Effects Of Dicer Knockout On Development And RNAi Efficiency In Plutella Xylostella(L.)

Dicer is an evolutionarily highly conserved endonuclease that belongs to the RNase III family and specifically recognizes and cleaves double-stranded RNA(dsRNA).In most insects,there are two homologous Dicer genes,Dicer-1(Dcr-1)and Dicer-2(Dcr-2),which are involved in the miRNA pathway and siRNA pathway of RNAi,respectively.Meanwhile,Dcr-1 participates in the regulation of growth and development of organisms.However,the function of Dicer remains unknown in the diamondback moth(DBM),Plutella xylostella.Therefore,P.xylostella Dcr-1(PxDcr-1)and Dcr-2(PxDcr-2)were knocked out by using the CRISPR/Cas9 technique to clarify their function.PxDcr-1 and PxDcr-2 were cloned and sequenced.The ORF of PxDcr-1 was of 7524 bp,encoding 2507 amino acids,and PxDcr-2 was of 5085 bp,encoding 1694 amino acids.Similar domains were identified in two Dicer genes of P.xylostella,with PxDcr-2 lacking two domains of Dicer-PBD(partner-binding domain of the endoribo-nuclease Dicer)and dsRBD(double-stranded RNA binding domain),which may lead to the difference in function.The PxDcr-1 and PxDcr-2 genes were knocked out by the CRISPR/Cas9 technique to obtain mutants.PxDcr-1 knockout obtainedtwo mutants,one of which had 5-nucleotide insertion followed by one-nucleotide deletion one nucleotide after the PAM structure(NGG),marked as GGTAT-;the other mutant had two-nucleotide deletion after the PAM structure,marked as--.PxDcr-2 knockout also obtained two mutants with 5-nucleotide insertion after the PAM structure,marked as AACGA and GATCC.Analysis of these mutants at the genetic level revealed that protein translation was terminated beforehand.Compared with the wild-type DBM,the PxDcr-1 mutants had not only higher mortality,lower emergence rate,lower egg production,lower hatching rate,but also smaller ovary,smaller dry ovarian tube and lower mature egg mass.However,the PxDcr-2 mutants had normal growth and reproduction ability.Two target genes,Pxyellow and Pxwhite were selected for RNAi,and their dsRNA were injected into DBM larvae,separately.The results showed that the expression of the target genes was significantly lower in the wild type DBM than that in the control,and the expression of the target genes in PxDcr-2 mutants was not significantly different from that in the control,indicating that the RNAi pathway in DBM was blocked by knocking out PxDcr-2.The mutants of PxDcr-1 and PxDcr-2 were obtained by using the CRISPR/Cas9 technique to clarify the function of Dicer in DBM.The results showed that PxDcr-1 was involved in the regulation of the reproduction and development of DBM,and PxDcr-2 played animportant role in the siRNA pathway of RNAi in DBM.