红鳍东方鲀(Takifugu rubripes)Vtg基因实时荧光定量PCR分析的引物设计与评估
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摘要
为应用实时荧光定量PCR(qPCR)技术分析红鳍东方鲀(Takifugu rubripes)卵黄蛋白原(Vitellogenin,Vtg)基因的组织表达谱,利用Primer5.0软件设计了扩增红鳍东方鲀Vtg基因片段的引物并进行了qPCR评估。结果显示:引物对VtgF-VtgR在qPCR扩增时,熔解曲线为单一尖锐峰;标准曲线分析显示:引物的扩增效率为105.32%,R~2值为0.999。上述结果说明该对引物具有良好的扩增特异性和扩增效率,满足了qPCR扩增的要求。
引文
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[24] 王效维,刘海霞,杨颖丽,等.低表达H蓝花药发育相关基因小体积荧光定量反应体系的建立和可靠性分析[J].西北农业学报,2011,20(4):111-115.
[25] 沈维祥,陈春峰,张小燕,等.基于突变阻滞扩增系统的实时荧光PCR技术检测幽门螺杆菌23S rRNA基因突变及其评价[J].中国医药生物技术,2017,12(2):123-128.
[2] Yong Y S,Quek L S,Lim E K,et al.A case report of puffer fish poisoning in Singapore[J].Case Reports in Medicine,2013,(2013-12-4),2013,2013(2013):206971.
[3] Narahashi T.Pharmacology of Tetrodotoxin[J].J Toxicol-Toxin Review,2001,20(1):67-84.
[4] Geffeney S L,Ruben P C.The Structural Basis and Functional Consequences of Interactions Between Tetrodotoxin and Voltage-Gated Sodium Channels[J].Marine Drugs,2006,4(3):143-156.
[5] Vaishali B,Mary L,Madhurima D,et al.Tetrodotoxin: Chemistry,Toxicity,Source,Distribution and Detection[J].Toxins,2014,6(2):693-755.
[6] Tamao N,Osamu A .Tetrodotoxin – Distribution and Accumulation in Aquatic Organisms,and Cases of Human Intoxication[J].Marine Drugs,2008,6(2):220-242.
[7] Yamamori K,Kono M,Furukawa K,et al.The toxification of juvenile cultured kusafugu Takifugu niphobles by oral administration of crystalline tetrodotoxin[J].Journal of the Food Hygienic Society of Japan,2004,45(2):73-75.
[8] Kono M,Matsui T,Furukawa K,et al.Accumulation of tetrodotoxin and 4,9-anhydrotetrodotoxin in cultured juvenile kusafugu Fugu niphobles by dietary administration of natural toxic komonfugu Fugu poecilonotus liver[J].Toxicon,2008,51(7):0-1273.
[9] Matsumoto T,Nagashima Y,Kusuhara H,et al.Pharmacokinetics of tetrodotoxin in puffer fish Takifugu rubripes by a single administration technique[J].Toxicon,2008,51(6):0-1059.
[10] Tatsuno R,Shikina M,Shirai Y,et al.Change in the transfer profile of orally administered tetrodotoxin to non-toxic cultured pufferfish Takifugu rubripes depending of its development stage[J].Toxicon,2013,65(2):76-80.
[11] Yin X,Kiriake A,Ohta A,et al.A novel function of vitellogenin subdomain,vWF type D,as a toxin-binding protein in the pufferfish Takifugu pardalis ovary[J].Toxicon Official Journal of the International Society on Toxinology,2017,136.
[12] Ikeda K,Emoto Y,Tatsuno R,et al.Maturation-associated changes in toxicity of the pufferfish Takifugu poecilonotus[J].Toxicon,2010,55(2):289-297.
[13] Romano M,Rosanova P,Anteo C,et al.Vertebrate yolk proteins: A review[J].Molecular Reproduction & Development,2004,69(1):109-116.
[14] Finn R N .Vertebrate yolk complexes and the functional implications of phosvitins and other subdomains in vitellogenins[J].Biology of Reproduction,2007,76(6):926-935.
[15] Ghosh P,Thomas P.Binding of metals to red drum vitellogenin and incorporation into oocytes[J].Marine Environmental Research,1995,39(1–4):165-168.
[16] Smolenaars M M,Madsen O,Rodenburg K W,et al.Molecular diversity and evolution of the large lipid transfer protein superfamily[J].Journal of Lipid Research,2007,48(3):489.
[17] Nicot N,JeanFrancois Hausman,Hoffmann L,et al.Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress[J].Journal of Experimental Botany,2005,56(421):2907-2914.
[18] Jain M,Nijhawan A,Tyagi A K,et al.Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR[J].Biochemical & Biophysical Research Communications,2006,345(2):646-651.
[19] Camarena L,Bruno V,Euskirchen G,et al.Molecular Mechanisms of Ethanol-Induced Pathogenesis Revealed by RNA-Sequencing[J].Plos Pathogens,2010,6(4):1257-1262.
[20] 孙赛红,王玉芳,姜志强,等. 通过性腺形状和颜色鉴定红鳍东方鲀的性别[J].河北渔业,2014,2:13-15.
[21] Takashi Kamiya1, Wataru Kai1, Satoshi Tasumi1, et al. A Trans-Species Missense SNP in Amhr2 Is Associated with Sex Determination in the Tiger Pufferfish, Takifugu rubripes (Fugu) [J]. Plos Genetics,8(7): e1002798.
[22] 刘阳,杨淑霞,李敏惠,等.引物浓度与退火温度不当导致巢式PCR非特异性扩增[J].成都医学院学报,2008,3(2):111-114.
[23] 王香君,李梦茹,段杉.实时荧光定量 PCR 法定量微生物的条件及在鱼露和虾油微生物检测中的应用研究[J].食品与发酵工业,2018,44(2):194-201.
[24] 王效维,刘海霞,杨颖丽,等.低表达H蓝花药发育相关基因小体积荧光定量反应体系的建立和可靠性分析[J].西北农业学报,2011,20(4):111-115.
[25] 沈维祥,陈春峰,张小燕,等.基于突变阻滞扩增系统的实时荧光PCR技术检测幽门螺杆菌23S rRNA基因突变及其评价[J].中国医药生物技术,2017,12(2):123-128.
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