Cloning And Characterization Of 3 Genes Involved In Juvenile Hormone Degeneration In Leptinotarsa Decemlineata (SAY)

In insect,juvenile hormone(JH)plays critical roles in larval development,metamorphesis and reproduction.JH titer in haemolymphe is regulated by both synthesis and degradation.JH degradation mainly relies on three enzymes:juvenile hormone esterase(JHE),juvenile hormone epoxide hydrolase(JHEH)and juvenile hormone diol kinase(JHDK).Based on the transcriptomic data of L.decemlineata and sequences in the NCBI(National Center for Biotechnology Information)GenBank database,full-length cDNAs of JHEH and JHDK were obtained,theire functions had been characterized by RNA interference.At the same time,a putative juvenile hormone esterase was cloned.The main results were listed as follows.1.Cloning and characterization of juvenile hormone epoxide hydrolaseTwo full length LdJHEH cDNAs were cloned in L.decemlineata,and were provisionally designated LdJHEHl and LdJHEH2.Both mRNAs were detected in several tissues of the day 3 fourth-instar larvae,and adult ovary and male reproductive organs.Moreover,both LdJHEH1 and LdJHEH2 were expressed throughout all larval stages,with the highest peaks occurring 32 hours after ecdysis of the final instar larvae.Ingestion of double-stranded RNA(dsRNA)of LdJHEH1 LdJHEH2 or the mixture of the two dsRNAs successfully knocked down corresponding target genes,significantly affected JH degradation and increased LdKr-h1 mRNA levels.Knockdown of LdJHEHl,LdJHEH2 and both genes slightly reduced larval weight and delayed larval development,and significantly impaired adult emergence.Thus,we provide a line of experimental evidence in L.decemlineata to support the concept that both LdJHEH1 and LdJHEH2 encode function proteins involved in JH degradation.2.Cloning and characterization of juvenile hormone diol kinaseJuvenile hormone diol kinase(JHDK)is an important enzyme involved in the JH degradation pathway.Leptinotarsa decemlineata,JHDK cDNA(814 bp)contains an open reading frame encoding a 184-amino acid protein.LdJHDK shows a high degree of identity to the previously reported JHDKs.LdJHDK has three conserved sequence elements involved in purine nucleotide binding,contains three EF-hand motifs,and resembles other calcium-binding proteins.LdJHDK mRNA was mainly detected in hindgut and Malpighian tubules.Besides,a trace amount of LdJHDK mRNA was found other tissues.Moreover,LdJHDK was expressed throughout all developmental stages.Ingestion of double-stranded RNA(dsRNA)of LdJHDK successfully knocked down the target gene,affected JH degradation and significantly increased LdKr-hl mRNA levels.Knockdown of LdJHDK slightly reduced larval weight and delayed larval development,and significantly impaired adult emergence.Thus,LdJHDK has been proven to encode function protein involved in JH degradation.3.Identification of carboxylesterase genesBased on the Leptinotarsa decemlineata transcriptome dataset and the GenBank sequences,70 novel carboxylesterases and 2 acetylcholinesterases were found.The 72 members belong to a multifunctional carboxylesterase/cholinesterase superfamily(CCE).A phylogenetic tree was constructed including the 72 LdCCEs.After a series of analysis,we got the full length cDNA of a putative juvenile hormone esterase KM220550 by RT-PCR and RACE PCR,which was similar with other juvenile hormone esterase according to its characteristics and tissular-temporal expression patterns.In addition,among the 70 novel CCE genes,KM220566,KM220530,KM220576,KM220527 and KM220541 were fipronil-inducible,and KM220578,KM220566,KM220542,KM220564,KM220561,KM220554,KM220527,KM220538 and KM220541 were cyhalothrin-inducible.They were the candidates involving in insecticide detoxification.Moreover,our results also provided a platform to understand the functions and evolution of L.decemlineata CCE genes.