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五龄飞蝗不同发育时间实时定量PCR内参基因的筛选
引用本文:崔淼,刘晓健,李涛,郭亚平,马恩波,张建珍.五龄飞蝗不同发育时间实时定量PCR内参基因的筛选[J].昆虫知识,2014(3):733-740.
作者姓名:崔淼  刘晓健  李涛  郭亚平  马恩波  张建珍
作者单位:山西大学应用生物学研究所,太原030006
基金项目:国家自然科学基金资助项目(31272380,31201710);山西省高等学校留学回国人员科研资助项目(201163)
摘    要:【目的】筛选5龄飞蝗不同发育时间的最适内参基因,为相关研究提供基础数据。【方法】本文选取β-肌动蛋白(β-actin)、延长因子(EF-1α)、3-磷酸甘油醛脱氢酶(GAPDH)、核糖体蛋白49(RP49)、α-微管蛋白(α-Tubulin)和18S核糖体RNA(18S rRNA)基因作为候选内参基因,运用实时定量PCR(qPCR)方法研究各基因在5龄飞蝗不同发育时间的相对表达量,用geNorm与Normfmder软件分析这6个基因表达稳定性。【结果】geNorm分析结果显示6个内参基因表达稳定度M值顺序为:β-actin(0.3720)>RP49(0.3750)>α-Tubulin(0.4030)>18S rRNA(0.4270)>EF-1α(0.4970)>GAPDH(0.6040)。M值越小表示基因表达稳定度越高,同时geNorm软件以标准化因子配对差异值(Pairwise variations)0.15默认为取舍值,由于V2/3=0.098<0.15,所以最适内参基因数目为2个。运用NormFinder软件也得出相似的结果。【结论】β-actin与RP49为5龄飞蝗不同发育时间的最适内参基因。

关 键 词:飞蝗  实时定量PCR  内参基因  geNorm  NormFinder

Selection of reference genes on different days during the development of the fifth-instar nymph of Locusta migratoria with quantitative real-time PCR
Institution:CUI Miao LIU Xiao-Jian LI Tao GUO Ya-Ping MA En-Bo ZHANG Jlan-Zhen (Research Institute of Applied Biology, Shanxi University, Taiyuan 030006, China)
Abstract:Objectives] To identify the most appropriate reference genes on different days during the development of fifth-instar nymphs of Locusta migratoria to pave the way for relevant studies. Metllods] Six genes were selected as candidate reference genes, including the beta actin gene (β-actin), the Elongation factor la gene (EF-la), the Glyeeraldehyde-3-phosphate dehydrogenase gene (GAPDH), the Ribosomal protein 49 gene (RP49), the Alpha tubulin gene (α-Tubulin) and the 18S ribosome RNA gene (18S rRNA). The relative expression of these genes on different days during the development of L. migratoria was detected with quantitative real-time PCR (qPCR). The expression stability of these genes was then investigated using geNorm and Normfinder software. Results] Based on the results of geNorm analysis, the average expression stability (M) of the six genes was, in descending order; fl-actin (0.3720) 〉 RP49 (0.3750) 〉 α-Tubulin (0.4030) 〉 18S rRNA (0.4270) 〉 EF-la (0.4970) 〉 GAPDH (0.6040). The less the M value the more stable the gene expression. The software took 0.15 as a constant value for pairwise variations. The appropriate number of reference genes was 2 because V2/3=0.098 〈 0.15. Similar results were obtained using NormFinder software. Conclusion] β-actin and RP49 are the most suitable reference genes during the development of fifth-instar nymph ofL. migratoria.
Keywords:Locusta magratoria  quantitative real-time PCR  reference genes  geNorm  NormFinder
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