Wang Xubo

Selection of reference genes for reverse transcription quantitative real-time PCR normalization in black rockfish (Sebastes schlegeli)

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a technique widely used for quantification of mRNA transcription. Data normalization is an indispensable process for RT-qPCR and reference genes are most commonly used to normalize RT-qPCR and to reduce possible errors generated in the quantification of genes among several proposed methods. To date, RT-qPCR has been used in terms of gene expression studies in black rockfish (Sebastes schlegeli) but the majority of published RT-qPCR studies still lack proper validation of the reference genes. In the present study, mRNA transcription profiles of eight putative reference genes (18S rRNA, ACTB, GAPDH, TUBA, RPL17, EF1A, HPRT, and B2M) were examined using RT-qPCR in different tissues and larvae developmental stages of black rockfish. Three common statistical algorithms (geNorm, NormFinder, and BestKeeper) were used to assess expression stability and select the most stable genes for gene normalization. Two reference genes, RPL17 and EF1A showed high stability in black rockfish tissue analysis, while GAPDH was the least stable gene. During larvae developmental stages, EF1A, RPL17 and ACTB were identified as the optimal reference genes for data normalization, whereas B2M appeared unsuitable as the reference gene. In summary, our results could provide a useful guideline for reference gene selection and enable more accurate normalization of gene expression data in gene expression studies of black rockfish.

牙鲆( Paralichthys olivaceus ) CD59 基因的原核表达与功能探究

本研究分析了CD59基因在牙鲆(Paralichthys olivaceus)感染鳗弧菌(Vibrio anguillarum)前后的表达模式。结果显示，感染前，CD59基因在检测的4种免疫器官中均不表达；感染后，该基因均有不同程度的表达，其中，肾脏的表达量最高。对牙鲆CD59基因进行原核表达，构建原核表达载体pET-32a-CD59，并将其转化入大肠杆菌(Escherichia coli) BL21，使用异丙基-β-D-硫代半乳糖苷(IPTG)诱导，获得约为29 kDa的重组蛋白pET-32a-CD59。该蛋白以包涵体形式存在，通过His亲和层析柱纯化和超滤管浓缩目的蛋白后，经SDS-PAGE检测得到单一条带，纯化效果较好。Western blotting分析结果显示，重组蛋白pET-32a-CD59可以与抗His单克隆抗体发生特异性反应，说明牙鲆CD59基因在大肠杆菌系统中成功表达。对重组蛋白进行透析复性，复性结束后，蛋白无析出或絮状沉淀，初步表明复性成功。选取嗜水气单胞菌(Aeromonas hydrophila)、鳗弧菌、金黄色葡萄球菌(Staphylococcus aureus)和大肠杆菌4种致病指示菌，测定复性重组蛋白pET-32a-CD59的抑菌活性。研究表明，重组蛋白pET-32a-CD59对嗜水气单胞菌具有一定的抑菌活性。本研究旨在探究牙鲆免疫调节机制，并为提高牙鲆养殖效率提供一定的分子理论基础。