DNA Interference Of Bursicon Gene Inplutella Xylostella

A DNA fragment of the same genome sequence can inhibit the expression of specificgene and is identified as DNA interference(DNAi).So far,however,research about DNAi is relatively limited and none in insects.In this study,bursicon a subunit(PxBursα)in diamondback moth(DBM),Plutella xylostellawas targeted to explore whether DNAi exists in DBM.Fourteen single-stranded DNA(ssDNA)fragments 24 nt in length from different regions of exons or introns of PxBursa were designed and injected into the fourth instar larvae.Among the designed sequences,six fragment(two from exons and four from introns)treatments showed significantly effective results with higher pupae mortality rates and lower gene expression of PxBursa than the control treatment.The E2-5 DNA fragment resulted in the highest mortality rate in the exon treatment group,reaching 38.3%,and the I1-2 in the intron treatment group yielded a 36.9%mortality.Additionally,further investigations of prolonging or shortening the length of ssDNA indicated that the mortality ratewas dramatically reduced to 15.9%when the length of ssDNA from exon was shorted to 12 nt.As the length was elongated,the efficiency increased and climbed to 45%when the length was extended to 35 nt.However,the trend of ssDNA from introns was different.Although there was a significant reduction when the fragments were shortened to 18 nt and 12 nt,no obvious difference was observed between these two groups.Moreover,extending the fragment length did not change the interference efficiency.The E2-5 fragment was chosen as a template to compare the interference efficiency of ssDNA,double-strained DNA(dsDNA)and double-strainedRNA(dsRNA)in DBM and demonstrated that dsDNA had the highest mortality(52%).Two approximately 120-bp DNA sequences,one from the second exon and the other from the second intron,were cloned,purified and injected into the fourth instar DBM larvae at different concentrations of 100 ng/μL,170 ng/μL,280 ng/μL and 400 ng/μL to test whether long dsDNA had similar effects.This study showed that treatment with 100 ng/μL had the lowest mortality rate,less than 20%,and as the concentration was increased,the mortality rate was increased as well,reaching above 50%by treatment with 400 ng/μL.However,there was no significant difference between the treatments of 170 ng/μL and 280 ng/μL.RT-qPCR analysis indicated that the expression of PxBursa was decreased notably in the relevant treatment groups.In summary,this study demonstrated that the DNA fragments of exons and introns could lead to the death of pupae as well as inhibit expression of the corresponding gene.To the best of our knowledge,this was the first report on DNAi in insects.The characterization of DNAi in DBM was also uncovered in our current work,which might contribute to the development of new nucleic acid agents for DBM control.