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【目的】克隆小菜蛾Plutella xylostella(L.)小分子非编码RNA U6的cDNA序列,并评价其是否适合作为定量检测小菜蛾microRNA(miRNA)表达量的内参基因。【方法】本研究采用RT-PCR克隆获得了小菜蛾4龄幼虫核小RNA(small nuclear RNA,snRNA)U6的cDNA序列,并用定量PCR法检测了U6及8种miRNAs在小菜蛾不同发育阶段及不同杀虫药剂处理后的表达稳定性。【结果】小菜蛾U6的cDNA序列全长94 bp,与其他昆虫U6的核苷酸序列一致性达98.9%。用geNorm和RefFinder软件分析荧光定量PCR结果表明,U6在小菜蛾卵、1-4龄幼虫、蛹和成虫7个不同发育阶段表达稳定;用马拉硫磷、毒死蜱、辛硫磷、灭多威、呋喃虫酰肼、高效氯氰菊酯、氯虫苯甲酰胺、溴虫腈和Bt 9种不同作用机理的杀虫药剂处理3龄末幼虫48 h,对U6的表达水平无显著影响。【结论】小菜蛾U6表达水平不受不同发育阶段和不同杀虫药剂处理的影响,符合作为内参基因的基本特点,可作为定量PCR法评价小菜蛾miRNA或其他非编码小分子RNA表达水平的内参基因。研究结果为小菜蛾miRNA表达水平的准确定量奠定了基础。
【Objective】 The objective of this study was to clone the cDNA sequence of non-coding RNA U6 from Plutella xylostella (L), and to evaluate whether it is suitable as an internal reference for quantitative analysis of diamondback moth microRNA (miRNA) expression. 【Method】 The cDNA of U6 from the 4th instar larvae of Plutella xylostella was cloned by RT-PCR. U6 and 8 miRNAs were detected by quantitative PCR at different developmental stages of Plutella xylostella Expression stability of different insecticides after treatment. 【Result】 The results showed that the cDNA sequence of U6 was 94 bp in length and 98.9% identical to that of U6 in other insects. Fluorescent quantitative PCR analysis using geNorm and RefFinder software showed that U6 expressed steadily in 7 different developmental stages of the diamondback moth, eggs 1-4, larvae, pupae and adults. Malathion, chlorpyrifos, phoxim, methomyl , Furazolidone hydrazide, beta-cypermethrin, chlorantraniliprole, chlorfenapyr, chlorfenapyr and Bt. 48 h treatment of end-stage larvae did not affect the expression of U6. 【Conclusion】 The expression level of U6 in Plutella xylostella is not affected by different developmental stages and different insecticides, which accords with the basic characteristics of the reference gene. It can be used as an internal control to evaluate the expression of miRNAs or other non-coding small RNAs in Plutella xylostella by quantitative PCR gene. The results laid the foundation for accurate quantification of miRNA expression in Plutella xylostella.