Bacillus thuringiensis (Bt) is a ubiquitous gram- positive bacterium, which produces parasporal crystal protein during sporulation that has insecticidal action (δ-endotoxin). It attracts increasing attention due to its insecticidal properties to agricultural pests(Li et al., 1991; Knowles and Ellar, 1986). Bt toxin Cry1Ac22 insecticidal crystal proteins are isolated from Bacillus thuringiensis W015-1 in the intestines of diapausing larvae of silkworm (Bombyx mori). It is known that Bt W015-1 has higher insecticidal activity than HD73 on some lepidopteran insects such as Clanis bilineata Walker, Helicoverpa armigera, Spodoptera litura, Plutella xylostella (Xie et al., 2010). 

Cry1Ac22 insecticidal crystal protein, encoded by cry1Ac22 gene, is constituted by 1 178 amino acids, It is different from Cry1Ac1 protein (isolated from HD73 (Adang et al., 1985)) in amio acid sequence at sites of 233 (T/R), 448 (M/I) and 1158 (K/E) (Xie et al., 2010). cry1Ac22 gene can express 133 kD proteins in Escherichia coli, and the purified proteins show high insecticidal activity to Plutella xylostella (Xie et al., 2010; Liu et al., 2010). Taking into account that W015-1 was isolated from the intestines of diapausing larvae of silkworm and its cry gene shows apparent diversity with HD73, the strain and its insecticidal proteins could be used as candidate strain for integrated control and resistance management of agricultural pests.

Figuring out the expression and localization of Cry1Ac22 in Eukaryotic cell is of great significance to the study regarding the expression of cry1Ac22 in higher plant. In this research, we constructed GFP-labeled Cry1Ac22 fusion protein by applying the GFP of Aequrea victoria as reporter molecules, and investigated preliminarily the expression and localization distribution of the fusion protein in yeast, expecting to provide theoretical guidance for the research of Bt transgenic plants utilizing cry1Ac22 gene. 

1 Results and Analysis
1.1 Construction and identification of pGFP- Cry1Ac22 vector
The GFP gene used in this study is from mammalian expression vector pGFP. BamHⅠ and KpnⅠ were inserted into the amphi of the linear vector through the enzyme digestion and ligation. pMD18- T-Cry1Ac22 was constructed with the primers designed according to the cry1Ac22 gene sequence, digested by BamHⅠ and KpnⅠ, and obtained a 2 178 bp fragment (Figure 1B); pGFP vector is digested by the BamHⅠ and KpnⅠ and obtained a 3 400 bp pGFP linear fragment (Figure 1A). The targeted fragments were recycled, ligated at 16℃, and then transformed into JM109 competent cells. Identification and screening of the positive clones were performed by BamHⅠ and KpnⅠ digestion (Figure 2). From figure 2, we can see that lane No. 1,2,3,4,5,6,7,9,10 are positive recombinants showing a 2 178 bp cry1Ac22 target fragment and a 3 400 bp pGFP vector fragment. The results indicated that the pGFP-Cry1Ac22 is constructed successfully.

 

Figure 1 Enzyme digestion of pGFP (A) vector and pMD18- T-Cry1Ac22 (B) with BamHâ…  and Kpnâ…

Figure 2 Identification of transformants using BamHâ…  and Kpnâ…

1.2 Construction and identification of pYES2- Cry1Ac22-GFP yeast expression vector
Positive clones was digested by BamHⅠ and NotⅠ and obtained a 3 000 bp gene fragment, which is a fusion gene of cry1Ac22 gene and GFP gene (Figure 3). pYES2 vector was digested by BamHⅠ and NotⅠand obtained a 5.9 kb pYES2 vector fragment (Figure 3). The targeted fragments were recycled, ligated at 16℃, and then transformed into JM109 competent cells. The recombinant was digested with BamHⅠ and NotⅠ, and obtained a 3 000 bp target gene fragment and a 5.9 kb pYES2 vector fragment (Figure 4). These results indicated that No. 1,2,3,4 were positive recombinants and the yeast expression vector pYES2-Cry1Ac22-EGFP was constructed successfully.
 

Figure 3 Enzyme digestion of pGFP-Cry1Ac22 and pYES2 with BamHâ…  and Notâ…

1.3 Transformation of pYES2-Cry1Ac22-GFP vector
In order to study the expression and localization of Cry1Ac22-GFP in Eukaryotic cell, transformation into yeast was carried out. Theoretically, GFP- Cry1Ac22 would expressed in yeast after transformation. pYES2-Cry1Ac22-GFP vector was transformed into the Saccharomyces cerevisiae INVScl strains. Observation by fluorescence micro- scope shows that flourescence was distributed in the form of puncta, which indicated that Cry1Ac22- GFP protein can be expressed in yeast cells (Figure 5).
 

1.4 Expression of Cry1Ac22-EGFP fusion gene in yeast
The recombinant yeast induced by galactose was collected at different times, and detected by SDS- PAGE (Figure 6). A new protein was detected in the yeast crude protein induced by the galactose, with a molecular weight of about 130 kD. However, no such protein was detected in the crude protein that is not induced by the galactose (lane one) .The results primarily indicate that the recombinant pYES2-Cry1Ac22-GFP expressed Cry1Ac22-GFP fusion protein under the induction of galactose, and the expression was of high performance. The expression level tend to be stable as the elongation of induction.
 

1.5 Localization of cry1Ac22 gene in yeast
PYES2-Cry1Ac22-GFP vector was transformed into the yeast, induced by the galactose, and directly observed under confocal laser scanning microscopy. After induction for 12 h, 60% yeast cells are observed with green fluorescent under inversion fluorescence microscope. Fluorescent proteins were distributed in the form of dispersion in the whole yeast cell transformed with pYES2-GFP vector (Figure 7), while in the yeast transformed with pYES2-Cry1Ac22-GFP, fluorescent proteins were distribute in plasma membrane and cytoplasm, but not in nucleus. Domainâ… in the N-terminal of cry1Ac22 gene is constituted by a group of α helical bundle formed by six or seven amphiphilic α helixes surrounding a hydrophobic α helix. The hydrophobic part acts as the function domain of specific virulence and participates in the perforation of cell membrane. The result is in accordance with the localization prediction.
 

2 Discussion
PYES2 (5 900 bp) used in this study is a yeast-Escherichia coli multicopy shuttle plasmid. It is a secretion type expression vector controlled by T7 promoter, and can replicate autonomously in S. cerevisiae and E. coli. When multiclone was inserted to recombinant vector, it can be screened by the insertional inactivation of LacZ gene, which also contains resistance gene.

PYES2 contains yeast URA gene, and can act as the auxotroph screening tag. Taking the auxotroph saccharomyces cerevisiae as model organism, pYES2-Cry1Ac22-EGFP shuttle vector containing reporter gene and enhanced green fluorescent protein (EGFP) gene was constructed successfully (Misteli and Spector, 1997). The vector can replicate in the E. coli and express in the S. cerevisiae. It forms fusion protein in the same open reading fragment. EGFP proteins display green fluorescence under the excitation of ultraviolet light. And its ligation with target gene will not affect the expression of target protein, Besides, it could fuse with target gene and label the expression status of target gene.

After the transformation of recombinant plasmid into the auxotroph S. cerevisiae, the expression of yeast in different times was observed under fluorescence microscope. If the recombinant plasmid can replicate in the cell, the EGFP will be expressed displaying green fluorescence under the excitation of ultraviolet light. The result indicated that green fluorescence can be observed in the transformed yeast under fluorescence microscope, which tended to be most powerful after induction for 12 h. No fluorescence was detected in the control yeast.

In this study, we constructed the Cry1Ac22 fusion protein marked with green fluorescent protein (GFP), and investigate the expression and localization of this fusion protein in Saccharomyces cerevisiae. This study would provide the theoretical guidance for studying the function of Bt insecticidal proteins, and presents insights for the trail of tracing transgenes protein.

3 Materials and Method
3.1 Plasmid and strains
Strains and plasmids used in this study were stored in Haide Institute of Tropical Agricultural Resources (HITAR) (Table 1).
 

3.2 Culture media
The culture media for yeast growth is SD: yeast nitrogen-containing bases 0.67%, glucose 2.00%, compounds derived from an-uracil amine acid 0.13%; The culture media for inducing the expression of GFP is SC (yeast nitrogen-containing bases 0.67%, Galactose 2.00%, compounds derives from an-uracil amine acid 0.13%). LB culture media was used for E. coli, according to Sambrook et al (2002). 

3.3 Reagents and devices
Yeast nitrogen base without amino acids is from Difco. Restriction enzymes, Taq DNA polymerase, denatured characinlike Single-Stranded DNA, dNTP were bought from TaKaRa. Lysozyme, RNaseA, Recycling kit of DNA agarose gel, Amino Acids, DMSO, PEG3350, Raffinose, Galactose were bought from Amresco. Tris (pH 8.0) phenol, chloroform and such kind of reagents were national A.R. PCR instrument used in this study is ABI 9600. Perpendicular-plate-electrophoretic apparatus is products of USA Bio-Rad firm. Microscope Olympus BX50 is used for fluorescence observation.

3.4 The construction of recombinant plasmid pGFP-Cry1Ac22
The pGFP vector and pMD18-T-Cry1Ac22 vector were digested by BamHâ… and Kpnâ… . The products were separated by 1.0% agarose gel electrophoresis. The target fragments were recycled by DNA agarose gel recycling kit, and were ligated with vector at 16℃ for overnight. 4 μL of the ligation products were added into E. coli JM109 competent cells (kept on ice). The tube was flicked slightly for uniform distribution, kept on ice for 30 min, then kept at 42℃for 90 s for thermal shock, and then add into 400 μL LB culture media, followed by Shaking culture at 37℃for 1h. Then 100 μL broth was spread on LB screening plate (containing 100 μg/mL ampicillin), and culture at 37℃ for overnight. The colony plasmid was extracted using boiling method and the identification of positive clones was performed through enzyme digestion.

3.5 The construction of recombinant plasmid pYES2-Cry1Ac22-EGFP
The pGFP-Cry1Ac22 identified positive and pYES2 were digested by BamHⅠ and NotⅠ for 3 h at 37℃. Separated the products by 1.0% agarose gel electrophoresis, recycled the target fragments using DNA agarose gel recycling kit, and ligated the two target fragments with vector at 4℃ for overnight. Insert the ligation products into E. coli JM109 competent cells, and choose the positive bacterial colony for expanding and extracting recombinant plasmid. The identification of positive clones was performed by enzyme digestion.

3.6 Transformation of pYES2-Cry1Ac22-EGFP into S. Cerevisiae
The Saccharomyces cerevisiae competent cells were obtained through the method of LiAc/SS2 DNA/PEG according to Invitrogen. Add 500 μL competent cells into 100 μL Buffer 1. mixed with 1~2 μg recombinant plasmids identified positive and 2 μL characinlike DNA, shake lightly. Then add 600 μL Buffer 2 and shaked for 30 min at 30℃. After that, add 70 μL DMSO and treated at 42℃ for 15 min. Centrifugated the solution for 10 s, add 300~500 μL TE Buffer to wash and precipitate for 30 min at 30℃, spread on SD-ura plate, and cultured for 3 days at 30℃. The bacterial colony become transformant recon; The bacterial colony was picked and observed under microscope for further identification.

3.7 The induced expression of recombinant plasmid in Saccharomyces Cerivisiae
The positive colony was vaccinated into SC-U fluid medium with 1% raffinose and cultured at 170 r/min and 30℃ for overnight. The product was centri- fuged by 12 000 r/min at 4℃ for 5 min. The thallus were precipitated by SC-U fluid medium with 2% galactose until its OD₆₀₀ becomes 0.4, then cultured under the condition of 30℃,170 r/min. Collect galactose to culture bacterial liquid for 6 h, 12 h, 24 h, then grind it by liquid nitrogen. After that, collect supernatant fluid for SDS-PAGE, and identify it by 10% PAGE. 

3.8 The localization of cry1Ac22 gene in yeast cell
The bacterial colony was transferred from plate into SC screening medium, cultured at 30℃ for overnight. 50% of the solution was washed 3 times, then transferred to SC induction medium (20% galactose instead of glucose) and cultured for 20 h. Collect induced and non-induced thallus, wash 3 times, add 2% low-gelling temperature agarose, shake slightly, and placed on microscope slide pretreated at 50℃,  and then placed in dark for 5 min, followed by observation under fluorescence microscope.

Authors’ contributions
Shenkui Liu and Zhuoming Liu design and execute this experiment. Youzhi Li participates the experiment design and data analysis; Xuanjun Fang is the person in charge of this project, conducting experiment design, data analysis, writing and modifying of the manuscript. All authors have read and approved the final manuscript.  

Acknowledgements
This research is funded by the project of China National Bt Collection Initiative and National 863 plans (Project No. 2004AA2111112). Authors appreciate Dr Xinxin Zhang from the ASNESC of Northeast Forestry University, Mr Wenfei Zhang and Liu Xie from HITAR for technological supports and helpful advice on the experiment. Thanks for peer reviewers for there useful advice and revising suggestion to this paper. And we mentioned some reagent suppliers and sequencing suppliers in this work, that doesn’t mean we would like to recommend or endorse their products and services.

References
Adang M.J., Staver M.J., Rocheleau T.A., Leighton J., Barker R.F., and Thompson D.V., 1985, Characterized full length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta, Gene, 36(3): 289-300 doi:10.1016/0378-1119(85)90184-2

Knowles B.H., and Ellar D.J., 1986, Characterization and partial purification of a plasma membrane receptor for Bacillus thuringiensis δ-endotoxin with different insect specificity, Biochimica et Biophysica Acta, 924: 509-518

Li J., Carrol J., and Ellar D.J., 1991, Crystal structure of insecticidal δ-endotoxin from Bacillus thuringiensis at 2.5Å resolution, Nature, 353(6347): 815-821 doi:10.1038/353815a0 PMid:1658659

Liu Z.M., Liu S.K., Li Y.Z., and Fang X.J., 2010, Expression and purification of Bacillus thuringiensis Cry1Ac22 protein in Escherichia coli, Bioscience Methods, Vol. 1, No. 2 (doi: 10.5376/bm.2010.01.0002)

Misteli T., and Spector D.L., 1997, Applications of the green fluorescent protein in cell biology and biotechnology. Nat. Biotechnol., 15(10): 961-964 doi:10.1038/nbt1097-961 PMid:9335045

Sambrook J.E., Fritsch F., and Maaiatis T., 2002, Experiment guidance of Molecular clone, Science press, Beijing, China, pp.483-485

Xie L., Zhang W.F., Liu Z.M., Cai Y.G., Li Y.Z., and Fang X.J., 2010, Characterization of a new highly toxic isolate of Bacillus thuringiensis from the diapausing larvae of silkworm and identification of cry1A 22 gene, Bt Research, Vol.1 No.1 (doi: 10.5376/bt.2010.01.0001)