方格星虫实时荧光定量PCR内参基因的选择与验证
彭冰冰1 , 宾东超1 , 周于娜2 , 杨志会3 , 蔡小辉1 , 彭银辉1
1.北部湾大学 海洋学院,广西北部湾海洋生物多样性养护重点实验室,广西 钦州 536011;
Selection of Reference Genes for Real-Time Quantitative PCR of Peanut Worm Sipuculus nudus
PENG Bingbing1 , BIN Dongchao1 , ZHOU Yuna2 , YANG Zhihui3 , CAI Xiaohui1 , PENG Yinhui1
1. Guangxi Key Laboratory of Beibu Gulf Marine Biodiversity Conservation, Ocean College, Beibu Gulf University, Qinzhou 536011, China;
摘要 目前研究基因定量表达的方法主要为实时荧光定量PCR,选择合适的内参基因可以提高实时荧光定量PCR分析的准确性。选取方格星虫的体壁肌肉、吻、肾管、脑、肠道、收吻肌、食道、血淋巴细胞为材料,应用实时荧光定量PCR(qRT-PCR)技术分析EF1-α2、α-Tub1、RPL13-b、Actin1、H3-b、GAPDH1共6个候选内参基因的表达稳定情况,并以方格星虫的免疫球蛋白组织分布情况对内参基因的稳定性进行验证。结果显示,通过BestKeeper软件计算候选内参基因稳定性顺序为:RPL13-b＞H3-b＞EF1-α2＞GAPDH1＞Actin1＞α-Tub1;Normfider软件分析候选内参基因稳定性顺序为:RPL13-b＞EF1-α2＞GAPDH1＞H3-b1＞Actin1＞α-Tub1;geNorm软件分析候选内参基因稳定性顺序为:RPL13-b=H3-b＞EF1-α2＞GAPDH1＞Actin1＞α-Tub1。采用最稳定和最不稳定内参基因对免疫球蛋白基因在不同组织的表达水平变化进行校准时发现,其表达谱呈现不同模式。RPL13-b内参基因在方格星虫全组织中的表达最稳定,可作为最适单内参基因。
关键词 ：
方格星虫 ,
实时荧光定量PCR ,
内参基因
Abstract ：At present, the main method to study the quantitative expression of genes is real-time quantitative PCR(qRT-PCR), and the accuracy of qRT-PCR analysis can be improved by selecting the appropriate internal reference genes. In this study, EF1-a2,a-Tub1, RPL13-b, Actin1, H3-b and GAPDH1 were analyzed by qRT-PCR from the rostrum, somatic muscles, renal duct, brain, intestine, adductor muscle, esophagus, and blood lymphocyte of peanut worm Sipunculus nudus , the expression of six candidate internal reference genes was stable, and the stability of internal reference genes was verified by IgGFc-binding protein. BestKeeper software revealed that the descending order of the stability of internal reference genes was expressed as:RPL13-b＞H3-b＞EF1-α2＞GAPDH1＞Actin1＞α-Tub1; Normfider software analysis selected the stability of the internal reference gene, with the descending order of RPL13-b＞EF1-α2＞GAPDH1＞H3-b1＞Actin1＞α-Tub1; The descending order of stability of internal reference genes selected by geNorm software analysis was described as: RPL13-b=H3-b＞EF1-α2＞GAPDH1＞Actin1＞α-Tub1. When the stable internal reference gene RPL13-b was used to calibrate the qRT-PCR data, the expression trend of IgGFc-binding protein gene was basically the same as in different tissues. The most unstable internal reference gene-Tub1 was used to calibrate the qRT-PCR data, with different change trend of expression level of IgGFc-binding protein gene. The expression of RPL13-b internal reference gene was the most stable in the whole tissues of peanut worm and was used as the optimal single internal reference gene.
Key words ：
Sipuculus nudus real-time quantitative PCR
reference gene
收稿日期: 2020-05-18
基金资助: 广西自然科学基金资助项目(2018GXNSFAA281208);广西科技开发项目(桂科AB18221112);广西高校中青年教师科研基础能力提升项目(2019KY0436);北部湾大学高层次人才科研启动项目(2019KYQD17);广西北部湾海洋珍稀物种养护自治区重点实验室项目(2018ZB11,2019ZB07).
通讯作者:
彭银辉(1981—),男,副研究员,硕士;研究方向:水产动物病原微生物学. E-mail:pyinhui@163.com.
作者简介 : 彭冰冰(1998—),女,本科生;研究方向:水产动物病原微生物学. E-mail:1490315542@qq.com.
引用本文:
彭冰冰, 宾东超, 周于娜, 杨志会, 蔡小辉, 彭银辉. 方格星虫实时荧光定量PCR内参基因的选择与验证[J]. 水产科学, 2022, 41(2): 304-310.
Sipuculus nudus . 水产科学, 2022, 41(2): 304-310.
链接本文:
http://www.shchkx.com/CN/10.16378/j.cnki.1003-1111.20098 或 http://www.shchkx.com/CN/Y2022/V41/I2/304
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