Temperature And Juvenile Hormone Coordinately Regulate Vg Expression In Helicoverpa Armigera Via HSF

The cotton bollworm,Helicoverpa armigera belongs to the family of Noctuidae in the order of Lepidoptera.It is an important agricultural insect pest attacking more than 200 crops including cotton,corn and pepper and causing considerably economic loss every year.H.armigera is known with its strong fecundity.An adult female of H.armigeraC-C.Vitellogenin(Vg)synthesis,the prerequisite for egg prodcution is one of key scientific issues in the study of insect reproduction.In many insects including H.armigera,Vg synthesis is regulated by juvenile hormone(JH).Under JH action,Vg is massively synthesized in the fat body,transported into the ovary by hemolymph,and deposited in oocytes in the form of vitellin(Vn).However,the molecular mechanism underlying coordinate regulation of Vg expression and vitellogensis by temperature and JH remians unclear.Our preliminary studies have demosntrated that heat shock factor(HSF)is involved in the coordinate regulation of Vg expression by environmental temperature and JH,but the molecular mechanism needs to be explored.In this thesis research,the adult females of H.armigera were used as experimental materials,and the fat body samples were collected at 6 h,12 h,24 h,48 h and 72 h post adult emergence.Western blot showed that the expression of HSF and Vg was positively correlated.Treatment of a JH analog(JHA),methoprene induced Vg expression in the fat body.After HSF RNAi,the levels of Vg mRNA and protein significantly decreased.As a consequence,ovarian development and oocyte maturation were blocked.In addition,HSF RNAi significantly inhibited JH-induced Vg expression.The above results indicate that HSF is likely involved in JH-stimualted Vg expression.When HSF was overexpressed in Drosophila S2 cell lines,application of JH led to increased phosphorylation of HSF.When the PKA inhibitor,H89 was added into S2 cells prior to JH treatment,HSF phosphorylation was inhibited,indicating that JH stimulates HSF phosphorylation via PKA.C,HSF was only detected in the nuclei.Interestingly,further treatment of JH had no effect on HSF subcellular localization.Analysis of Vg upstream promoter region revealed six potential HSF binding sites.Further dual luciferase reporter assay demonstrated that a sequence containing the DNA element,ATTCCATTCA was recognized by HSF.The Vg promoter activity assay showed that the expression levels of RFP-His increased along with elevated JH titer and treatment duration.Overexpression of HSF significantly enhanced JH-induced RFP-His expression,suggesting the induction of HSF in Vg transcription.This dissertation research demonstrated for the first time that HSF was involved in the coordinate regulation of Vg expression by environmental temperature and JH.JH induced HSF phosphorylation via PKA,whereas higher temperature such as 27? stimulates HSF sublocalization in the nuclei.HSF was likely to bind the upstream DNA element,ATTCCATTCA of Vg and stimulated Vg transcription.The findings provide new insights into our understanding in JH-and environmental temperature-dependent Vg expression and fecundity.