Construction and analysis of Sip1Aa insecticidal protein random recombination library |
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Authors: | Ming-Yue Ding Jing Wang Lin Wang Ji-Guo Gao Rong-Mei Liu Hai-Tao Li |
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Institution: | 1. College of Life Science, Northeast Agricultural University, Harbin 150030, China;2. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China |
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Abstract: | The Sip1Aa protein from Bacillus thuringiensis is highly toxic to Colaphellus bowringi Baly. In order to obtain mutant proteins with higher insecticidal activity, a random recombinant library of Sip1Aa protein was constructed using error-prone PCR. A total number of 100 positive transformants were randomly selected for sequence determination, and 25 mutants (M1 to M25) were selected and expressed the respective Sip1Aa mutants. These Sip1Aa variants had a total of 29 base mutations, with an average of 1.2 base mutations per mutant. Compared with that of the wild-type Sip1Aa protein, the insecticidal activity of the mutants M1 (A31G, Y118C, D227E), M5 (K168R) and M21 (I307T) was significantly decreased, with and LC50 values 4 to 6 times higher than the Sip1Aa protein. The mutant M8 (R174S) showed increase in the insecticidal activity against the Colaphellus bowringi Baly was obtained, with an LC50 value 4-fold less than the Sip1Aa protein. The results of this study provide reference for the molecular modification of Sip1Aa protein and the study of key sites of its insecticidal activity. |
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Keywords: | Sip1Aa Random recombination library Directed evolution |
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