Cloning And Characterization Of Genes Involved In Juvenile Hormone Biosynthesis In Leptinotarsa Decemlineata(Say)

Sesquiterpenoid juvenile hormones(JHs)are produced and released by corpora allata,and regulate insect growth,development,metamorphosis and reproduction.The biosynthesis pathway of JH is divided into two parts.The first is the classical mevalonate pathway that utilizes acetyl-CoA to form farnesyl diphosphate.The second is a particular way in arthropod that utilizes farnesyl diphosphate to form JH.In present study,we obtained 11 full length cDNAs encoding putative JH biosynthesis enzymes using bioinformatics and gene cloning methods.The critical role of LdJHAMT in JH biosynthesis was elucidated by RNA interference.Expression changes of ten mevalonate enzyme-encoding genes in response to juvenile hormone levels were analyzed.The main results were shown as fellows.1.Cloning of genes involved in JH biosynthesisBased on transcriptome and genomic data of Leptinotarsa decemlineata,we obtained EST sequences of 11 target genes.Using RT-PCR,we cloned full length cDNAs of these genes which encoded acetoacetyl-CoA thiolase(LdAACTl and LdAACT2),hydroxymethylglutaryl(HMA)-CoA synthase(LdHMGS),HMG-CoA reductase(LdHMGR1 and LdHMGR2),mevalonate kinase(LdMevK),phospho-mevalonate kinase(LdPMK),mevalonate diphosphate decarboxylase(LdMDD),isopentenyl-diphosphate isomerase(LdIDI),farnesyl pyrophosphate synthetase(LdFPPS)and juvenile hormone acidmethyl transferase(LdJHAAMT).Multiple sequence alignment and phylogenetic relationship analysis suggeste that these genes may encode functional enzymes.2.Functional analysis of LdJHAMT by RNA interferenceMethyl transfer of JHAMT is one of the final steps of JH biosynthesis.In thid chapter,we analysed the temporal and spatial expression patterns of LdJHAMT.LdJHAMT was highly expressed during each molt and almost specificly expressed in brain-corpora cardiaca-corpora allata complex.Two dsRNA(dsJHAMTl and dsJHAMT2)were used to interfere LdJHAMT,feeding either of dsRNAs could successfully knock down LdJHAMT,reduced JH titer and downregulated the mRNA level of a JH early response gene LdKr-h1.Knock down of LdJHAMT in second and fourth instar resulted in larval lethality,reduced weight,shorten development duration and inhibited pupation.Moreover,feeding dsJHAMT reduced eclosion rate,caused small-sized adult females and failure of oviposition.All of these defective phenotypes resulted from dsJHAMT could be rescued by application of pyriproxyfen.These data suggest that LdJHAMT encod functional juvenile hormone acid methyl transferase that plays a critical role in JH biosynthesis in L.decemlineata,and LdJHAMT may be a potential target for L.decemlineata control.3.Expression profiles of mevalonate enzyme-encoding genes in response to JH levelsUsing qRT-PCR,the temporal and spatial patterns of ten mevalonate enzyme-encoding genes were tested.Nine of these genes(except for LdAACTl)were mainly expressed in the larval brain-corpora cardiaca-corpora allata complex,and adult ovary and testis.The 9 genes were transcribed at high levels right after each ecdysis,and at low levels in the mid instar.It is suggested that these nine genes are involved in JH biosynthesis.Moreover,knockdown of LdJHAMT or injection of JH suppressed the transcripts of these nine genes.These data showed that accumulation of JH precursor or injection of JH may cause negative feedbacks to repress the expression of the 9 mevalonate enzyme-encoding genes(excluding LdAACTl)to balance the enzyme quantity in L.decemlineata.