Study On The Immunity Of Plutella Xylostella LncRNA Against Paecilomyces Cicadae

Plutella xylostella,as a pest in cruciferous plants,is harmful and causes great loss.Due to unreasonable use of chemical pesticides,P.xylostella has evolved resistance against various insecticides,and it is necessary to develop biological control technology.The use of entomopathogenic fungi or virulence factors is an important mean of pest biological control,and the key to success lies in understanding the immune mechanism of insect.Long non-coding RNA(LncRNA)has been found to play an important role in the regulation of insect physiological processes.In order to study the immune mechanism of P.xylostella LncRNA against entomopathogenic fungi,we constructed a library of LncRNA from the larvae infected by Paecilomyces cicadae and sequenced it.LncRNAs were screened and identified,and the differential expression of LncRNAs were screened,and the immune related target genes of LncRNA regulation were predicted.We also study the regulation of Lnc40766 on the expression of Toll-like 6 by RNAi.The main results are as follows:(1)A total of 33403 novel LncRNAs and 55982 m RNAs were identified by high-throughput sequencing from P.xylostella larvae infected by P.cicadae.92.07% of LncRNAs were intergenic LncRNAs.464 LncRNAs were up-regulated and 443 LncRNAs were down regulated.The results of RT-qPCR of four differentially expressed LncRNAs were consistent with results of high-throughput sequencing.(2)A total of 5235 LncRNA cis regulatory target genes and 682 LncRNA trans regulatory target genes were obtained.The cis regulatory immune related target genes include Toll-like receptor 6,Dscam,Lysozyme-like,PGRP-like,PGRP-lb,SCR and Catalase.The target of Lnc40766 is Toll-like receptor 6(Toll-like 6).The expression of Toll-like related genes including Toll-like 6 was up-regulated after infection by P.cicadae.Result of Inta RNA 2.0 analysis showed that the 186～207 nucleotide sequence of Lnc40766 was partially complementary to the 877～895 nucleotide sequence of Toll-like 6.(3)The full length of Lnc40766 is 679 bp.RT-qPCR results showed that the expression level of Lnc40766 in the third instar larvae of P.xylostella was significantly higher than that in other developmental stages,and the expression level of Lnc40766 in midgut was significantly higher than that in epidermis,malpighian tubules,fat bodies and hemocytes.The expression level of P.xylostella Lnc40766 was induced by P.cicadae,Staphylococcus aureus and Escherichia coli.(4)The cDNA sequence of Toll-like 6 has 2313 bp,encoding 770 amino acids,and have a transmembrane domain,leucine rich domains and Toll receptor domain.RT-qPCR results showed that the expression of P.xylostella Toll-like 6 was high in the second and third instar larvae and midgut tissues,which was consistent with the expression level of Lnc40766.Compared with 0 h,the transcription level of Toll-like 6 decreased by 76.24% 6h post ds Toll-like 6 injection.The survival rate of larvae injected with ds Toll-like 6 was only 46.67% after infection for 48 h.(5)The results of RNAi showed that the expression level of Lnc40766 decreased by84.63% and the expression level of Toll-like 6 decreased by 79.03% 6 h post injection of ds Lnc40766.The survival rate of larvae injected with ds Lnc40766 was 51.11% after infection for 48 h.This study will help us understanding the molecular mechanism of LncRNA in innate immune response,and provides theoretical basis and possible targets for new pest control methods in application.