Construction of Plant Expression Constructs Harboring Full-length Bt Cry1Ac22 Toxin Gene and Truncated Functional Domains of Bt Cry1Ac22 Toxin and Arabidopsis Transformation  

Zhuoming Liu1,2 , Yan Zhou1,3 , Youzhi Li3 , Shenkui Liu2 , Xuanjun Fang1,2,3
1. Hainan Institute of Tropical Agricultural Resources, Sanya, 572025, P.R. China
2. Alkali Soil Natural Environmental Science Center (ASNESC), Northeast Forestry Uinversity, Harbin, 150040, P.R. China
3. College of Life and Technology Science, Guangxi University, Nanning, 530004, P.R. China
Author    Correspondence 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
© 2011 BioPublisher Publishing Platform
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.
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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, Vol.2 No.2 (doi:10.5376/bm.2011.02.0003)


 The full-length Bt toxin gene (3 534 bp) and truncated functional domains of Bt toxin gene (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.

Bt Cry1Ac22 toxin; Bt toxin functional domain; Plant expression construct; Arabidopsis thaliana; Genetic transformation

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 â…  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 â…¡ 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 â…¢ 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 hydrolysed to the 65 kD core protein (Lenin et al., 2001). Therefore, it is essential  to carry out the research to express the total length cry1Ac22 and the functional domain from Bt W015-1.

In the experiment, we cloned the full length of cry1Ac22 gene of 3 534 bp in lenth and its functional domains of 1 959 bp by the PCR, and constructed them to plant expression vector pBI121 to make the constructs of pBI121-Cry1Ac22F and pBI121-Cry1Ac22T both carrying with Kanamycin selection marker.

Arabidopsis thaliana was transformed during the flowering stage mediated by Agrobacterium tumufaciens. Lots of transgenic Arabidopsis thaliana seeds with positive Kanamycin resistance were harvested in this research that facilitate the understanding of Bt toxin functions in plant transgenic breeding.

1 Results
1.1 Cloning of the full length cry1Ac22 gene and its function domain
cry1Ac22 full length gene of 3 534 bp (Figure 1A) and cry1Ac22 function domain of 1 959 bp (Figure 2A) were amplified by specific primers. Both of the target genes were ligated to pMD18-T vector and positive clones were identified by enzymetic digestion of BamHⅠand SalⅠ, respectively (Figure 1B). And then named as pMD18-T-Cry1Ac22F for full length gene (Figure 1C) and pMD18-T-Cry1Ac22T for truncated gene (Figure 2B), respectively. Finally, we sequenced the positive clones to validate the sequence of Cry 1Ac22 by GenBank database.

 figure 1 PCR amplification of cry1Ac22 and digestion of pMD18- T-Cry1Ac22F by restriction enzymes

 figure 2 PCR amplification of cry1Ac22T and digestion of cry1Ac22T with BamHâ… and Salâ… 

1.2 Plant expression construct of pBI121-Cry1Ac22F and pBI121-Cry1Ac22F
Full length gene cry1Ac22 cutting from recombinant plasmid pMD18-T-Cry1Ac22 was ligated into plant expression vector pBI121 to make a plant expression construct named as pBI121-Cry1Ac22F, and then was transformed into Escherichia coli JM109. While the construct of pBI121-Cry1Ac22T for truncated gene was constructed by the same way. Both of the constructs were validated by digesting with BamHâ…  and Salâ… . A 3.5 kb target fragment and a 18 kb vector fragment was cut for pBI121-Cry1Ac22F (Figure 3). Whereas a 1.9 kb target fragment and a 18 kb vector fragment for pBI121-Cry1Ac22T (Figure 4).

 figure 3 Digestion of pBI121-Cry1Ac22F with BamHâ… and Salâ… 

 figure 4 Digestion of pBI121-Cry1Ac22T with BamHâ… and Salâ… 

1.3 Identification of Agrobacterium recombinant plasmid by PCR
The constructs were transformed into Agrobacterium EHA105, and the plasmids were extracted by using commercial extract kits. The positive clones were amplified and identified by the same specific primers as for the constructs. A 3.5 kb target band for pBI121- Cry1Ac22F (Figure 5), and a 1.9 kb target band for pBI121-Cry1Ac22T (Figure 6), were amplified that indicated both of them have been transformed into Agrobacterium EHA105 successfully.

 figure 5 PCR identification of pB1121-Cry1Ac22F

 figure 6 PCR identification of pB1121-Cry1Ac22T

1.4 Identification of of transgenic Arabidopsis thaliana
The cry1Ac22 full length gene and cry1Ac22 truncated gene were transformed into the genome of Arabidopsis thaliana plants mediated by Agrobacterium, respectively. We identified that the plants of No.3, No.4, No.6, No.7, No.8 and No.10 were exsiting the 3.5 kb cry1Ac22 full length fragment, (Figure 7).

 figure 7 Identification of transgenic cry1Ac22F plants

Whereas for truncated cry1Ac22T transformation, we found that cry1Ac22T gene has been integrated into Arabidopsis thaliana plants with the plants of No.3, No.5, No.6, No.9, No.10 (Figure 8).

 figure 8 Identification of transgenic cry1Ac22T plants

2 Discussions
Bacillus thuringiensis cry1A toxin genes are the most commonly used as insecticidal targeted gene, among them, cry1Ac gene was extensively used for developing transgenic Bt cotton. There are numbers of reports that the full length cry1A genes were transformed into many crops including cotton, rice, potato, corn, canola, soybean, sugarcane, peanut etc. but the amounts of Bt toxins expressed in these transgenic plants were considerable low and were not enough toxic dose to the targeted pests (Romeise et al., 2006). These researches indicated that the full length sequence of the cry1A gene be not suitable for developing transgenic crops. Therefore, enhancement of Bt toxicity in transgenic plants would be essential goals in Bt transgenic breeding. 

In this research, we designed two kinds of plant expression constructs, pBI121-Cry1Ac22F and pBI1 21-Cry1Ac22T, and transformed into the  model of dicot plant, Arabidopsis thaliana. Both of the gene were transformed into Arabidopsis genome. The preliminary studies on the expressed toxin in Arabidopsis plant indicated that the expression of truncated domain be higher than that of full length gene (data not showed). We believed that the Bt toxin shoud be well designed for transgenic plants than completely employed from those deposited in GenBank.

3 Materials and Methods
3.1 Materials and reagent
Escherichia coli JM109, pMD18-T vector, pBI121 plant expression vector were deposited in our lab. DNA restriction endonucleases Sacâ… , BamHâ… , DNA Marker, T4 DNA ligase, Taq DNA polymerase and DNA extraction kit were bought from TAKARA company. Antibiotics, biochemistry and molecular biological reagents were bought from Shanghai Bioen gineering Company, Harbin Demei Biology Company etc. Primers were synthesized by Beijing Aoke Biotechnology Company. Other reagents were all domestic analytically purity.

3.2 Extraction of plasmid, purification and preparation of competent cell for E. coli
Plasmid DNA of E. coli and Agrobacterium were extracted by alkali lysis method (Zhang et al., 2000); the competent cells for the E. coli were prepared by the CaCl2 method followed by the method of Sambrook et al (1992). 

3.3 Primer designed and synthesized
Two pairs of specific primers were designed based on the conserved cry1Ac22 gene sequence and function domain (Table 1), which were synthesized by the Nanjing Jinsite Biotechnology Company. The restriction enzyme site BamHâ…  was added in the forward primer, the Salâ…  was added in the reverse primer.

 Table 1 PCR primers

3.4 Cloning of cry1Ac22F and cry1Ac22T
We respectively amplified the cry1Ac22F gene and cry1Ac22T using the W015-1 genomic DNA as templates with specific primer. PCR procedure is as following: 94℃ for 5 min in advance, 30 cycles of 94℃, 1 min, 54℃, 1 min and 72℃, 30 s, 72℃, 10 min for extension and then 4℃ until moved to use. The PCR products were electrophoretically separated in the agarose gel and then were recovered to ligate to the pMD18-T vector. Two sequencing clones of cry1Ac22F and cry1Ac22T were completed to be transformed into the competent cell. The positive recombinants were identified by the BamHâ… and Salâ… digestion and then were sequenced. 

3.5 Construct of pBI121-Cry1Ac22F and pBI121- Cry1Ac22T
pBI121 vector is a commonly plant expression vector driven by the CaMV35S promoter with kanamycin resistant gene. The recombinant plasmid of pMD18-T-Cry1Ac22F and pBI121-Cry1Ac22T were digested by the restriction enzymes BamHâ…  and Salâ… . The digested fragments were ligated into the pBI121 vector that was also digested with the same restriction enzyme, and then were transformed into E. coli JM109 and identified by the restriction enzyme. The positive clones of  pBI121-Cry1Ac22F and pBI121-Cry1Ac22T were screened by the enzyme digestion. Plant expression constructs of pBI121-Cry1Ac22F and pBI121-Cry1Ac22T were showed as the figure 9 and figure 10.

 figure 9 Structure of the construct of pBI121-Cry1Ac22F

 figure 10 Structure of the construct of pBI121-Cry1Ac22T

3.6 Identification of Agrobacterium recombinant costructs by PCR
These two constructed plant expression vectors were transformed into the Agrobacterium competent cells. The Agrobacterium plasmid DNA was extracted using the alkali split kits, the positive clones were identified by using the primers that were used to constructs of pBI121-Cry1Ac22F and pBI121-Cry1Ac22T in the Table 1. PCR procedure is followed as: 94℃, 5 min for pre-denaturation, 30 cycles of 94℃, 1 min, 54℃, 1 min and 72℃, 30 s, finally 72℃, 10 min for extension and then 4℃ for ever. The PCR products were electrophoretically separated in the agarose gel.

3.7 Transformation of  Arabidopsis 
Arabidopsis (Arabidopsis thaliana) was transformed by the approach of inflorescence infiltration, followed as the procedures of Clough with slight modifications (Clough et al., 1998). Agrobacterium baterials with constructs of pBI121-Cry1Ac22F and pBI1 21-Cry1Ac22T respectively were inoculated in 10 mL LB liquid medium, and shaked incubation at 28℃ over a night, and then added the incubating liquid into 500 mL LB liquid medium on the ratio of 1% bacteria. While the density of Agrobacterium reached OD600=1.0~1.2 at 28℃, the strains were collected by centrifuged  with 3 000 r/min at 4℃ for 15 min , After removing the supernatant liquid and the precipitate  was dissolved into medium with 1/2 MS with 5% (w/v) Sucrose and 0.044 µmol/L Benzylamine purine to suspend Agrobacterium and then make its final density reach 5 µL/L by adding surfactant SilwetL-77.

The inflorescence of Arabidopsis thaliana was immersed into the infiltration utensils filled with 500 mL suspensions by soaking in Agrobacterium liquid medium for 10~15 min and then continuing to culture in darkness for 2 days. Transformed plants were cultivated in the normal growth conditions.

3.8 Validation of positive transgenic Arabidopsis by kanamycin selection and PCR detection
Kanamycin resistant selection and PCR detection were carried out according to the experimental methods of Clough et al (1998). Putative transgenic Arabidopsis seeds were soaked by Agrobacterium, treated by the 95% ethanol and 3.0% sodium hypochlorite containing 0.05% Tween and then rinsed by 4~5 times in the sterile water. Steriled seeds were suspended in 2 mL 0.1% steriled agarose and seeded into 9 mm dish in diameter,  4 mg seeds per dish which contained 1/2×MS with 50 µg/mL kanamycin (KM) and 0.8% agorose. Culture conditions were as follows: Firstly, these dishes were treated at 4℃ 2 days, then treated at 24℃ for 23 h light and 1 h darkness cultured 7~10 days. Finally, The status of plantlet colour and root developmental condition was easily judged whether the plantlets harbored resistant gene.

The resistant plantlets were transplanted into culture pan. The transgenic Arabidopsis genomic DNA were extracted from the Arabidopsis young leaves carrying the kanamycin resistant gene by using modified CTAB method. Then employing non-transgenic Arabidopsis genomic DNA as negative control, pBI121-Cry1Ac22F and pBI121-Cry1Ac22T plasmid as positive control, we used transgenomic DNA as template to identify the target genes by using specific primers for cry1Ac22F and cry1Ac22F, the expected amplified products were 3 534 bp and 1 959 bp respectively. PCR procedure was as follows: 94℃ 5 min pre-denaturation, 30 cycles of 94℃ 1min, 54 ℃ 1min and 72℃ 30 s, final cycle , 72℃ 10 min for extension and then 4℃ for ever. The amplified products were separated by agarose gel electrophoresis.

Authors' contributions
ZL is the person who designed and conducted this experiment; ZL and YZ, finished the data analysis and paper preparation; SL conducted experimental design and result analysis; XF is the PI of this project involving in project design, data analysis, paper modification. All authors had read and consented the final text.

This research is partly sponsored by the National 863 Project ( No. 2004AA2111112). Authors would like to appreciate Dr X Zhang who provided solid technological supports and helpful advice during the experiment in ASNESC of Northeast Forestry University. Thanks for two anonymous reviewers with their critical comments. In this paper we mentioned some reagent suppliers and sequencing service providers, that doesn't mean we would like to recommend or endorse the production of theirs.

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