Cloning And Characterization Of Genes Related To Juvenile Hormone Biosynthesis And Signalling In Leptinotarsa Decemlineata (Say)

Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), is the economically most important insect pest of potatoes throughout North America, Europe, and parts of Asia. It is the major quarantine pests and important invasive alien species in China. The outbreak of the beetle results in large quantity of yield loss. Nowadays, traditional chemical control is the major method to control the beetle. However, this led to the gradually enhanced resistance. It is necessary to develop efficient alternative control technologies for the pest.As the new scientific research means, RNAi not only is used in gene function studies, but also can be applied to produce genetically-modified plants or bacteria to develop a promising approach for management of agricultural pests.Juvenile hormone controls development, metamorphasis and reproduction in Insect. It is a specific pathway only in insects and a few other creatures. In this paper, genes related to juvenile hormone synthesis and signal transduction pathway were cloned in Leptinotarsa decemlineata. Double-stranded RNAs of several genes were synthesized to confirm their physiological functions, and to evaluate their potential application for pest control. The results were summarized as follows:1. Cloning of genes related to juvenile hormone synthesis in Leptinotarsa decemlineataBased on the corresponding gene sequences in the genome of Bombyx mori and Tribolium castaneum,3unigenes related to juvenile hormone biosynthesis were obtained from the transcriptomic data of Leptinotarsa decemlineata. These genes are homologous to juvenile hormone acid methyltransferase, farnesoic acid methyltransferase and adenosylhomocysteine. After these unigenes were verified by RT-PCR, their full length cDNA sequences were obtained by5’and3’RACE technology. Multiple alignment of the amino acid sequence shows that these genes have higher similarities with those from other insect species. Moreover, the mRNA levels of the3genes were determined by qPCR. They exhibited similar temporal expression patterns coinciding with juvenile hormone fluctuation in the haemolymph.2. RNA interference-mediated knockdown of juvenile hormone biosynthesis related-genes in Leptinotarsa decemlineataDietary introduction of bacterially expressed dsRNA successfully silenced the3juvenile hormone biosynthesis related-genes. Knockdown of these genes caused larval lethality and abnormal phenotype, affected pupation and adult emergence. Juvenile hormone analog pyriproxyfen could partially rescue the negative impact caused by gene silencing.3. Cloning of JH signaling-related genes and the influence of JH biosynthesis gene dsRNAs on their expression levelSix unigenes associated with juvenile hormone signal transduction pathway are found. They are putative transcriptional intermediary factor2, methoprene-tolerant, Kruppel-homolog1and3broad members. Their full length cDNA sequences were obtained by5’and3’RACE technology. Ingestion of JH biosynthesis gene dsRNAs had obvious influence on the expression of these genes. Moreover, juvenile hormone analog pyriproxyfen could partially restore the expression.In total, our data revealed that dietary delivery of bacterially expressed dsRNA is an efficient method in Leptinotarsa decemlineata. This may be a promising bioassay approach for genome-wide screens to identify effective target genes, and for potential application as novel RNAi-based insecticides.