Page 4 - BM 2011 Vol.2 No.3

Bioscience Methods
BM 2011, Vo.2, No.3 http://bm.sophiapublisher.com
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Research Article Open Access
Construction of Plant Expression Constructs Harboring Full-length Bt Cry1Ac22
Toxin Gene and Truncated Functional Domains of Bt Cry1Ac22 Toxin and
Arabidopsis Transformation
Zhuoming Liu 1,2 , Yan Zhou 1,3 , Youzhi Li 3 , Shenkui Liu 2 , Xuanjun Fang 1,2,3
1. Hainan Institute of Tropical Agricultural Resources, Sanya, 572025, P.R. China
2. Alkali Soil Natural Environmental Science Center (ASNESC), Northeast Forestry University, Harbin, 150040, P.R. China
3. College of Life and Technology Science, Guangxi University, Nanning, 530004, P.R. China
Corresponding author: xuanjunfang@hitar.org; Author
Bioscience Methods 2011, Vol.2 No.3 DOI:10.5376/bm.2011.02.0003
Received: 27 Dec., 2010
Accepted: 19 May, 2011
Published: 30 May, 2011
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article as:
Liu et al., 2011, Construction of Plant Expression Constructs Harboring Full-length Bt Cry1Ac22 Toxin Gene and Truncated Functional Domains of Bt
Cry1Ac22 Toxin and Arabidopsis Transformation, Bioscience Methods, doi:10.5376/bm.2011.02.0003
Abstract The full-length Bt toxin gene (3 534 bp) and truncated functional domains of Bt toxin (1 959 bp) of cry1Ac22 were
amplified by PCR from Bt strain W015 -1. The PCR products were ligated into plant expression vector pBI121 cutting off the
ß- glucuronidase gene to make the constructs of pBI121- Cry1Ac22F and pBI121- Cry1Ac22T. The constructs carrying with
Kanamycin resistant marker were transferred into T-DNA vector and then validated by restricted enzyme digestion and PCR
identification. Arabidopsis transformation with the transfered T-DNA vector were performed during the flowering stage mediated by
Agrobacterium tumufaciens. Transgenic Arabidopsis seeds with positive Kanamycin resistance were harvested in this research that
facilitate the understanding of Bt toxin functions in plant transgenic breeding.
Keywords Bt Cry1Ac22 toxin; Bt toxin functional domain; Plant expression construct; Arabidopsis thaliana; Genetic
transformation
Background
Bt-toxin protein gene is the most commonly used
insect-resistant gene, which is widely applied in crops
such as transgenetic Bt cotton, Bt rice and woods like
poplar eucalyptus to kill the pests of Lepidoptera,
Coleoptera and so on (van Wordragen et al., 1993).
cry1Ac is a insect disinfestation gene which has been
extensively used in transgenetic cotton exhibited high
toxicity against Lepidopteran. According to the
research, toxicity peptide of insecticidal crystal
protein Cry1Ac is composed of three typical structural
domains. Structural domain Ⅰ locating in peptide
chain at N-terminus, which is an alpha helical bundle,
formed by six or seven amphipathic alpha helix
enclosing a hydrophobic alpha helix, participated
plasmalemmal perforation.
The domain Ⅱ is located in the middle of peptide
chain. It is an anti-parallel β- pleated sheet consisted
of three Greek key topological structures. And its
apical loop participate in the integration of toxin and
receptor protein. While the domain Ⅲ at C-terminus
is a sandwich structure which consisted of two
anti-parallel β-pleated sheets, lining up in jelly roll
topological structure, and can prevent protease having
over degradation on toxin molecule (Schnepf et al., 1998).
cry1Ac22 gene was cloned from the strain W015-1
isolated from the dissected guts of diapausing larvae
of the silkworm (Xie et al., 2010), which can be
efficiently expressed in the E. coli (Liu et al., 2010)
and yeast (Liu et al., 2010). The expressional products
have showed high insecticidal activity against larvae
of Plutella xylostella. The expressed Cry1Ac22
toxicity protein in E. coli can be hydrolysed into a
molecular weight of about 65 kD protein, which still
showed insecticidal activity (Xie et al., 2010). And it
is consistent with the former report that the Cry1Ac
protein expressed in the Bacillus thuringiensis can be