Expression Profiles and Functions of EcR, USP and FTZ-F1of Helicoverpa Armigera
|School||Nanjing Agricultural College|
|Keywords||Helicoverpa armigera EcR USP FTZ-F1 RNAi|
For the holometabolous insects, periodically molting occurs during larval development. The hormones regulate many physiological events throughout the insect life cycle. Based on the research of the molting related genes, can not only understand growth of the insect and the molecular mechanism of insect molting, but also can be found on the novel molecular targets to effectively control the pest. Insect ecdysone receptor (EcR) and ultraspiracle proteins (USP) is the molecular target of the ecdysone in the onset position of the insect molting cascade, which have great significance in insect growth, development and breeding. In this study, RNAi technology and real-time quantitative PCR were applied to research the expression profile and the functions of EcR and USP during the two last instar stages. The expression levels of molting related genes E74, JHE, HR3and FTZ-F1were also analyzed after EcR, USP was knocked down to determine the relationship between these genes. FTZ-F1has been identified as a transcriptional activator of the fushi tarazu gene in Drosophila melanogaster, which participation larvae to larvae molt, the larvae to pupal transformation and adult development. The βFTZ-F1fragments in larvae of H. armigera was also cloned for the first time, and the expression characteristics of FTZ-Fl in the last instar stages were investigated.qRT-PCR was used to analysis the expression profile of EcR, USP, E74, JHE, HR3and FTZ-F1mRNA in H. armigera during the two last instar stages, results showed that the six molting related genes were expression in5th instar larvae and6th instar larvae, which appeared cyclical changes. The expression profile of EcR and USP have the similar trend, both of them have a peak in the fifth instar and two peaks in the final instar larvae, EcR expression is8-16h prior to USP. The expression of E14appears an expression peak in both fifth instar and the final instar respectively. The expressions of JHE and HR3have the similar trend, both of them have a peak in the fifth instar and two peaks in the final instar stage. The expressions of FTZ-F1have two peaks in the fifth instar and two peaks in the final instar larvae. According to the expression levels and the time of expression peaks appeared, we can conclude that EcR, USP, E14, HR3, FTZ-F1involved in insect larvae molt, the formation of prepupae and the transformation of prepupae to pupal, JHE involved in the larvae molt and prepupae to pupal transformation of H.armigera.EcR and USP silence respectively led to down-regulation of the expression levels of EcR, USP and other molting related genes. RNAi of EcR led to reduction of the E74, JHE, HR3and FTZ-F1mRNA expression levels in a certain degree, which meaned that EcR might be at the up-stream of E74, JHE, HR3, FTZ-F1and involved in the expression regulation of E74, JHE, HR3, FTZ-F1mRNA. The similar phenomenon was observed in the larvae by RNAi of USP, injection of USP dsRNA led to the reduction of the USP, E74, JHE, HR3and FTZ-F1mRNA expression levels, which meaned that USP might be at the up-stream of E74, JHE, HR3, FTZ-F1and involved in the expression regulation of E74, JHE, HR3, FTZ-F1mRNA. FTZ-F1RNAi caused the down-regulation of gene expression of FTZ-F1, at the same time it influences the HR3expression. HR3showed the rise and fall of expression levels after FTZ-F1RNAi. It was reported that HR3is responsible for high level expression of the FTZ-F1gene in the mid-prepupal period, we hypothesized that between FTZ-F1and HR3existed inhibition phenomena.RNAi technology was used to analysis the functions of EcR, USP and FTZ-F1. The RNAi of EcR in the5th instar larvae, nearly20%larvae died due to unsuccessful molt to the next stage with the exuviae attached to the body. Injection of EcR dsRNA in the6th instar larvae, larvae appeared three types of abnormal phenotypes:i.e. prepupal formation failed, failed to undergo larval-pupal metamorphosis and stopped as larval-pupal intermediates. According to the the abnormal phenotypes by RNAi of EcR, it may be inferred that EcR is required for the ecdysising, prepupal formation and pupal transformation. Injection of USP dsRNA into the5th and6th instar larvae, the abnormal phenotypes are similar to the phenotypes caused by EcR dsRNA.I In the5th larvae, the the injected larvae can not molted to the next stage unsuccessful with the exuviae attached to the body. In the6th instar larvae, the abnormal phenotype is failure of prepupal formation and larval-pupal metamorphosis. According to the RNAi results, it can be speculated that USP is required for the ecdysising, prepupal formation and pupal transformation.The co-injection of EcR dsRNA and USP dsRNA, the abnormal phenotypes are similar to that by separate injections. According to the qRT-PCR the silence of USP led to a downgrade of EcR expression. After the silence of FTZ-F1, the fifth instar larvae can not proceed the normal molting to enter the next stage and express as molting stagnation. The final instar larvae (six instar) injected with FTZ-F1 dsRNA can not become prepupal, which might mean that FTZ-F1is required for the ecdysising and prepupal formation.