Scientia Agricultura Sinica ›› 2012, Vol. 45 ›› Issue (18): 3755-3763.doi: 10.3864/j.issn.0578-1752.2012.18.008

• PLANT PROTECTION • Previous Articles     Next Articles

Cloning, Expression and Activity Analysis of Carboxylesterase Gene from Aphis glycines (Hemiptera: Aphididae)

 YANG  Shuai, WANG  Ling, ZHAO  Kui-Jun, HAN  Lan-Lan   

  1. 1.东北农业大学农学院，哈尔滨 150030
  • Received:2012-01-04 Online:2012-09-15 Published:2012-04-01

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Abstract: 【Objective】 The objective of this study is to clone carboxylesterase gene and provide a molecular basis for insecticide resistance of Aphis glycines. 【Method】 A full-length cDNA was cloned by using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods, and the carboxylesterase gene was named AgCarE, and inserted into Escherichia coli. The carboxylesterase activity of AgCarE was characterized with the α-NA as a substrate and its enzyme activity was analyzed. 【Result】 The AgCarE is 1 946 bp in length, and the open reading frame encodes 526 amino acids (GenBank accession number is JF970181). The predicted isoelectric point of AgCarE is 6.08. The AgCarE possesses a catalytic triad, consisting of a Ser, a His and a Glu residue (Ser186, Glu313 and His434), which is characterized by carboxylic esters (EC: 3.1.1.-). The AgCarE was recombined into pET21b vector. Then the HIS fusion protein was expressed by induction with IPTG. The result of SDS-PAGE and Western-blot analysis showed that the AgCarE protein from A. glycines was expressed in E. coli BL21 which induced by IPTG, and its MW was found to be about 59 kD, similar as the predicted result. The activity for AgCarE is 0.036 mmol/100 μL enzyme buffer. 【Conclusion】 A cDNA encoding carboxylesterase from A. glycines was cloned and expressed. The results will further explore the three-dimensional structure, hydrolysis of carboxylesterase and provide a possibility for the design of new types of pesticides.

Key words: Aphis glycines, carboxylesterase, gene clone, activity analysis

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