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当前位置: 首页 > 文章 > 太平洋鳕RAG1和IgM基因荧光定量PCR方法的建立及初步应用 水产学报 2015,39 (4) 475-484
Position: Home > Articles > RAG1 and IgM detection in Pacific cod( Gadus microcephalus)using real-time PCR Journal of Fisheries of China 2015,39 (4) 475-484

太平洋鳕RAG1和IgM基因荧光定量PCR方法的建立及初步应用

作  者：
李幸;温施慧;毛明光;姜志强;蒋洁兰
单  位：
大连海洋大学农业部北方海水增养殖重点实验室
关键词：
太平洋鳕;RAG1;IgM;绝对荧光定量PCR
摘  要：
为研究太平洋鳕发育早期特异免疫系统形成的机制,通过RAG1和Ig M基因的转录水平衡量特异免疫系统的发育特点。根据Gen Bank中RAG1和Ig M的序列信息,分别设计1对特异引物,从太平洋鳕头肾中扩增得到RAG1和Ig M的基因片段。将所获基因片段分别插入到克隆载体p MD18-T中,从而构建太平洋鳕RAG1和Ig M基因的质粒标准品。建立并优化太平洋鳕RAG1和Ig M基因绝对荧光定量PCR方法。为进一步验证该方法的可靠性,分别利用绝对定量和相对定量检验目的基因在太平洋鳕早期发育过程不同组织内的表达差异。以优化后的绝对荧光定量PCR方法检测不同发育时期太平洋鳕RAG1和Ig M的表达情况。结果显示,RAG1的回归方程为y=-3.266x+33.77,回归系数R2=0.996;Ig M的回归方程为y=-3.119x+27.61,回归系数R2=0.998。绝对定量和相对定量结果在基因转录趋势上显现出一致性,即RAG1基因在胸腺和头肾中表达,且在胸腺中的表达量显著高于头肾中的表达量,在肝脏和脾脏中无表达;Ig M基因在胸腺、头肾、肝脏和脾脏中均有表达,其中脾脏中表达量最高,其次是头肾。RAG1基因在太平洋鳕发育早期的表达水平很低,到61日龄(days posthatching,dph)至95 dph表达量显著提高;Ig M基因在早期表达水平同样很低,到33 dph至61 dph才有明显表达,在95 dph时表达量显著提高。研究表明,本实验方法可靠,特异性较强,可成功对目标基因转录水平进行检测。
译  名：
RAG1 and IgM detection in Pacific cod( Gadus microcephalus)using real-time PCR
作  者：
LI Xing;WEN Shihui;MAO Mingguang;JIANG Zhiqiang;JIANG Jielan;Key Laboratory of Mariculture & Stock Enhancement in North China's Sea,Ministry of Agriculture,Dalian Ocean University;
关键词：
Gadus macrocephalus;;RAG1;;IgM;;absolute quantification PCR
摘  要：
To study the mechanisms of immune system formation at the early developmental stages of the Pacific cod,G adus macrocephalus,transcriptional levels of RAG 1 and Ig M were measured to characterize the immune system development. According to the sequences of RAG 1 and Ig M in Gen Bank,pairs of specific primers were designed separately to amplify both genes from the head kidney of Pacific cod. Then the fragments of RAG 1 and Ig M genes were inserted into p M D18-T cloning vector to construct the standard plasmids of RAG 1 and Ig M genes. Both absolute( A-q PCR) and relative quantification PCR( R-q PCR) were established and optimized. The results showed that a linear regression equation of RAG 1 is y =- 3. 266 2x +33. 772,and the regression coefficient( R2) of the standard curve is 0. 996 1; a linear regression equation of Ig M is y =- 3. 119 7x + 27. 612,and the regression coefficient( R2) of the standard curve is 0. 998 1. The results of A-q PCR and R-q PCR revealed a consistent trend in gene transcription. The RAG 1 was only detected in the thymus and head kidney,and the expression level in thymus was higher than that in the head kidney. The Ig M was detected in the thymus,kidney,liver and spleen,mainly in the spleen and head-kidney.The expression level of RAG 1 did not increase until 61 to 95 days post-hatching( dph),while the expression level of Ig M started to increase from 33 to 65 dph and significantly increased on 95 dph. Further analysis showed that the method of A-q PCR is reliable and specific to analyze the expression of target genes.

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