Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (8): 1493-1503.doi: 10.3864/j.issn.0578-1752.2018.08.007

• PLANT PROTECTION • Previous Articles     Next Articles

Cloning and Expression of Phosphatidylethanolamine Binding Protein in Helicoverpa armigera

ZHAO Jie1, REN SuWei1, LIU Ning2, AI XinYu1, MA Ji1, LIU XiaoNing1   

  1. 1College of life science and technology, Xinjiang University, Urumqi 830046; 2Institute of Crop Variety Resources, Xinjiang Academy of Agricultural Sciences, Urumqi 830091
  • Received:2017-09-15 Online:2018-04-16 Published:2018-04-16

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Abstract: 【Objective】The objective of this study is to clone and analyze phosphatidylethanolamine binding protein (PEBP) gene from Helicoverpa armigera, investigate the temporal and spatial expression profile in H. armigera, and test the expression of HaPEBP after 2-tridecanone treatment in the 6th instar larval midgut of H. armigera. The results will provide a theoretical basis for further studying the function of HaPEBP and select the gene as a molecular target to regulate the population of H. armigera. 【Method】 The cDNA sequence of HaPEBP was obtained from midgut of 6th instar larvae by using RACE technique, and its amino acid sequence and protein structure were analyzed. The recombinant vector pET32a-HaPEBP was constructed and transformed it into Escherichia coli BL21 (DE3) strain. The fusion protein was induced by IPTG and identified by SDS-PAGE to confirm its distribution. the His-HaPEBP was purified using Ni-NTA affinity chromatography. The HaPEBP expression profile at different developmental stages, tissues and under 2-tridecanone treatments was determined by qRT-PCR. 【Result】 The HaPEBP cDNA sequence is 760 bp, and its ORF is 588 bp, encoding 195 amino acids. The predicted molecular weight and isoelectric point of the protein are 21.76 kD and 5.93, respectively. The HaPEBP is a cytoplasmic monomer protein without signal peptide, transmembrane region and disulfide bond, which consists of 4 α-helixes and 9 β-sheets. The soluble fusion protein, which was about 40 kD consistent with predicted 39.1 kD, was synthesized in BL21-pET32a-HaPEBP strain by 1 mmol·L-1 IPTG induced 4 h at 37℃. And then the pure His-HaPEBP (183.3 ng·μl-1) was obtained through Ni-NTA column and imidazole gradient buffers. HaPEBP was expressed at all larval stages (1st-6th instar larvae and prepupa), and the highest expression level was observed in the 6th instar larvae. It was also expressed in the fat body, midgut, head and integument of the 6th instar larvae and the highest expression was found in the fat body. The expression of HaPEBP in the midgut of 6th instar larvae decreased after treatment with different concentrations of 2-tridecanone. In the low concentration groups (2.5 and 5 mg·g-1), the expression of HaPEBP significantly decreased at 6 h and gradually increased over time. However, the expression of HaPEBP decreased at 12 h in the high concentrations (10 and 15 mg·g-1), the mRNA content decreased firstly and then increased over time. 【Conclusion】The HaPEBP cDNA was cloned and analyzed. The fusion protein His-HaPEBP was synthesized and purified using the prokaryotic expression system. The results of temporal and spatial expression showed that the HaPEBP was highly expressed in 6th instar larvae and fat body. The expression of HaPEBP significantly decreased after treatment with 2-tridecanone in the midgut of 6th instar larvae.

Key words: Helicoverpa armigera, phosphatidylethanolamine binding protein, prokaryotic expression, temporal and spatial expression, 2-tridecanone

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