不同实验条件下大口黑鲈 (Micropterus salmoides) 基因表达定量实时 PCR 分析的内参基因选择,Aquaculture Research

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不同实验条件下大口黑鲈 (Micropterus salmoides) 基因表达定量实时 PCR 分析的内参基因选择
Aquaculture Research ( IF 1.9 ) Pub Date : 2022-10-11 , DOI: 10.1111/are.16124
Jing Qian , Junying Zhang , Yajie Mei , Mingyan Shi , Fan Wang

大口黑鲈基因功能的研究需要使用合适的内参基因进行准确的归一化。采用实时荧光定量PCR（qPCR）方法评估了β-肌动蛋白、EF1-α、cytb和12SrRNA 4个候选内参基因在不同组织、两个不同发育阶段、不同实验条件下大口黑鲈的表达水平。通过geNorm、NormFinder和BestKeeper软件计算分析4个候选内参基因的表达稳定性。结果表明，cytb是大口黑鲈不同组织和两个不同发育阶段的最佳内参基因。细胞色素EF1-α分别是10 g和55 g不同盐浓度下称重的大口黑鲈鳃内参基因的最佳选择。在重达55 g的大口黑鲈中经脂多糖（LPS）、嗜水气单胞菌和迟缓爱德华氏菌攻击后， EF1-α被推荐为脾脏最合适的内参基因，而cytb被评为头肾最佳内参基因。由EF1-α和cytb归一化的CaM表达水平在重 55 g 的大口黑鲈的脾脏和头肾中，LPS 组比对照组显着上调。因此，这些结果表明cytb和EF1-α分别是不同组织、两个不同发育阶段和不同实验条件下合适的内参基因。本研究将为大口黑鲈在不同组织、不同发育阶段、不同实验条件下选择合适的内参基因提供有说服力的信息。

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Selection of internal reference gene for quantitative real-time PCR analysis of gene expression in largemouth bass (Micropterus salmoides) under different experimental conditions

The study of gene function in largemouth bass requires accurate normalization by use of suitable internal reference genes. The expression levels of four candidate internal reference genes including β-actin, EF1-α, cytb and 12SrRNA in different tissues, two different developmental stages, and different experimental conditions of largemouth bass were evaluated by quantitative real-time PCR (qPCR) method. The expression stabilities of four candidate internal reference genes were analysed by geNorm, NormFinder and BestKeeper software calculation. The results showed that cytb is the best internal reference gene in different tissues and two different developmental stages of largemouth bass. The cytb and EF1-α was the best choice for internal reference gene in gill of largemouth bass 10 g and 55 g weighing under different salt concentrations respectively. After being challenged by Lipopolysaccharide (LPS), Aeromonas hydrophila and Edwardsiella tarda in largemouth bass weighing 55 g, EF1-α was recommended as the most suitable internal reference gene in spleen, but cytb was ranked as the best internal reference gene in head kidney. The expression levels of CaM normalized by EF1-α and cytb in spleen and head kidney of largemouth bass weighing 55 g, and which showed the significant upregulation in LPS group than those in the control group. Therefore, these results indicated that cytb and EF1-α are the suitable internal reference genes in different tissues, two different developmental stages and different experimental conditions respectively. This study will provide convincing information for the selection of suitable internal reference genes in different tissues, different developmental stages and different experimental conditions of largemouth bass.

更新日期：2022-10-11

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