Scientia Agricultura Sinica ›› 2012, Vol. 45 ›› Issue (20): 4187-4196.doi: 10.3864/j.issn.0578-1752.2012.20.008

• PLANT PROTECTION • Previous Articles     Next Articles

Cloning, Expression Analysis and Binding Characteristics of Chemosensory Protein Gene AlucCSP1 in Apolygus lucorum (Meyer-Dür)

 HUA  Jin-Feng, ZHANG  Shuai, CUI  Jin-Jie, WANG  Dao-Jie, WANG  Chun-Yi, LUO  Jun-Yu, LU Li-Min   

  1. 1.中国农业科学院棉花研究所棉花生物学国家重点实验室，河南安阳455000
    2.河南大学植物逆境重点实验室，河南开封475000
  • Received:2011-10-08 Online:2012-10-15 Published:2012-02-21

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Abstract: 【Objective】The objective of this study is to clone a chemosensory protein gene AlucCSP1 from Apolygus lucorum, and to test it’s tissue-specific expression and binding characteristics.【Method】The AlucCSP1 was cloned from A. lucorum by RT-PCR. Using semi-quantitative RT-PCR and Real-time PCR methods, the expression pattern of AlucCSP1 in different adult tissues were determined. AlucCSP1 was expressed using pGEX-4T-1 vector and BL21 (DE3) prokaryotic expression system, then purified using GSTrap FF. The binding characteristics of AlucCSP1 with fifty-eight standard odorant samples and five cotton secondary metabolites were investigated using bis-ANS as fluorescence ligand.【Result】The open reading frame full length of AlucCSP1 (GenBank accession: JN573217) was 393 bp, encoding 130 amino acids, including 17 aa of signal peptide in the N-terminal. Sequencing and analysis indicated that AlucCSP1 was characterized by four conservative Cys. It was found that AlucCSP1 was dominantly expressed in the antenna of the female. The molecular weight of recombinant protein of pGEX /AlucCSP1 was about 39 kD. After purification with affinity chromatography and treatment with thrombin protease to cut off GST, the AlucCSP1 protein was obtained. AlucCSP1 had high affinity with gossypol, tannin, quercetin and rutin hydrate, the binding constant was 13.4, 32.7, 19.7 and 38.5 μmol•L-1, respectively .【Conclusion】AlucCSP1 was cloned from A. lucorum and it was dominantly expressed in the female antenna. This may be valuable for studying the role in A. lucorum chemical sensors of AlucCSP1.

Key words: Apolygus lucorum, chemosensory protein, expression profiles, prokaryotic expression, purification, competitive binding

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