sea tangle (ST) with tunic of sea squirt (SS) in diet on growth and carcass composition of juvenile abalone was determined. One thousand four hundred and seventy abalones were distributed into 21 containers. Six formulated diets in triplicate were prepared. A 200 g/kg ST was included into the ST0 diet. The 200, 400, 600, 800 and 1000 g/kg of ST were substituted with the same amount of tunic of SS, referred to as the ST200, ST400, ST600, ST800 and ST1000 diets, respectively. Finally, Undaria was prepared to compare effect of the formulated diets on performance of abalone. The experimental diets were fed to abalone for 16 weeks. Weight gain of abalone fed the ST400 diet was higher than that of abalone fed the ST0, ST600, ST800 and ST1000 diets and Undaria. Weight gain of abalone fed the formulated diets was higher than that of abalone fed the Undaria. The chemical composition of the carcass of abalone was affected by dietary substitution of ST with tunic of SS. In conclusion, ST could be completely substituted with tunic of SS without retardation in performance of abalone. Abalone fed the ST400 diet substituting 400 g/kg ST with tunic of SS achieved the best growth. 相似文献
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康拉克湖区叶尔羌高原鳅繁殖特性的初步研究 总被引：1，自引：0，他引：1
试验研究了康拉克湖区叶尔羌高原鳅的繁殖特性,包括雌、雄鱼的外部特征和内部构造及繁殖习性。雌雄鱼从体型、胸鳍形态上均有所不同;卵巢为单一囊状,以1根输卵管通到泄殖孔;精巢分为左右两叶,表面光滑无皱褶;成熟卵的颜色为浅黄色,粘性,沉性卵。生殖高峰期在5月中旬到6月底。成熟雌鱼性成熟系数高,平均35.23,最高达67.83。回归分析显示,繁殖群体全长与体质量呈极显著的幂函数关系;绝对繁殖力与全长、体重呈正相关;相对繁殖力、成熟系数与全长和体质量不存在显著的函数关系。 相似文献
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Rayette Souza da Silva Juliana Ralica Tavares Lopes Roberto Vilhena do Espírito Santo Marcos Antnio Souza dos Santos Carlos Alberto Martins Cordeiro Eliane Tie Oba Yoshioka Roselany Corrêa Heitor Martins Júnior Jos Brito de Loureno Júnior 《Aquaculture Research》2020,51(8):3358-3366
Two experiments were carried out to evaluate the effects of palm kernel meal (Elaeis guineensis) as corn (Zea mays) substitutes in tambaqui (Colossoma macropomum) diets based on zootechnical performance, health, characteristics of the muscle, yield and economic efficiency. The first one was carried out in a recirculation aquaculture system for 75 days wherein tambaquis received diets with 0% (T0), 25% (T25), 50% (T50), 75% (T75) and 100% (T100) of corn replacement by palm kernel meal. The second experiment, conducted in ponds for 184 days, tambaquis were fed T0 and T25 diets (selected because showed greater weight gain without health damage). Final weight and weight gain of tambaquis were impaired by the increasing levels of this ingredient on diets, and the feed conversion ratio was worse. The protein efficiency ratio and specific growth rate gradually reduced, while the total number of leucocytes and neutrophils gradually increased. Cholesterol and triglycerides decreased in T75 and T100. Variables analysed in experiment 2 were not affected by treatment, except muscle lipids. Palm kernel meal can replace up to 25% of corn in tambaqui diets since it maintains a balance in economic and zootechnical performance of production. 相似文献
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建立同时检测鲫(Carassuys auratus Linnaeus)血浆和肌肉中邻苯二甲酸二甲酯(DMP)和邻苯二甲酸二乙酯(DEP)的高效液相色谱方法,进行初步的代谢动力学研究.血浆样品经液-液萃取,肌肉样品经液-液萃取和固相小柱处理后,以乙腈:水= 40:60(V/V)为流动相,流速1.0 mL/min,采用SHIMADZU ODS柱(150 mm×4.6 mm; i.d., 5 μm)色谱柱分离.测定鲫经口染毒后血浆和肌肉中DMP和DEP的浓度.结果表明,标准曲线线性范围为0.05～10.0 μg/mL,线性关系良好(r>0.99),定量限为0.05 μg/mL,回收率95%以上,日内变异系数和日间变异系数均低于15%.经口染毒48 h内,鲫血浆和肌肉中均能检测到DMP和DEP的残留.该法操作简便、快速、灵敏、准确,已经初步用于鲫血浆和肌肉中DMP和DEP的代谢动力学和残留研究. 相似文献