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1.
Metacaspase是在原生动物、真菌和植物中发现的一种具有底物精氨酸/赖氨酸特异性的半胱氨酸蛋白酶。根据蛋白质结构特征可将metcaspase分为typeⅠ与typeⅡ两种类型,均参与调控多种植物与原生动物程序性细胞死亡。本研究根据GenBank数据库莱茵衣藻(Chlamydomonas reinhardtii) type Ⅰ metacaspase基因(GenBank No. XM_001696904)序列,采用巢式PCR克隆获取type Ⅰmetcaspase基因开放阅读框(ORF)序列并命名为CrMC1。CrMC1 ORF全长987 bp,推测编码1个包含405个氨基酸的蛋白质。通过与已知物种type Ⅰ metacaspase氨基酸序列进行同源序列比对发现,CrMC1具有高度保守的p20、p10、连接区结构域以及精氨酸、半胱氨酸活性中心位点。研究显示,在H_2O_2诱导的莱茵衣藻程序性细胞死亡过程中,CrMC1表达量显著提高(P0.05),2 h后达到峰值,3 h时下降至对照组同一水平。结果表明,莱茵衣藻typeⅠ metacaspase基因CrMC1参与H_2O_2诱导的莱茵衣藻程序性细胞死亡调控。 相似文献
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Metacaspase是在原生动物、真菌和植物中发现的一种具有底物精氨酸/赖氨酸特异性的半胱氨酸蛋白酶。根据蛋白质结构特征可将metcaspase分为typeⅠ与typeⅡ两种类型,均参与调控多种植物与原生动物程序性细胞死亡。本研究根据GenBank数据库莱茵衣藻(Chlamydomonas reinhardtii) type I metacaspase基因(GenBank No. XM_001696904)序列,采用巢式PCR克隆获取type I metcaspase基因开放阅读框(ORF)序列并命名为CrMC1。CrMC1 ORF全长987 bp,推测编码1个包含405个氨基酸的蛋白质。通过与已知物种type I metacaspase氨基酸序列进行同源序列比对发现,CrMC1具有高度保守的p20、p10、连接区结构域以及精氨酸、半胱氨酸活性中心位点。研究显示,在H2O2诱导的莱茵衣藻程序性细胞死亡过程中,CrMC1表达量显著提高(P<0.05),2 h后达到峰值,3 h时下降至对照组同一水平。结果表明,莱茵衣藻type I metacaspase基因CrMC1参与H2O2诱导的莱茵衣藻程序性细胞死亡调控。 相似文献
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缺刻缘绿藻ω3脂肪酸去饱和酶基因的特性及在氮饥饿过程中相对转录量的分析 总被引：2，自引：1，他引：1
根据莱茵衣藻和普通小球藻ω3脂肪酸去饱和酶氨基酸序列(GenBank登录号分别为ABL09485和BAB78717)的保守区(TMFWALF和HHDIGTH)设计简并引物,以缺刻缘绿藻H4301总RNA为模板,通过反转录PCR和cDNA末端快速克隆技术(RACE),克隆了缺刻缘绿藻ω3脂肪酸去饱和酶基因的cDNA全长序列(GenBank登录号:EU658930),它与莱茵衣藻的这个基因具有69%相似性。该基因cDNA序列长2330bp,其中5'-非翻译区长107bp;3'-非翻译区912bp,且具有明显的poly(A)尾巴;开放阅读框1311bp,编码一个436个氨基酸的蛋白质,分子量为49.06ku,等电点7.85;该基因编码的蛋白为膜结合蛋白,含有2个疏水区域和3个保守的组氨酸簇基序。将该基因的cDNA序列与其DNA序列比对后,发现该基因在其编码区存在6个内含子,剪切位点均符合GT-AG规则。实时荧光定量PCR结果显示,在氮饥饿培养过程中,缺刻缘绿藻ω3脂肪酸去饱和酶基因的相对转录量在4h时可能因休克显著提高(P<0.05),然后开始下调,12h开始显著下降(P<0.05),并在20h降到最低(约为完全培... 相似文献
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为进一步研究刺参甘露糖结合凝集素(AJ-MBL)的功能及提高刺参自身免疫力,对AJ-MBL基因的CDS区进行原核表达、纯化并初步鉴定其生物学活性。采用RT-PCR法扩增AJ-MBL基因编码区,经酶切鉴定、序列分析后,将其CDS区498 bp片段克隆至原核表达载体pET28a中,得到重组质粒pET-AJ-MBL。重组质粒转化大肠杆菌BL21(DE3)感受态细胞并经IPTG诱导表达出分子量约17 ku的融合蛋白。该蛋白以包涵体形式存在,用Ni2+离子亲和柱纯化。结果显示,获得高表达量的重组纯化蛋白。纯化的17 ku AJ-MBL进行红细胞凝集试验检测其生物学活性。凝集试验结果显示,AJ-MBL凝集最小浓度为10 μg/mL。本实验成功构建了AJ-MBL原核表达质粒,并在大肠杆菌中高效表达了17 ku AJ-MBL蛋白,该蛋白具有良好的生物学活性。 相似文献
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为发掘适用于基因工程抗病育种的鱼类启动子,通过实时荧光定量PCR实验对鲤Rab GTP酶(Ras-associated binding-GTPases 1a3,Rab1a3)基因的表达模式进行了分析,证实该基因在鳃、头肾等与机体免疫防御功能密切相关的组织内转录水平较高,且免疫激活后转录显著增强,符合基因工程抗病育种所需的外源免疫基因转录模式。从鲤细菌人工染色体文库中,使用Rab1a3基因特异引物筛选获得包含该基因区域的文库克隆,测序获得该基因完整序列,以及上下游调控序列。通过生物信息学手段,预测到长度为1014 bp的鲤Rab1a3基因启动子序列,该启动子不具有典型的TATA盒或CpG岛特征,存在多个免疫相关转录因子结合位点。在草鱼肾组织细胞系内验证该启动子活性,结果显示,绿色荧光蛋白基因和萤火虫荧光素酶基因都能够在该启动子驱动下表达,证实该片段具有启动子活性,且启动子活性在受到免疫诱导后增强,双荧光素酶报告基因检测结果显示,该启动子活性在免疫刺激后增强至免疫刺激前的8.67倍。研究表明,鲤Rab1a3基因启动子有望被开发成为免疫诱导型的基因工程元件,驱动外源免疫基因在鱼体内适时表达,抵御外界病原感染,同时避免非必要条件下的过度表达形成生长负担。 相似文献
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根据报道的PM70菌株的PlpB基因序列,设计PlpB基因一对寡核苷酸引物。以C48-1菌株为实验材料,成功扩增PlpB基因的全序列,该片段共831bp。将扩增基因插入原核表达载体pGEX-KG,成功构建了重组表达质粒pKG-PlpB,并对pKG-PlpB重组表达质粒进行序列测定,结果表明:扩增序列与GenBank上所报道的PM70株的PlpB基因序列核苷酸同源性在99%以上。将该重组质粒转化大肠杆菌BL21(DE3)感受态细胞,经诱导获得大小约56KDa的表达蛋白。Western blot检测表明,该表达蛋白具有良好的反应原性。 相似文献
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为研究草鱼抗菌肽NK-lysin基因的组成结构、在组织中的表达差异及其抑菌活性,实验根据已知鱼类NK-lysin基因保守区序列设计上下游引物,采用RT-PCR和RACEPCR技术克隆草鱼NK-lysin基因(Cinkl),RT-PCR进行各组织间的表达分析,并构建Cinkl成熟肽的原核表达载体,采用琼脂糖孔穴扩散法检测重组蛋白的抑菌活性。结果显示,Cinkl c DNA全长768 bp,编码121个氨基酸;基因组DNA全长3 361 bp,包含4个外显子和3个内含子。经多序列比对分析和结构域预测表明,Ci Nkl氨基酸序列具有SAPLIP家族的特征:含有6个保守的半胱氨酸和saposin B蛋白结构域,与斑马鱼Nklc、Nkld的相似性分别为57.98%和63.03%。构建NK-lysin氨基酸序列系统进化树,发现Ci Nkl与斑马鱼Nklc、Nkld聚为一支。RT-PCR检测结果显示,Cinkl m RNA在所有检测的草鱼组织中均有表达,在脾脏中表达量最高,心脏、鳃、中肾和头肾的表达量次之,皮肤、脑、肝脏和肠有微量表达。p ET-28b-MBP与Ci Nkl成熟肽序列构建的原核表达质粒在宿主菌Rosetta(DE3)表达出约57 ku的可溶性融合蛋白,利用Ni-NTA His·Bind树脂进行纯化,Ci Nkl重组蛋白对G–细菌如大肠杆菌M15株、嗜水气单胞菌,以及G+细菌如金黄色葡萄球菌均具有抑菌活性。基于草鱼NK-lysin的组织表达特征和重组蛋白的抑菌活性,推测NK-lysin在草鱼的先天性免疫防御中发挥调节作用。 相似文献
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MicroRNA(miRNA)是一类内源性的非编码RNA,参与调节病毒复制、细胞增殖或凋亡以及肿瘤发生等生物反应。本研究探讨了miR-130c-5p在乌鳢水泡病毒(snakehead vesiculovirus, SHVV)感染中潜在靶基因g的靶向关系以及对病毒复制的影响。本研究以斑点叉尾鮰卵巢(Channel catfish ovary, CCO)为实验材料,通过qRT-PCR和Western blot测定SHVV不同感染时间和感染剂量条件下,病毒基因水平和蛋白水平以及miR-130c-5p变化情况。此外,将SHVV的g基因上miR-130c-5p对应的靶序列克隆到质粒pmirGLO,构建质粒pmirGLO-G用于双荧光素酶报告实验进行靶基因验证。结果显示,随着SHVV感染时间及剂量的不断增加,miR-130c-5p和g基因的表达水平都显著上调。进一步实验证明,miR-130c-5p类似物和pmirGLO-G质粒共转染可显著抑制荧光素酶活性强度,而转染miR-130c-5p抑制剂则明显上调了pmirGLO-G报告载体的荧光信号。此外,miR-130c-5p的过表达显著降低了病毒G基因的mRNA及蛋白表达,而抑制miR-130c-5p的表达则上调了g基因的mRNA及蛋白的表达水平。研究结果表明,miR-130c-5p通过靶向SHVV的g基因,引起G蛋白的降解,从而抑制SHVV的增殖。本研究结果为理解microRNA调控SHVV的致病机制提供了重要基础,为抗SHVV疫苗等药物的研发提供了理论支持。 相似文献
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中国明对虾抗菌肽基因应答WSSV侵染的表达及其SNP分析 总被引：1，自引：1，他引：0
通过白斑综合征病毒(WSSV)感染实验,利用实时定量PCR技术研究了中国明对虾(Fenneropenaeus chinensis)应答病毒侵染后,已知的3种抗菌肽(对虾肽)在肝胰腺、肌肉、肠和鳃4种组织中的差异表达情况.结果显示,虽然3种抗菌肽表现出明显的组织表达特异性,即在不同组织中的表达趋势和表达丰富度存在明显的差异,但是就同一个组织而言,3种抗菌肽在1～120 h WSSV侵染区间内的表达趋势基本一致,在0 h(未侵染病毒)时,3种抗菌肽的表达量极低(为0);在6～24 h期间,检测到明显的表达量;48～120 h期间,3种抗菌肽的表达量总体呈现下降的趋势.这暗示3种抗菌肽在对虾机体内可能具有相似的生物学功能.在此基础上,本研究对各类型中国明对虾抗菌肽的SNP位点进行了筛选,进一步对不同SNP类型与抗WSSV或易感WSSV的关联程度进行了分析,结果显示3种抗菌肽基因的SNP位点很少,且在抗性和易感对虾群体内不存在明显的偏向分布. 相似文献
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Association between the interannual variation in the oceanic environment and catch rates of bigeye tuna (Thunnus obesus) in the Atlantic Ocean 
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下载免费PDF全文 The environmental processes associated with variability in the catch rates of bigeye tuna in the Atlantic Ocean are largely unexplored. This study used generalized additive models (GAMs) fitted to Taiwanese longline fishery data from 1990 to 2009 and investigated the association between environmental variables and catch rates to identify the processes influencing bigeye tuna distribution in the Atlantic Ocean. The present findings reveal that the year (temporal factor), latitude and longitude (spatial factors), and major regular longline target species of albacore catches are significant for the standardization of bigeye tuna catch rates in the Atlantic Ocean. The standardized catch rates and distribution of bigeye tuna were found to be related to environmental and climatic variation. The model selection processes showed that the selected GAMs explained 70% of the cumulative deviance in the entire Atlantic Ocean. Regarding environmental factors, the depth of the 20 degree isotherm (D20) substantially contributed to the explained deviance; other important factors were sea surface temperature (SST) and sea surface height deviation (SSHD). The potential fishing grounds were observed with SSTs of 22–28°C, a D20 shallower than 150 m and negative SSHDs in the Atlantic Ocean. The higher predicted catch rates were increased in the positive northern tropical Atlantic and negative North Atlantic Oscillation events with a higher SST and shallow D20, suggesting that climatic oscillations affect the population abundance and distribution of bigeye tuna. 相似文献
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In this experiment, a feeding trial was performed to determine the effects of fructooligosaccharide (FOS) on growth performance, digestive enzyme activity and immune response of Japanese sea bass, Lateolabrax japonicus juveniles (initial weight 38.3 ± 0.5 g), and the fish were examined following feeding with six levels of FOS (0, 0.5, 1, 2, 4 and 6 g/kg) for 28 days. Significant enhancement of weight gain (WG) and specific growth rate (SGR) was found in fish fed 1 g/kg FOS incorporated diets (p < .05), while the feed conversion ratio (FCR) in the 1, 2 g/kg FOS groups reduced significantly compared with the control (p < .05). Besides, the crude lipid in the 4, 6 g/kg FOS groups increased significantly compared with the control (p < .05). On the other hand, the erepsin and lipase activities significantly elevated in intestine of fish fed 2 g/kg FOS (p < .05) and the lysozyme activity in serum of fish fed 2 g/kg FOS were significantly higher than that in the control (p < .05). Moreover, the alkaline phosphatase activities in serum of fish fed 0.5, 1, 2 g/kg FOS were significantly higher than in control (p < .05). Regression analysis showed that the relationships between dietary FOS levels and either SGR, FCR, erepsin or lysozyme activities were best expressed by regression equations, and the optimal inclusion levels are 1.37, 1.80, 3.06, 3.11, 1.93 and 1.80 g/kg for SGR, FCR, erepsin, lipase, lysozyme and total superoxide dismutase activities, respectively. Overall, this study revealed that FOS incorporated diets could beneficial for L. japonicus culture in terms of increasing the growth, digestion and immune activities. Under the present experimental condition, the optimal supplementary level of FOS in the diet of L. japonicus is 1–3 g/kg. 相似文献
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Liping Li Rui Wang Wanwen Liang Xi Gan Ting Huang Yan Huang Jian Li Yunliang Shi Honglin Luo Ming Chen 《广西水产科技》2014,(1)
Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China. 相似文献
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Iciar Martinez Amaya Velasco Ricardo Pérez-Martín Eskil Forås Michiaki Yamashita Carmen G. Sotelo 《Journal Of Aquatic Food Product Technology》2013,22(4):289-297
The purpose of this work was to evaluate the suitability of a method based on the presence of two restriction sites (for Hae III and Hindf I) in the mitochondrial NADH dehydrogenase subunit 5 (mt ND5) gene to identify Scomber species. The evaluation was performed on 144 reference and market samples by sequencing of the entire 505-bp fragment of the mt ND5 gene and of a 464-bp fragment of the Kocher fragment of the cytochrome b gene (mt Cytb). Sequence analysis of any of the two fragments allows the identification of each of the four Scomber species, but S. japonicus and S. colias had the same restriction sites at the ND5 amplicon and would not have been differentiated by this analysis. Similarly, loss of the Hae III site in some S. scombrus individuals would have misidentified them as not being Scomber. All the market products were correctly labeled except one acquired in Spain labeled as originating in the Atlantic and containing S. japonicus. 相似文献
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Growth hormone (GH) and reproduction: a review 总被引：5，自引：0，他引：5
Florence Le Gac Odile Blaise Alex Fostier Pierre-Yves Le Bail Maurice Loir Brigitte Mourot Claudine Weil 《Fish physiology and biochemistry》1993,11(1-6):219-232
Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations. 相似文献
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Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL?1 (1.25 ng well?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation. 相似文献
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Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection 下载免费PDF全文
下载免费PDF全文 G Sharon M Pimenta‐Leibowitz M C L Vilchis N Isakov D Zilberg 《Journal of fish diseases》2015,38(1):67-74
Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality). 相似文献
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