Identification And Characterization Of CncC And Keap1 In Leptinotarsa Decemlineata(Say)

Drosophila cap 'n' collar isoform C(CncC)and Kelch-like ECH associated protein 1(Keap1)regulate metamorphosis by coordinate transcriptional control of a subset of genes involving in ecdysteroidogenesis,20-hydroxyecdysone(20E)signaling,and juvenile hormone(JH)degradation.Up to now,however,the roles of CncC and Keap1 in the control of metamorphosis has not been confirmed in other insect species.By mining the transcriptomic and genome data of L.decemlineata,and performing RT-PCR and RACE,6 full-length cDNAs of CncC and Keapl were obtained.The role of CncC and Keapl was further studied by RNA interference.Our results revealed that LdCncC/LdKeapl signaling regulated larval-pupal metamorphosis through triggering ecdysone biosynthesis.The main results were listed as follows.1-Molecular cloning and phylogenetic analysis of CncC and Keapl in L.decemlineataBased on the transcriptomic and genome data of L.decemlineata,6 full-length cDNAs of CncC and Keapl were obtained.In L.decemlineata,LdCnc had four splicing isoforms.LdCncA and LdCncB were truncated forms.LdCncCl and LdCncC2 differed in the first exon but shared the following 10 exons.LdKeapl gene possessed two splicing isoforms(LdKeaplA and LdKeaplB)which differed in the first exon.Phylogenetic analysis revealed that both CncC-and Keapl-like proteins formed order based separate clades.Obviously,LdCncC and LdKeapl belonged to coleopteran clade.Using primers from the common sequences of LdCncC and LdKeapl isoforms,we determined their temporal expression patterns.Two genes were expressed in all larval stages.At the first,second and third larvae instar,the expression levels were higher before and after molting,and were lower in the intermediate instar.In the fourth larval instar,the two genes reached their highest expression levels between 24 and 60 hours after ecdysis.The tissue expression profiles of LdCncC and LdKeapl genes were also tested.Their mRNAs were easily detectable in all tested tissues including brain-corpora cardiaca-corpora allata complex,prothoracic gland,foregut,midgut,hindgut,Malpighian tubules,epidermis,fat bodies and hemocytes of the day 2 fourth-instar larvae.The templates were also present in female ovaries and male testis of the adults.LdCncC was highly expressed in larval midgut and prothoracic gland,and adult ovaries,whereas LdKeapl was transcribed at the highest level in the larval brain-corpora cardiaca-corpora allata complex,and at higher levels in the larval foregut and prothoracic glands,and the adult ovaries.2.Effect of silencing of CncC and Keapl on the metamorphosis in L.decemlineataTo dissect the physiological roles of LdCncC and LdKeap1 in larval development,we dietarily introduced each of the two dsRNAs of the common sequences of LdCncC and LdKeap1 into the newly-molted fourth-instar larvae.Combined data revealed that continuous ingestion of a dsCncC or a dsKeapl for 3 days significantly downregulated its target gene.Ingestion of dsCncC or dsKeapl significantly downregulated the expression of Ldspo,Ldphm,Lddib and Ldsad,but did not affect the transcription of Ldshd.As a result,20E titers in the treated larvae were significantly lowered.Moreover,consumption of dsCncC and dsKeapl significantly lowered the transcripts of five 20E response genes(LdEcR-A,LdUSP,LdE75,LdHR3 and LdFTZ-F1).In L.decemlineata,two Jheh genes(LdJheh1 and LdJheh2)have been cloned.We found the expression of LdJheh1 and LdJheh2,and the JH titers were not significantly affected in the LdCncC and LdKeap1 RNAi larvae.Feeding of 20E recovered the developmental period to the normal length.At the same time,it rescued the larval and pupal body mass rises.Moreover,consumption of 20E rescued or even overcompensated the expression levels of LdEcR-A,LdUSP,LdE75,LdHR3 and LdFTZ-F1 in the LdCncC and LdKeap1 RNAi hypomorphs.3.Effects of LdCncC and LdKeap1 on detoxification in L.decemlineataIt has been reported that the CncC/Keapl signal is involved in the transcriptional regulation of a series of detoxification genes in Drosphila melanogaster.In the present paper,we detected the expression levels subsets of representative genes belonging to the glutathione S-transferase(GST)family gene,carboxylesterase(CarE)and cytochrome P450 monooxygenase(P450)by qRT-PCR.Out of 14 LdGSTd and LdGSTe members,the expression of 11 ones was significantly suppressed in the dsCncC-fed larvae,whereas the expression of 4 was significantly activated and 5 was dramatically repressed in the dsKeapl-fed larvae.Similarly,among 9 cyp genes tested,a subset of 6 were repressed in the dsCncC-fed larvae and another subset of 6 cyp genes were surpressed in the dsKeap1-fed larvae.Among 11 CarE genes determined,the expression of a subset of 8 was inhibited in the dsCncC-fed larvae and the transcription of another subset of 7 CarE genes was downregulated.Therefore,the transcriptional regulation of a series of detoxification genes by LdCncC and LdKeap1 is evolutionarily conserved in L.decemlineata.