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本研究以河川沙塘鳢(Odontobutis potamophila)为研究对象,采用RACE、实时荧光定量PCR等技术,首次对该鱼的TLR2基因进行了克隆和表达模式分析。主要研究结果如下:TLR2基因的c DNA全长序列为3 473 bp,包括2 631 bp开放阅读框(ORF)、75 bp的5’UTR区和767 bp的3’UTR区,并且具有脊椎动物典型的加尾信号AATAAA和26 bp的poly A尾巴,推测该序列编码876个氨基酸。蛋白结构预测表明TLR2蛋白的TIR结构域和LRR基序符合TLR家族的共同特征,其蛋白分子也存在多个功能位点。系统树中河川沙塘鳢与所有鱼类聚类为独立的分支,与鲈形目亲缘关系最近。应用RT-PCR法检测健康河川沙塘鳢鱼体中TLR2基因的m RNA组织表达差异情况。结果显示,TLR2基因在检测的12种组织中均有表达,在肝、肾、肠、血液和胃中都有较高的表达水平。人工感染嗜水气单胞菌后,对河川沙塘鳢3种主要免疫组织(脾、肾、肝)中进行不同时段表达量变化的检测,结果表明,TLR2基因在脾中感染后4 h表达水平发生下降,随后的24~48 h又上升,48 h时达到顶峰,72~96 h维持在略低于24 h水平上。在肾中,感染后表达水平持续上升,且24~96 h间保持相对稳定的表达水平。在肝组织中,感染后一直呈现上升趋势,72 h时达到顶峰,其他时段表达水平相差不大。可见TLR2在河川沙塘鳢抵御外源微生物侵染的先天免疫中发挥重要作用。
In this study, Odontobutis potamophila was used as the research object, and the TLR2 gene of the fish was cloned and analyzed for the first time using RACE and real-time fluorescence quantitative PCR. The main results are as follows: The full-length cDNA of TLR2 gene was 3 473 bp in length, including 2 631 bp open reading frame (ORF), 75 bp 5’UTR region and 767 bp 3’UTR region, Of the tailed signal AATAAA and a 26 bp poly A tail, suggesting that the sequence encodes 876 amino acids. Protein structure prediction indicates that the TIR domain of TLR2 protein and LRR motif are in line with the common features of the TLR family, and their protein molecules also have multiple functional sites. Systematic tree river 塘 pond 鳢 clustered with all the fish as an independent branch, and the Percrosses relatives closest. RT-PCR method was used to detect the expression of TLR2 mRNA in healthy river sand mullet. The results showed that TLR2 gene was detected in all of the 12 tissues tested, and had a higher level of expression in liver, kidney, intestine, blood and stomach. After artificial infection of Aeromonas hydrophila, the expression of TLR2 gene was detected in the three main immune tissues (spleen, kidney and liver) of Siberian tiger pond. The results showed that TLR2 gene was expressed 4 h after infection in the spleen The level then decreased, and then increased again from 24 to 48 h, reached its peak at 48 h and remained slightly below 24 h at 72 to 96 h. In the kidney, the expression level continued to rise after infection, and maintained a relatively stable expression level between 24 and 96 h. In the liver tissue, the infection showed an upward trend after 72 h, reaching the peak at other time points, and the expression levels remained the same at other times. Thus, TLR2 plays an important role in the innate immunity of R. solanacearum against microbial infection.