Identification And Function Analysis Of Non-coding RNAs Involved In Chlorantraniliprole Resistance In Plutella Xylostella(L.)

The diamondback moth(DBM),Plutella xylostella(L.),is a major pest of cruciferous vegetables.Due to the long-term use of chemical control,P.xylostella has developed resistance to various types of insecticides including chlorantraniliprole.Previous studies have reported several protein-coding genes involved in chlorantraniliprole resistance,such as ryanodine receptor(RyR),cytochrome P450,esterase(CarE)and glutathione S-transferase(GST).Now,this is the first attempt to clarify the effect of non coding RNAs(miRNA and lncRNA)on the DBM chlorantraniliprole resistance from the post transcriptional level.The main research results are as follows:1.Two chlorantraniliprole-resistant strains were constructed for this study,including a laboratory-resistant strain CHR and a field-resistant strain ZZ.Using RN A sequencing(RNA-seq),a total of 199 known and 54 novel miRNAs were identified.Among them,23 miRNAs were differentially expressed between CHR and CHS,and 90 miRNAs were differentially expressed between ZZ and CHS,of which 11 differentially expressed miRNAs were identified in both CHR and ZZ.2.Using RNA-seq,1309 lncRNAs were identified,including 877 intergenic lncRNAs,190 intronic IncRNAs,76 anti-sense lncRNAs and 166 sense-overlapping lncRNAs.64 lncRNAs were differentially expressed between CHR and CHS and 83 were differentially expressed between ZZ and CHS,of which 22 were differentially expressed in both CHR and ZZ.3.A near-isogenic line DBM strain(NIL-CHR)was constructed using a high chlorantraniliprole-resistance strain(DBM-CHR)and a laboratory-susceptible strain(DBM-CHS).GSTu1 gene was up-regulated in all the three chlorantraniliprole-resistance strains used in this chapter.Using RNAi method in DBM-CHS,NIL-CHR and DBM-CHR,we determined that GSTu1 was involved in the chlorantraniliprole-resistance.4.Alternative polyadenylation(APA)was found in GSTu1 gene.Using RT-PCR and RACE methods,a total of 4 transcripts were finally identified,and miR-8530 was found to be able to act on the 3 long GSTul transcripts.There is a long non-coding RNA(lnc-PxGSTu1-AS)located on the antisense strand of GSTu1 gene,and lnc-PxGSTu1-AS could maintain the stability of GSTu1-long transcripts by inhibiting the role of miR-8530.5.In addition,some unpublished DBM miRNAs or RNA binding proteins may also inhibit the expression of the 4 GSTu1 transcripts(PxGSTu1-long and PxGSTu1-short),but lnc-PxGSTu1-AS could also maintain the stability of PxGSTu1-long and PxGSTu1-short in the resistant strains,thus mediating the resistance of the P.xylostella to chlorantraniliprole.In conclusion,a batch of miRNAs and lncRNAs associated with chlorantraniliprole resistance were obtained,and it is preliminarily elucidated that the non-coding RNAs(miR-8530 and lnc-PxGSTu1-AS)are involved in the chlorantraniliprole-resistance by regulating the expression of GSTu1 gene in DBM.These results provide important theoretical supports for non-coding RNA mediated insecticide-resistance.