›› 2014, Vol. 57 ›› Issue (3): 286-292.doi:

• 研究论文 • 上一篇    下一篇

小菜蛾U6 snRNA基因的克隆及作为miRNA定量表达分析内参基因的评价

封冰, 梁沛*, 高希武   

  1. (中国农业大学昆虫学系,北京 100193)
  • 出版日期:2014-03-20 发布日期:2014-03-20

Cloning and evaluation of U6 snRNA gene as a reference gene for miRNA quantification in Plutella xylostella (Lepidoptera: Plutellidae)

FENG Bing, LIANG Pei*, GAO Xi-Wu   

  1. (Department of Entomology, China Agricultural University, Beijing 100193, China)
  • Online:2014-03-20 Published:2014-03-20

摘要: 【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。

关键词: 小菜蛾, U6 snRNA, 定量PCR, 内参基因, 杀虫剂, miRNA

Abstract: 【Aim】 To clone the cDNA sequence of small nuclear RNA (snRNA) U6 from Plutella xylostella (L.) and evaluate the possibility of using the U6 as a reference gene in quantification of microRNAs (miRNAs). 【Methods】 In this study, the full-length cDNA of U6 was cloned from the 4th instar larvae of P. xylostella by using the reverse-transcription PCR (RT-PCR) method. The expression stability of U6 and other eight miRNAs of P. xylostella was evaluated in different developmental stages and after different pesticide treatments with qRT-PCR.【Results】The full-length cDNA of U6 is 94 bp and shares 98.9% nucleotide sequence identity with U6 from other known insects. The qRT-PCR results analyzed by geNorm and RefFinder softwares showed that the expression of U6 was quite stable among eggs, the 1st-4th instar larvae, pupae and adults of P. xylostella, as well as in the 3rd instar larvae of P. xylostella at 48 h after treatment with nine insecticides (malathion, chlorpyrifos, phoxim, methomyl, fufenozide, beta-cypermethrin, chlorantraniliprole, chlorfenapyr and Bt CrylAc) that possess different mode of actions. 【Conclusion】U6 of P. xylostella is highly conserved among species, and its expression level is not affected by developmental stages or insecticides treated. Therefore, U6 can be taken as a qualified reference gene to evaluate the expression level of miRNA or non-coding snRNA by quantitative RT-PCR. The results pave the way for the accurate determination of snRNA expression in P. xylostella.

Key words: Plutella xylostella, U6 snRNA, quantitative RT-PCR, reference gene, insecticide, miRNA