Characterization Of Cytochrome P450 Genes And Mirna Regulation Of Cs-spook In Rice Striped Stem Borer, Chilo Suppressalis

1.SSB P450 gene family analysis at genome level The rice stem stripped borer(SSB),Chilo suppressalis Walker,is one of the most important insect pests for rice production in China and the rest of Southeast Asia.In this work,we performed an exhaustive search from both the draft genome and transcriptome,yielding 77 CYP genes in C.suppressalis.To the best of our knowledge,this is the first catalog of CYP genes in the rice stripped stem borer.All of these genes were confirmed by RT-PCR sequencing.The full-length transcripts of twenty CYPs were successfully amplified with RACE.In total,more than 30 CYP genes have intact open reading frames(ORF).The CYP genes of C.suppressalis were classified into four clans,CYP2,CYP3,CYP4 and mitochondrial.Phylogenetic and evolutionary analysis showed that there was an apparent expansion of the CYP3 clan in the rice stripped stem borer.In this study,we identified CYP321F,a new P450 subfamily,located in the genome as tandem repeats,which is exclusive to the rice stripped stem borer.2.Amplification and expression of Cs-phantom gene reported the characterization of the Cs-phantom(CYP306A1)in the rice striped stem borer,C.suppressalis.A full-length transcript of Cs-phantom with an open reading frame of 478 amino acids shows five conserved P450 motifs,i.e.,Helix-C,Helix-I,Helix-K,PERF and heme-binding motifs.The phylogenetic analysis showed the evolutional conservation in insects with a high similarity in Lepidoptera.The relative spatial and temporal transcript profile revealed that Cs-phantom was highly expressed in the prothoracic glands.It appears throughout the larval development,starting out low at the beginning of the larval instar,then peaking on day 3-4,and becoming low again before the larvae molt.In conclusion,our results suggest that,Cs-phantom is functionally conserved in the rice striped stem borer C.suppressalis.3.The function of Spook gene in SSB Spook gene is a member of P450 family,which participates in the ecdysteroid biosynthesis.We studied the function of Spook in SSB(Cs-spook),showing that Cs-spook has conserved P450 motifs such as Helix-C,Helix-I,Helix-K and PERF motif(PxxFxPxRF).The expression of Cs-spook was higher in late instar larvae than newly molted larvae.It was highly expressed in prothoracic glands.Cs-spook was knocked down by dsRNA treatments.Compared with the control,the gene expression level was reduced to 9%at 24 h post-injection(PI),to 33%at 48 h PI,and to 24%at 72 h PI.The ecdysteroid titer decreased significantly in the dsRNA-treated group(P<0.05),resulting in delayed larval development.The delayed development in dsRNA-treatment group was rescued by treating with 20-E.Our work demonstrates that Cs-spook participates in the biogenesis of 20-E and regulates the molt of SSB,as seen in other species.4.Four miRNAs might be involved in the biogenesis of 20-E MicroRNAs(miRNAs)are small noncoding RNAs that serve as post-transcriptional regulators of gene expression.In order to study the roles of miRNAs in regulating genes in the ecdysone cascade,we found seven miRNAs(Bantam,miR-8,miR-080,miR-089,miR-124,miR-154 and miR-257)that were predicted to regulate the Cs-spook by at least four miRNA prediction software programs.Luciferase experiments confirmed that Bantam,miR-080,miR-089,miR-154 and miR-257 can target on Cs-spook gene.When co-transfected these five miRNAs,the expression abundance of target gene with Cs-spook 3'UTR was reduced approximately 36%,19%,14%,45%,and 57%,respectively.In contrast,miR-8 and miR-124 did not show any significant effect.This result suggested that at least four miRNAs could target Cs-spook.We study on the expression of miR-257 in different development stages,demonstrating its expression correlates inversely with those of Cs-spook during larval development.Our work demonstrates that miR-257 regulate the Cs-spook enzyme,which might participate in the biogenesis of 20-E and regulates the molt of C.suppressalis.