Acta Entomologica Sinica ›› 2019, Vol. 62 ›› Issue (6): 672-684 .doi: 10.16380/j.kcxb.2019.06.003
• RESEARCH PAPERS •
ZHAO Jie1,2, WEI Qian1, REN Su-Wei1, LIU Xiao-Ning1,*
【Aim】 This study aims to clone and analyze the forkhead box protein A (FoxA)-like protein gene HarmFoxAl from Helicoverpa armigera, and to investigate its expression profiles after the larval midgut was subjected to 2-tridecanone treatment, so as to provide a theoretical basis for further studying the function and regulatory pathway of FoxA involved in the growth and developmental process of H. armigera. 【Methods】 The cDNA sequence of HarmFoxAl was cloned from the larval midgut of H. armigera, and its amino acid sequence and protein structure were analyzed. The recombinant vector pET32a-HarmFoxAl was constructed and transformed into Escherichia coli Transetta strain. The fusion protein His-HarmFoxAl was induced by IPTG in Transetta (pET32a-HarmFoxAl) strain, and was purified using the Ni-NTA affinity chromatography. The expression profiles of HarmFoxAl in different developmental stages (1st-6th instar larva and prepupa), tissues (fat body, midgut, integument and head) of the 6th instar larva, and midguts of the 10 mg/g 2-tridecanone treated 6th instar larvae of H. armigera at different time post treatment were detected by qPCR. 【Results】 The open reading frame of HarmFoxAl (GenBank accession no.: XM_021331806) is 669 bp, encoding 222 amino acids. The predicted molecular weight and isoelectric point of HarmFoxAl protein are 25.03 kD and 6.34, respectively. HarmFoxAl is a monomer protein without signal peptide, transmembrane region and disulfide bond, and its core sequence consists of four α-helixes and three β-sheets. After the Transetta (pET32a-HarmFoxAl) strain was induced by 0.5 mmol/L IPTG for 5 h at 25℃, a ~45 kD soluble fusion protein His-HarmFoxAl was expressed in the strain, which is consistent with the predicted molecular weight (42.8 kD). Developmental stage-specific expression profiles revealed that HarmFoxAl was expressed in the 1st, 2nd, 3rd and 6th instar larva and prepupa, with the highest expression level in the prepupa. Tissue expression profiles revealed that HarmFoxAl was expressed in the fat body, midgut and integument of the 6th instar larva, with the highest expression level in fat body, while not expressed in the head. When the 6th instar larvae were exposed to 10 mg/g 2-tridecanone, the expression level of HarmFoxAl in the midgut was significantly decreased first, and then gradually increased with the exposure time, being significantly higher than that of the control group at 48 h after treatment. 【Conclusion】 HarmFoxAl is highly expressed in the prepupal stage and larval fat body of H. armigera. After the H. armigera larvae are exposed to 2-tridecanone, the expression level of HarmFoxAl significantly decreases first and then gradually rises in larval midgut. Therefore, we presume that HarmFoxAl may play an important role in metamorphosis and metabolic detoxification of H. armigera.
forkhead box protein,
temporal and spatial expression,
ZHAO Jie, WEI Qian, REN Su-Wei, LIU Xiao-Ning. Cloning and expression profiling of the forkhead box protein A-like protein gene HarmFoxAl in Helicoverpa armigera (Lepidoptera: Noctuidae)[J].Acta Entomologica Sinica, 2019, 62(6): 672-684 .
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