Cloning And Characterization Of Gene FTZ-F1 In Leptinotarsa Decemlineata(Say)

In insects,a combination of juvenile hormone(JH)and 20-hydorxyecdysone(20E)regulates metamorphosis.Both of JH and 20E bind with their receptors,and then regulate the expression of a series of downstream genes,and initiate the occurance of metamorphosis.These two hormone signal pathways interact with each other by some transcription factors.FTZ-F1,a nuclear receptor type transcription factor(NRs),is one among these transcription factors.Nuclear receptor type transcription factors may play important roles during metamorphosis,reproduction and diapause in animals.Researchs have shown that DmFTZ-F1 in Drosophila melanogaster has two protein isoforms:aFTZ-F1 and ?FTZ-F1.DmaFTZ-F1 is responsible for the formation of alternate body segments during early embryogenesis.Dm?FTZ-F1 regulates the timing of molting and is needed in larval,pupal and adult stage.Similar important function of FTZ-F1 has been observed in other insects investigated so far,including coleopteran,dipteran,lepidoptera,hymenoptera.Therefore,FTZ-F1s are very important in animal development and deserve further research.Leptinotarsa decemlineata,an notorious defoliator,is a major hazard to solanaceous plants.In the present paper,based on the transcriptomic data of L.decemlineata,two LdFTZ-F1 isoforms were obtained.By RNAi,their function during larval-pupal metamorphosis was characterized.We further explored the roles of LdFTZ-Fl in thejuvenile hormone and ecdysone signal pathways.The main results were listed as follows.1.Molecular cloning,phylogenetic analysis and temporal expression profiles of LdFTZ-Fl in L.decemlineataBased on the transcriptomic data of L.decemlineata,two fragments of LdFTZ-F1 were obtained.After verification of the correctness,two protein isoforms were obtained and named respectively as:LdFTZ-F1-1 and LdFTZ-F1-2.The predicted LdFTZ-F1-1 and LdFTZ-F1-2 proteins share an identical DNA-binding domain,hinge region and ligand-binding domain,but have distinct N-terminal domains.The phylogenetic tree showed that the two LdFTZ-F1 proteins and three from Tribolium castaneum respectively clustered together formed Coleoptera clade.The temporal expression profiles of LdFTZ-F1-1and LdFTZ-F1-2 were also determined at the egg,larval,pupal and adult stages.They were expressed throughout all developmental stages.LdFTZ-F1-1was highly expressed in eggs and third instars,whereas higher LdFTZ-F1-2 levels were found in the last instars,wandering larvae,pupae and adults.In the first and second instars,the expression levels of both LdFTZ-F1-1and LdFTZ-F1-2were lower.Moreover,both LdFTZ-F1-1and LdFTZ-Fl-2 were ubiquitously expressed in all tested tissues,especially in brain-corpora cardiaca-corpora allata complexes and epidermis.2.Hormone signaling pathways triggerring the expression of LdFTZ-F1The temporal expression profiles of LdFTZ-F1 isoforms indicate that the transciption of both isoforms is similar to the expression pattern of an ecdysteroidogenesis gene LdSHD.Silencing LdSHD successfully knocked down LdSHD,significantly inhibited ecdysteroidogenesis and decreased 20E titer,lowered the mRNA levels of LdFTZ-F1-1 and LdFTZ-F1-2.Thus,20E peak seems critical for the induction ofLdFTZ-F1 in L.decemlineata.Moreover,knockdown of LdILP2 significantly lowered the expression of LdFTZ-F1.However,neither treated with JHIII/Hal nor introduced dsLdAS-C and dsLdJHAMT into the larvae affected the expression of LdFTZ-F1.The results show that 20E and ILP signal pathways are involved in regulation of LdFTZ-F1.3.Effect of silencinggeneLdFTZ-Fl to L.decemlineata Dietary introduction of double-stranded RNAs(dsRNAs)targeting a common fragment of both LdFTZ-Fl-1and LdFTZ-F1-2successfully knocked down the target mRNAs.Ingestion during 3 consecutive days of dsLdFTZ-Fl did not affect the weight and survival of the resulting larvae,but impaired the pupation:96%of resulting larvae died before pupation.Moreover,ingestion of an ecdysteroid agonist halofenozide did not rescue the negative effect of dsLdFTZ-F1 on pupation.Thus,our results in L.decemlineata demonstrated that FTZ-F1 played a critical role during larval-pupal metamorphosis.Moreover,ingestion of dsLdFTZ-F1 significantly reduced mRNA levels of three Halloween gene LdPHM,LdDIB and LdSHD,decreasing 20E titer.At the same time,two 20E receptor genes LdEcR-A and LdEcR-B expression levels in the resulting larvae were significantly decreased.Thus,20E signal pathway was disrupted.Furthermore,knockdown of LdFTZ-Fl also disturbed juvenile hormone signal pathway:JH biosynthesis was activated,JH titer was increased,and a JH early-inducible gene Kr-hl was significantly downregulated.In summary,LdFTZ-F1s are involved in the regulation of pupation by modulating 20E and JH titers and mediating their signaling pathways in L.decemlineata.