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

• RESEARCH PAPERS • Previous Articles     Next Articles

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

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