Cloning of the gene encoding an insecticidal protein inPseudomonas pseudoalcaligenes
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- 作者：Jie Zhang (1)
Jian Zhao (1)
Daxu Li (1)
Sujun Liu (1)
Li Li (1)
Qun Sun (1)
Ming Huang (1)
Zhirong Yang (1) - 关键词：Pseudomonas pseudoalcaligenes ; insecticidal protein ; locust ; gene cloning
- 刊名：Annals of Microbiology
- 出版年：2009
- 出版时间：March 2009
- 年：2009
- 卷：59
- 期：1
- 页码：45-50
- 全文大小：691KB
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Jian Zhao (1)
Daxu Li (1)
Sujun Liu (1)
Li Li (1)
Qun Sun (1)
Ming Huang (1)
Zhirong Yang (1)
1. Life Sciences School, Key laboratory for Bio-resources and Eco-environment of Chinese Education Ministry, Sichuan University, 610064, Chengdu, Sichuan, P.R. China - ISSN：1869-2044
文摘
Pseudomonas pseudoalcaligenes strain PPA (first isolated from yellow-spined bamboo locust,Ceracris kiangsu, in 1991) is an effective biological control agent for locust.Escherichia coli DH5α was transformed using a partialSau3AI digest ofP. pseudoalcaligenes chromosomal DNA ligated withpUC19 vector cut byBamHI. The resulting genomic library was screened using an oligodeoxynucleotide probe derived from the N-terminal sequence of the purified insecticidal protein. Five positive clones were obtained after two rounds of screening. The 1.4 kbBamHI-KpnI fragment of the smallest clone (pSC1) contained the N terminal sequence of the insecticidal protein. Further sequencing identified an open reading frame encoding a polypeptide of 276 amino acid residues that is thought to contain a signal peptide of 22 amino acids and a mature protein of 254 amino acids. The calculated molecular weight of 26.7 kDa for the putative mature protein is close to that of the native insecticidal protein. The insecticidal protein lacked significant identity with toxic proteins from other bacteria upon comparison of amino acid sequences. ABamHI-KpnI fragment was cloned intopT7-6. A novel protein was expressed inE. coli BL21 (DE3) and its western blot similar to natural toxin.