As we all know, the feature of Bacillus thuringiensis (Bt) is able to generate spores, accompanied by a parasporal crystal protein, also known as insecticidal crystal proteins (ICP for short). The vast majority of ICP expression is dependent on spore formation, that is, only in the sporulation phase will produce insecticidal crystal proteins (ICPs) (Liu et al, 2010, 2011). However, Cry3Aa is an exception that expression of the Cry3Aa protein happens in the vegetative phase and end in sporulation (Schnepf et al, 1998). Studies have shown that promoter sequence of cry3Aa gene is different from other ICP gene’s promoter. Transcription of cry3Aa relies on unique ςA factor in the vegetative phase, whereas other ICP gene’s expression is dependent on the activated ςE factor and ςK factor in the sporulation phase (Schnepf et al, 1998). For this reason, whether can cry3Aa gene promoter be applied to drive the other cry genes expressing insecticidal crystal protein in order to achieve the expression of parasporal crystal protein in the vegetative phase of Bacillus thuringiensis?

In this study we attempt to design an expression vector that is capable to express insecticidal crystal protein in the vegetative phase of Bacillus thuringiensis. To this end, we designed a pair of primer (C3-5 and C3-3) based on the known cry3Aa gene promoter, and used Bacillus thuringiensis DNA extracted from Bt subsp. tenebrions (Btt) as a template, then we amplified a target DNA fragment of 575 bp to ligate into the vector pBU4 to build a vector, pBUC3. Subsequently, we inserted cry1Ac31 gene into the pBUC3 vector, finally, we transformed the expression vector containing cry1Ac31 gene into Bt non-crystal IPS mutant strain through electronic impulse transformation approach, which were verified by PCR, microscopy and SDS-PAGE, in order to further provide a new way to realize multi-gene expression in Bacillus thuringiensis.

1 Results and Analysis
1.1 To obtain cry3Aa promoter gene
We designed a pair of primer, C3-5/C3-3, to amplify the target fragment of 575 bp by using Pfu DNA polymerase and Btt DNA template (Figure 1). Sequencing analysis showed that -35 region (GATTAAGA) of the fragment exists between the site of 402 bp to 409 bp, and -10 region (TATAAATT) exists between the sites of 425 bp to 433 bp. While the transcription initiate site at 438 bp, STAB-SD sequence (GAAAGGAGG) appears between the sites of 443 bp to 451 bp, and ribosome binding site (RBS) (GAAAGGGAGG) between the site of 550 bp to 560 bp. It was clear that the obtained promoter fragment contains the complete promoter domain (Figure 2).

Figure 1 The promoter fragment amplified by PCR

Figure 2 Sequence analysis of the amplified promoter and its domain structure

1.2 To build expression vectors pBUC3 for Bt vegetable growth phase
We ligated the 575 bp length promoter sequence into pBU4 vector to make a new vector named pBUC3. The positive clones of new vector were identified by PCR that contain the 575 bp promoter fragment (Figure 3). The positive clones were further digested with Fokâ… , which the restriction digesting profile of No 1 clone was consistent with the expected result analyzed by Vector NTI software, there has been a 526 bp specific bands (Figure 4). Final sequencing results verified the No 1 clone met the design goals of this study that would be the expression vector expressing in the vegetable growth phase of Bt, the clones were named pBUC3.

Figure 3 Positive clones Screened by PCR

Figure 4 Restriction enzymetic analysis of recombinant plasmid (No.1) digested by Fokâ…

1.3 cry1Ac31 gene expression in Bt IPS
We inserted cry1Ac31 gene into pBUC3 vector to make an expression construct named pBUC3-1Ac31, that were transformed into Bt non-crystal mutant strain IPS by electronic impulse transformation method. Positive clones were screened by PCR amplification, and the results showed that selected three clones contain the target bands (Figure 5). By the observation of optical microscope, we can visually see that one of the clone, IPSC3-1, have been able to generate a large number of bidiamond-shaped insecticidal crystal, while wildtype strain BtS3299-1 and non-crystal mutant IPS has no crystal any more (Figure 6). Subsequent SDS-PAGE protein electrophoresis confirmed (Figure 7) that IPSC3-1 at 12 h after inoculation will be able to produce 130 kD protein band, whereas IPS and BtS3299-1 no protein band. It can be proved that cry1Ac31 gene expressed in the IPS in the vegetable growth phase.

Figure 5 PCR for screening positive clones

Figure 6 Light micrographs of the tested strains observed after 18 hours culture

Figure 7 Cry1Ac31 protein generated after 18 hours culture identified by SDS-PAGE

2 Discussion
The pBU4 vector, having multiple cloning restriction sites with EcoRâ… , Sacâ… , Kpnâ… , Xmaâ… , BamHâ… , Xbaâ… , Salâ… , Pstâ… , Sphâ… , and Hindâ…¢ and being resistance to Amp and Tc , can replicate in the species crossing E.coli and Bacillus strains. Therefore, the pBUC3 built in this research has the feature of pBU4, which can replicate self and express in Escherichia coliand Bacillus, this feature would be a prerequisite for the vector to be transformed into Bacillus strain to express the proteins. As pBUC3 has a strong promoter for expressing in Bacillus, and its expression does not rely on the spore formation of Bacillus, therefore, in practical applications, cell culture time of this new vector is much shorter than that relying on spore forming Bacillus strains.

Different ICP gene expression in the same strains is concentrated in the spores of each other between the competitive (Sanchis et al., 1996). Therefore, we can assume that two insecticidal protein genes expressing in the vegetative phase and spore forming phase, respectively, may have the better effect than that expressing in spore forming period together. Furthermore, if an ICP gene can express not only in the vegetable growth phase but also in the sporulation, the expression levels may be much higher than that only expressing in the vegetable growth phase or in the spore forming period.

3 Materials and Methods
3.1 Strains and plasmids
The strains and plasmids used in this research were shown in Table 1. The Bt strain S3299-1is a wild type strain harboring cry1Ac31 gene. It was collected by Hainan Institute of Tropical Agricultural Resources. IPS was Bt non-crystal mutant strain, which was presented by Dr. Neil Crickmore from University of Sussex in UK.

Table 1 Strains and Plasmids used in this research

3.2 Culture media, antibiotics and reagents
The Bt and E.coli was cultured in the LB liquid and solid culture medium, respectively. The SOC culture medium was used for cell recovery after transformation. The temperature for Bt culture was 30℃, whilst the temperature for E.coli was 37℃. The concentration of ampicillin and tetracycline was 100 μg/mL and 50 μg/mL, respectively. PCR reagents were purchased from TaKaRa Biotechnology (Dalian) Co., Ltd.; the PCR primers were synthesized by Beijing Sunbiotech Co., Ltd. The pfu DNA polymerase and T₄DNA polymerase were purchased from Sangon Biotech (Shanghai) Co., Ltd. Restriction enzyme and T₄DNA ligase were purchased from MBI Co., Taq DNA polymerase was provided by Hainan Institute of Tropical Agricultural Resources.

3.3 PCR amplification of cry3Aa promoter and recovery of its products
A pair of primers, C3-5/C3-3, was designed based on the cry3Aa promoter, the sequence was as follows, C3-5: 5’-AGCTTAATTAAAGATAATATCTTTGAA-3’; C3-3: 5’-GGATTCATTTTTCTTCCTCCCTTTCTT-3’.

Then, we employed DNA of Bt strains Btt as the template to amplify the cry3Aa promoter by using the primers C3-5/C3-3, as well as the pfu DNA polymerase. PCR products were recoveried and purified by using agarose gel DNA extraction kit. The PCR reaction conditions was referred to Song et al (1998)’s method.

The positive clones of E.coli carrying pBUC3 were selected also using primers C3-5/C3-3. We amplified the positive clones by using the plasmid DNA as the template and the Taq DNA polymerase. The IPS positive clones harboring pBUC3-1Ac31 were selected by employing total DNA as template, using primers C3-5/C3-3 and K2-5/K2-3, as well as Taq DNA polymerase.

3.4 Construction of expression vector pBUC3 in Bt vegetative stage
The plasmid pBU4 was completely digested by using restriction endonuclease EcoRâ…  at 37℃ in waterbath. The digested products were separated by 1.0% agarose gel electrophoresis. After gel recovery and purification, the sticky end of the purification products was filled with T₄DNA polymerase. And then the products were precipitated with an equal volume of isopropanol At -20℃ for 10 min. Recovered the precipitation by centrifuging at 12 000 r/min for 10 min, and then added 10 μL TE to dissolve. Subsequently, the cry3Aa promoter fragment obtained by PCR and the vector pBU4 filled with sticky end with T₄DNA ligase was connected, which was transformed into E.coli JM110. The recombinants were screened in LB medium containing ampicillin (100 μg/mL) and tetracycline (50 μg/mL). Positive clones were identified by PCR with C3-5/C3-3 primers and enzyme digestion. In order to further determine whether the promoter sequence insertion the pBU4 plasmid or not, we sequenced the positive clone. And the expression vector obtained was named as pBUC3.

3.5 The expressionof cry1Ac31 gene in IPS
The recombinant plasmids pCRY1Ac31 and the vector pBUC3 were completely digested with restriction endonucleases BamHâ…  and Salâ…  at 37℃ water. The digested products were separated by 1.0% agarose gel electrophoresis. After gel recovery and purification, the full-length of cry1Ac gene was connected to pBUC3 by Using T₄DNA ligase. Then the ligated products were electrotransformed into the expression host Bacillus thuringiensis mutant IPS, non-crystal. The recombinants were screened in LB medium containing ampicillin (100 μg/mL) and tetracycline (50 μg/mL), and then positive clones were identified by PCR. After the verified positive clones were inoculated into LB solid medium for 12 h, we observed the parasporal crystal with microscope. Finally, we validated its expression on 7.5% SDS-PAGE electrophoresis analysis.

The universal primers K2-5/K2-3 were used for identification of the cry1Ac gene, the sequences were as follows, K2-5: 5’-AGGACCAGGATTTACAGGAGG-3’; K2-3: 5’-GCTGTGACACGAAGGATATAGCCAC-3’. Preparation of electrotransformation competent and electrotransformation method of Bt strains were referred to Sambrook et al (1989). Microscope observe method referred to Xie et al (2010)’s method And the SDS-PAGE method refers to Liu et al (2010).

Authors' contributions
CL conceived the experimental design and carried out the experiments; WFZ and YZ analyzed the data, wrote and revised the manuscript; YZL conducted experimental design, data analysis and paper modification. All authors have read and approved the final manuscript.

Acknowledgments
This work was initiated by the China National Bt Collection Initiative Project. Part of the experiments was completed in Prof. Li Youzhi’s lab. We appreciated their technology support and useful suggestions. All the authors appreciated two anonymous reviewers for their useful critical comments and revising advice to this paper. Mention of trade names or commercial products in this paper is solely for the purpose of providing specific information and does not imply recommendation or endorsement by authors or institutes or University involved in this study.

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