微孢子感染东亚飞蝗实时定量PCR内参基因的筛选
Identification of stable reference genes for real-time quantitative PCR (RT-qPCR) in Locusta migratoria infected by Paranosema locustae
魏笑佳；何 岚；阿地拉·阿吉；季 荣；扈鸿霞
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DOI：10.7679/j.issn.2095-1353.2021.011
作者单位：新疆师范大学生命科学学院，中亚区域跨境有害生物联合控制国际研究中心，乌鲁木齐 830054
中文关键词：微孢子；东亚飞蝗；内参基因；实时定量PCR；表达稳定性
英文关键词：Paranosema locustae; Locusta migratoria; real-time PCR; reference genes; expression stability
中文摘要：
【目的】 筛选出微孢子Paranosema
locustae感染条件下东亚飞蝗Locusta
migratoria实时定量PCR最适内参基因。【方法】 本研究应用实时荧光定量PCR技术测定东亚飞蝗甘油醛-3-磷酸脱氢酶基因（Glyceralde-hyde-3-phosphate
dehydrogenase，GAPDH）、18s核糖体RNA（18sRNA）、微管蛋白基因（Tubulin，TUB）、肌动蛋白基因（Actin，ACT）和延伸因子1基因（Elongation
factor1，EF1）5个候选基因在3个微孢子浓度（1 × 104、1 × 106和1 × 108个孢子/头）感染12 d后的表达稳定性；应用geNorm、Bestkeeper和Normfinder
3个软件综合分析5个内参基因的稳定性。【结果】 geNorm软件对5个内参基因在不同微孢子虫浓度感染下稳定性分析结果表明：18S基因稳定性最低，平均表达稳定性值M值最高为0.658 4，TUB基因稳定性最高，M值最低为0.278 4。NormFinder软件分析在不同微孢子虫浓度感染下的5个内参基因的稳定值分别为0.152（EF1）、0.181（ACT）、0.212（TUB）、0.329（GAPDH）和0.395（18S），可知内参基因稳定性顺序为：EF1 > ACT > TUB > GAPDH > 18S。由BestKeeper软件分析表明5个候选内参基因的SD值均小于1.0，表达均较稳定。根据其变异系数值CV的大小可知，ACT基因最为稳定。【结论】 综合3种软件分析结果，在不同微孢子虫浓度感染下，可选择的较为稳定的内参基因ACT、EF1和TUB基因作为相对定量的参照基因。
英文摘要：
[Objectives] To identify stable reference genes in Locusta migratoria infected by paranosema locustae for real-time quantitative PCR (RT-qPCR). [Methods] In this study, five commonly used candidate L. migratoria reference genes (glyceralde-hyde-3-phosphate dehydrogenase，GAPDH. 18sRNA. tubulin，TUB. actin，ACT. And elongation factor1，EF1) were evaluated to assess their suitability for use in the normalization of RT-qPCR data in locusts infected with different concentrations of Paranosema locustae spores（1×104、1×106 and 1×108 spores/individual）. Gene expression profiles were analyzed using the geNorm, Normfinder, and BestKeeper software packages. [Results] geNorm predicted TUB to be the most, and 18S the least, stable reference genes based on their respective M-values (0.2784 and 0.6584). NormFinder estimates of the stability values (SV) of each candidate reference gene were EF1(0.152), ACT(0.181), TUB(0.212), GAPDH(0.329) and 18S(0.395), which allows these genes to be ranked in order of stability as follows; EF1 > ACT > TUB > GAPDH > 18S. BestKeeper evaluates the expression stability of reference genes by calculating and comparing their variance, including their coefficient of variance (CV) and standard deviation (SD). This analysis indicated that all five genes were stable on the basis of having SD values < 1.0. However, with respect to CV values, the ACT gene was the most stable. [Conclusion] These results indicate that the best reference genes for L. migratoria infected with P. locustae are ACT、EF1 and TUB.