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Cloning of the gene encoding an insecticidal protein inPseudomonas pseudoalcaligenes

Cloning of the gene encoding an insecticidal protein inPseudomonas pseudoalcaligenes Annals of Microbiology, 59 (1) 45-50 (2009) Cloning of the gene encoding an insecticidal protein in Pseudomonas pseudoalcaligenes Jie ZHANG, Jian ZHAO, Daxu LI, Sujun LIU, Li LI, Qun SUN, Ming HUANG, Zhirong YANG* Life Sciences School, Key laboratory for Bio-resources and Eco-environment of Chinese Education Ministry, Sichuan University, Chengdu, Sichuan, P.R. China, 610064 Received 31 October 2008 / Accepted 26 January 2009 Abstract - 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 DH5A was transformed using a partial Sau3AI digest of P. pseudoalcali- genes chromosomal DNA ligated with pUC19 vector cut by BamHI. The resulting genomic library was screened using an oligodeoxy- nucleotide 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 kb BamHI-KpnI fragment of the smallest clone (pSC1) contained the N terminal sequence of the insecti- cidal 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. A BamHI-KpnI fragment was cloned into pT7-6. A novel protein was expressed in E. coli BL21 (DE3) and its western blot similar to natural toxin. Key words: Pseudomonas pseudoalcaligenes; insecticidal protein; locust; gene cloning. INTRODUCTION for safety test of microbial insecticide of U.S. Environmental Protection Agency, a bacterial locusticide containing P. pseudo- alcaligenes and Bacillus thuringiensis had been tested in our A pathogen was isolated from natural dead yellow-spined bam- laboratory by standard testing procedure on vertebrates, such boo locust (Ceracris kiangsu Tsai, Orthoptera: Locustidae) in as rats, mice, rabbits, chickens, pigeons, quails, pigs, fish, etc. Geleshan farm of Chongqin. In Liu et al. (1995) its pathogenic- The results showed that the insecticide had neither toxicity nor ity was confirmed by the Koch’s postulates. It was identified as pathogenicity to the test animals. The bacterial locusticide had Pseudomonas pseudoalcaligenes according to its physiological the certificate for pesticide temporary registration of the Ministry and biochemical properties as well as the G+C content of DNA of Agriculture of China since 1997. (63.73 mol %). The results of preliminary bioassay showed As already shown (Zhang et al., 1997, 1998) an extracellular that the pathogen can infect the grasshoppers and C. kiangsu. insecticidal protein was discovered in P. pseudoalcaligenes. The Pseudomonas pseudoalcalingenes was found effective against insecticidal protein was purified by gel-filtration chromatogra- the major species of grassland locusts in Qinghai and Sichuan, phy on Sephadex G-100 and anion-exchange chromatography China. The fatality rate to Aulacobothrus sinensis was 87.3% 10 on DEAE-Sephadex A-50 from the suspension of P. pseudoal- when bred with corn leaf, which dipped with 10 CFU/ml of P. caligenes culture. Certain biophysical and biochemical properties pseudoalcalingenes 5 d. The fatality rates showed significant were also studied. The molecular weight of the subunit of the differences between different species of locust; the fatality rate insecticidal protein is about 26 kDa and pI is 5.16. Amino acid to Locusta migratoria mardlensis was 48% and the third instar composition analysis showed that it was an acidic protein. The locust was the most sensitive to the strain. The LC was 3.27 x 7 insecticidal protein is sensitive to heat, which has inactivity in 2 10 CFU/ml for third instar C. kiangsu, 5 d. The strain could also minutes at 80 °C, is stable at pH 6.6-10. It is sensitive to pepsin infect Gyrnaephora ruoergends, Lencarda separata and Maladera and insensitive to trypase. It does not cause haemolysis. orientails to some extent (Yang et al., 1996a, 1996b). The structure of the insecticidal protein and mechanism of According to the Ministry of Agriculture of China’s standards controlling locusts was discovered in P. pseudoalcaligenes in for safety test of insecticide, and referring to the requirements recent study. Laser Raman spectra analysis indicates that the molecule of the insecticidal protein has 58% B -sheet, 8% coil and has trans-gauche-trans configuration of C-C-S-S-C-C linkage * Corresponding Author. Phone: 86-028-85460487; (Luo et al., 2000). The majority of tyrosyl residues are exposed E-mail: bioyang@163.com 46 J. ZHANG et al. and a few are buried in the protein. The insecticidal protein acts Electrophoretic separation and detection of proteins. SDS- on the foregut, midgut, hindgut, Malpighian tubes and fat body, PAGE was performed in a vertical gel slab (Ausubel et al. 1998) and kills locusts as an intact molecule. After 24 to 48 h, it was by discontinuous buffer system on determination of insecticidal observed that all these tissues and cells showed pathological protein. The 12% separating gel and the 4% stacking gel were used. The gel slabs were 1.5 mm x 14 cm x 14 cm. The clearly changes in various degrees, and so did the host organelles, such visible bands of protein were obtained on the gel after fixation, as cytoblast, mitochondria, endoplasmic reticulum and ribo- staining and discoloration. By compared with the standard pro- some. Particularly, the changes of the mitochondria are much teins of specific molecular mass, the 26 kDa band was deter- more serious than those of others (Zhao et al., 2002). Under the mined, cut and kept. electron microscope it could be observed that many mitochondria The colorimetric test was performed by the method of expanded, partial cristae broke and blurred. Detection of oxygen Bradford (1976) to determine the protein content, with bovine measurement system shows the efficiency of oxidative phos- albumin as the standard protein. phorylation and the ATP synthesis decreases, while the activity of mitochondrial ATPase is almost not affected. That means the Determination of amino acid composition and N-terminal energy utilization of locusts is in gear, but the shortage of the sequencing. After the 1 mg purified insecticidal protein was supplying of energy results in their death. hydrolyzed in 6 M HCl 24 h, the Hitachi 835-50 high speed auto- Cloning insecticidal protein gene is essential to the con- matic amino acid analyzer was used to analysis the amino acid struction of transgenic grass or the optimization of structure composition. in subsequent structure-activity studies. In this study we used The Beckman LF3000 automatic protein sequencing machine the purified insecticidal protein to obtain rabbit antiserum, and was used for N-terminal amino acid sequencing (Matsudaira, sequenced the N-terminal amino acids, synthesized the cor- 1987). responding oligonucleotide probe as methods by Ding and Yang (2001). A genomic library of P. pseudoalcaligenes was construct- Preparation and characterization of antibody to insecticid- ed and the gene encoding the insecticidal protein was cloned and al protein from Pseudomonas pseudoalcaligenes. Antigen sequenced. was obtained from the 26 kDa protein band after repeated freezing and melting, homogenization and complete emulsifica- tion. Injection at the first time was performed on 10 locations MATERIALS AND METHODS on rabbit’s body, with each injected by 0.1 ml (about 600 μg/ml insecticidal protein) antigen. The first booster was on the fourth Bacterial strains, culture conditions, plasmid. The strain P. week, the second and third booster after 2 weeks in turn. Blood pseudoalcaligenes was isolated from a dead locust (C. kiangsu) was collected from the ear-vein of the rabbit, and stood for 4 h in 1991 and had been preserved in our lab. The enhanced before stayed overnight at 4 °C. We removed blood clot, collected beef-extract peptone medium (0.35%beef extract, 1%peptone, blood serum after centrifugation and determined the antibody 0.8%NaCl, 0.35%fructose; pH 7.2), LB medium containing 100 titer of blood serum. μg/ml ampicillin, SOB medium, and SOC medium were used for The indirect ELISA with protein G-alkaline phosphatase con- culture. Plasmids pUC19 and pT7-6 were utilized. jugate was applied to detect antiserum from rabbit (Mayer and Walker, 1980). Coated plate with 100 μl antigen solutions (50 The separation and purification of insecticidal protein. mM Na CO -NaHCO buffer pH9.6 with 0.05% NaN , 10 μg/ml 2 3 3 3 Pseudomonas pseudoalcaligenes was inoculated on the enhanced prude insecticidal protein) before incubated overnight at room beef-extract peptone medium and shaking-cultivated at 35 °C for temperature. Blocked residual binding capacity of plate with 48 h. The supernatant was collected after centrifugation at 6000 TTBS buffer, added 100 μl rabbit antiserum diluted in TTBS buffer x g for 20 min followed by ultrafiltration on the Millipore ultra- to each of the coated wells, incubated 3 h at room temperature. filtration system. The collected proteins solution of 10-40 kDa Added 100 μl goat anti-rabbit IgG-ap diluted in TTBS buffer with were dialyzed and condensed with PEG20000 powder to assay 1% BSA, and incubated 3 h at room temperature. The sample insecticidal activity, obtain the crude insecticidal protein. was detected with absorbance at 405 nm. The DEAE-cellulose-32 column chromatography was used to Western blot was applied to identify the specificity of anti- purify the protein. The impure protein was washed by elution serum (Simpson, 2004). SDS-PAGE-separated proteins were buffer, followed by equilibrium buffer containing NaCl (0-0.6 M) in blotted onto nitrocellulose. The TTBS block buffer and BCIP/NBT linear gradient. The collected fractions were subjected to analysis visualization solution were used. of insecticidal activity. The eluants of insecticidal activity were combined and concentrated after dialysis. Preparation and harvesting of genomic DNA fragments The concentrated solutions were separable by Sephadex after restriction enzyme digestion. The P. pseudoalcaligenes G-100 gel filtration. We collected the filtrated fractions and then genomic DNA was extracted by CTAB protocol (Ausubel et al., measured the purity by assaying the absorbance at 280 nm and 1998). Genomic DNA solution of 500 μl was divided into several the insecticidal activity. The fractions with insecticidal activity small parts and Sau3AI serially diluted was added respectively were combined and subjected to dialysis before concentrated for partial digestion. Digestion condition was optimized to make (Schagger and Von Jagow, 1987). the length of DNA fragments between 2-10 kb. The 2-10 kb DNA was recovered using low melting temperature agarose. Force microdrop feeding insect bioassay. Biological activ- ity of the protein was assayed by delivering 30 μl drops of Ligation of DNA fragment to plasmid. Bulk and small amount each protein dilution into mouth of third instar C. kiangsu of plasmid was extracted by the alkaline lysis method. The plas- with a microsyringe drive. The mortality of locust in 5 d was mid pUCl9 of 2696 bp with Amp and lacZa sites, digested with accounted to determine the insecticidal activity of the sample BamHI, was treated with calf intestine alkaline phosphatase (De Leon and Ibarra, 1995). (CIAP). Ann. Microbiol., 59 (1), 45-50 (2009) 47 Dephosphorylated linear plasmid pUCl9 of 0.1 μg was mixed Preparation and identification of the antibody to insecti- with 0.3 μg foreign DNA fragments and incubated to complete cidal protein from Pseudomonas pseudoalcaligenes the ligation reaction. The recombinant DNA was transferred to E. Immunodiffusion assay was used to identify the rabbit antiserum. coli DH5A competent cell (Lnoue et al., 1990). No precipitation lines were found in the reaction between preim- mune serum with crude insecticidal protein, it suggested that Radiolabeling of probe. According to the results of N-terminal there was no nonspecific reaction between the rabbit serum and sequence, designed an oligonucleotide probe. The oligonucle- the crude insecticidal protein. Antibody was detected from the otide sequence was end-labeled with [G - P] adenosine triphos- antiserum which was obtained after the second booster injection phate (Amersham Pharmacia, Piscataway, NJ, USA) using T4 and diluted 4 and 16 times. The precipitation lines appeared in polynucleotide kinase (Promega), incubated 45 min at 37 °C, crude insecticidal protein reacting with rabbit antiserum obtained DNA fragments was end-labeled with [A - P] cytosine triphos- after the third booster injection and even diluted 64 times, and phate using klenow fragment (Sambrook et al., 2002), incubated this suggested there be a high titer of the rabbit antiserum. 4 h at room temperature. The titer of rabbit antiserum after the third booster injection was tested by ELISA, Coated plate with 100 μl antigen solution In situ hybridization. The in situ lysis membrane of E. coli was with 10 μg/ml prude insecticidal protein, according to concentra- prepared at first. DNA was transferred from agarose gel onto tion change of protein, protein adsorption quantity was about nylon membrane, hybridized with the radiolabeled oligonucle- 0.32 μg each well. It was found that the maximum dilution factor otide probe in TMAC hybridization solution. After the intensity of the antiserum which appeared positive reaction was 12800. of radioactivity of background had detected the X ray film was To identify the specificity of the antiserum, the purified total exposed 6 d in -20 °C. bacterial protein, the total protein of supernatant from bacterial culture and crude insecticidal protein 10-40 kDa were subjected Expression of insecticidal protein in Escherichia coli. on electrophoresis, and then transferred onto pyroxylin filter Digested pSC1-1 with EcoRI and BamHI, recovered the 1.4 kb membrane with tank transfer systems, followed by the reaction fragment which contain insecticidal protein, and cloned into with the primary antibody and goat anti-rabbit IgG-AP in turn. EcoRI-BamHI-cleaved pT7-6, constructed pT7-6-IP, transferred The result showed that the antiserum can only reacted with into E. coli BL21 (DE3). When the culture reached the mid- insecticidal protein, suggested that the antiserum have high exponential phase, 1.0 mM IPTG was added to induce gene specificity (Fig. 1). expression. Cultivation was continued for an additional 2 h. Lysed bacteria with 1X SDS loading buffer, the supernatant were ana- Cloning of the gene encoding the insecticidal protein from lyzed on for SDS-page and Western blot. Pseudomonas pseudoalcaligenes The pUC19 fragment with dephosphorylated BamHI ends was ligated to 2-10 kb Sau3AI partial digested P. pseudoalcaligenes RESULTS genomic DNA fragments that were recovered from agarose slices using low-melting-point agarose gel. Then we transferred the Separation and purification of the Pseudomonas pseudoalcali- recombinant plasmid into E. coli DH5A , and about 10000 recom- genes insecticidal protein and the N-terminal sequencing binant colonies were obtained, which were genomic library of P. The insecticidal activity was assayed, the supernatant showed pseudoalcaligenes. toxic effect to grasshopper, while the pellet did not. The super- After screening the recombination colonies by oligonucle- natant lost its toxic effect completely after treatment by final otide probe, we obtained 5 positive clones. All positive clones concentration 100 μg/ml protease K at 37 °C for 4 h. That sug- had insert fragment, the longest fragment was about 10 kb, and gests that the insecticidal protein exist in the supernatant of bacterial culture. During the isolation and purification of the insecticidal protein M 1 2 3 1 2 3 from the bacterial culture, crude insecticidal protein of 10-40 kDa kDa was derived from the supernatant of bacterial culture by Millipore 97.6 ultrafiltration. The purified insecticidal protein was derived by DEAE-cellulose-32 column chromatography, and Sephadex 66.2 G-100 gel filtration. Only one single band was detected by SDS- 43.0 PAGE. The molecular weight of the subunit was approximate 26 kDa by calculation according to its mobility on SDS-PAGE, while the relative molecular weight calculated by Sephadex G-100 gel 31.0 filtration was 28 kDa. This proved that the insecticidal protein from P. pseudoalcaligenes was composed by one peptide chain. 20.1 The amino acid composition of the insecticidal protein was assayed. 14.4 Purified insecticidal protein powder was dotted on the PVDF membrane after being dissolved in the appropriate reagent. After fixating, it was sequenced using Beckman LF3000 auto- A B matic protein sequencing machine. The results suggested that its N-terminal sequence was Gly-Val-Trp-Gln-His-Gln-Ser-His-Ala- FIG. 1 - Identification of the specificity of antiserum by Western Ala, so we designed an 17 mer oligonucleotide probe based on blot analysis. A: SDS-PAGE, B: Western blot. Lane M: this sequence (5’-GGNGTNTGGCARCAYCA-3’, N means any basic protein molecular marker, lane 1: the total bacterial radical, Y means pyrimidine, N means purine). protein; lane 2: the total protein of supernatant from bacterial culture; lane 3: the crude insecticidal protein. 48 J. ZHANG et al. M 1 2 3 4 5 6 7 1 2 3 4 5 6 7 kb 21.23 5.15 4.27 3.63 2.03 1.58 1.38 A B FIG. 2 - A: double restriction endonuclease digested pSC1, B: hybridization with oligodeoxynucleotide probe. Lane M: L DNA/ EcoRI+HindIII, lane 1: EcoRI-BamHI, lane 2: SphI-BamHI, lane 3: SalI-BamHI, lane 4: SmaI-BamHI, lane 5: XbaI-BamHI, lane 6: KpnI-BamHI, lane 7: HindIII-BamHI. There are seven hybridization positive signals with oligonucleotide probe. The corresponding fragments were about 3.2 or 3.6 kb EcoRI-BamHI fragment, about 4.2 kb SphI-BamHI fragment, about 2.4 kb SalI-BamHI fragment, about 7.2 kb SmaI- BamHI fragment, about 4.0 or 4.5 kb XbaI-BamHI fragment, about 1.4 kb KpnI-BamHI fragment, about 2.8 kb HindIII-BamHI fragment. the shortest fragment was about 6.5 kb. All positive clones were TGA (GenBank accession no. DQ790644). In downstream of the digested with EcoRI completely. Hybridization with 17 mer oligo- terminate code, no other terminate code or reverse repeated nucleotide probe showed all of them had 5 kb positive hybridiza- sequence was found. The deduced amino acid sequence of tion signal fragment. this gene should contain Gly-Val-Trp-Gln-His-Gln-Ser-His-Ala- So the shortest recombinant plasmid pSC1 was used for fur- Ala (from 23 to 32 amino acids) which is same as N-terminal ther identification. Analysis with restriction endonuclease, only sequence of insecticidal protein. That indicated the ORF of 1.4 one recognition site by BamHI was found in plasmid pSC1, and kb fragment should contain insecticidal protein coding region the recognition site was in insert fragment. The plasmid pSC1, (Fig. 3). double digested with BamHI and other restriction enzyme, was There were 22 amino acid residues between initiation codon separated in agarose gel and transferred to nylon membrane. ATG of the ORF corresponding amino acid and N-terminal There are seven positive hybridization signals with oligonucle- sequence of insecticidal protein, for insecticidal protein was otide probe. Hybridization with oligonucleotide probe showed extracellular enzyme, so it should be a signal peptide. Ala Leu that the 1.4 kb BamHI-KpnI fragment was the smallest fragment, Ala (20 to 22 amino acids) should be cleavage site of signal which contained the gene encoding insecticidal protein (Fig. 2). peptide (Oliver, 1985). Most signal peptides have a tripartite structure: a positively Sequencing of the gene encoding the insecticidal protein charged N-domain, containing lysine and/or arginine residues; The BamHI-KpnI fragment was subcloned into pUC19, we named a hydrophobic H-domain; and a C-domain which specifies the recombinant plasmid pSC1-1. Sequencing the 1.4 kb frag- the cleavage site for specific signal peptidase (SPase). The ment by the ABI 310 Genetic Analyzer, we found that there N-terminal peptide of insecticidal protein only had one basic was an open reading frame of 828 bp, which coded the protein amino acids-Arg. The signal peptide had a C-domain, an atypical consist of 276 amino acid residues and had a terminate code of N-domain and lack of H-domain. FIG. 3 - Sequencing of the gene encoding the insecticidal protein. Ann. Microbiol., 59 (1), 45-50 (2009) 49 The deduced mature protein was 254 residues. According to Sequencing and the structural analysis of the gene encoding the sequence of the insecticidal protein gene, we deduced that insecticidal protein may help us unveil the structural charac- the molecular mass of the mature protein was 26.7 kDa and its teristics and the active domains of insecticidal protein, clarify isoelectric point was 5.57. This was consistent with the data of the regulation of gene expression, and construct high efficient purified protein. gene-engineered strains. Based on the N-terminal sequence of During the sequence analysis of the insecticidal protein insecticidal protein, we designed the oligonucleotide probe which gene, it was found that the codon usage had preference to G and was used to screen the gene library of P. pseudoalcaligenes. C, with the frequency of the third base of codon being G or C The coding region of insecticidal protein from P. pseudoalcali- reaching 71%. The amount of base in the coding chain contains genes has 828 bp that codes 276 amino acid residues. It was was high G and C of approximate 62.6%, and this coincided speculated that 22 amino acid residues of N terminus was for a with the high content of G and C in P. pseudoalcaligenes (Henry signal peptide. The signal peptide of insecticidal protein was dif- and Bessieres, 1984; Dumora et al., 1997; Bever and Iglewski, ferent from Secretory (Sec-type) signal peptides, Twin-arginine 1998). The 5’-end region of insecticidal protein gene had Shine- signal peptides are cleaved by one of the various type I SPases Dalgarno sequence AGGAAC, similarly to that of ribosome bind- of B. subtilis; Lipoprotein signal peptides, which are cleaved by ing site of E. coli (Stormo and Schuneuder, 1982). There was the lipoprotein-specific (type II) SPase; Type IV prepilin signal no sequence similar to -10 regions (TATAAT) or -35 regions peptides, which, in B. subtilis, are cleaved by the prepilin-spe- (TTGACA) in the promoter of E. coli were found. cific SPase ComC (Tjalsma et al., 2000). The sequence features By nucleotide-nucleotide BLAST (blastn) on NCBI, there resembled signal peptides of bacteriocins and pheromone which was no highly identical nucleic acid sequence with that of are exported by ATP-binding cassette (ABC) transporters. The insecticidal protein from P. pseudoalcaligenes, suggesting that signal peptides cannot be predicted by the regular algorithms for there was lack of identity between insecticidal protein from signal peptide prediction, such as SignalP, as it consist only of P. pseudoalcaligenes and other bacterial toxin. Furthermore, N- and C-domains and completely lack a hydrophobic H-domain compared with genes of insecticidal crystal protein of B. thur- (Nielsen et al.,1997). ingiensis, exotoxin A of P. aeruginosa, botulinum toxin A and B The gene coding insecticidal protein from P. pseudoal- of Clostridium botulinum, and diphtheria toxin, the identity was caligenes lacked significant identity with other bacterial toxins. lower than 15%. Therefore, insecticidal protein from P. pseudoalcaligenes was By NCBI conserved domain searching, 46.5% of the amino a novel insecticidal toxic protein discovered firstly in our lab. acid sequence of insecticidal protein was aligned with COG1804 Sequencing the cloned gene coding insecticidal protein revealed (CaiB, Predicted acyl-CoA transferases/carnitine dehydratase that it had the same N-terminal coding region as the natural [Energy production and conversion]), 42.6% of the sequence insecticidal protein. This region could code a mature protein of was aligned with KOG3957 (Predicted L-carnitine dehydratase/ 26.7 kDa whose molecular weight and amino acid composition alpha-methylacyl-CoA racemase [Lipid transport and metabo- was similar to that of the natural insecticidal protein. lism]), 58.9% of the sequence was aligned with pfam02515 The insecticidal protein gene was not expressed in E. coli, (CoA_transf_3, CoA-transferase family III). nor induced by IPTG. The results above might indicate that the promoter of insecticidal protein gene could not be recognized Expression of insecticidial protein gene in Escherichia by E. coli, or there were some fragments lost. Furthermore, the coli cloned fragments could not be expressed even with the help of The rabbit antiserum screening from the 5 positive clones in situ the lac promoter. lysis was negative neither induced or not by IPTG. The 1.4 kb The pT7-6 used to express genes under control of T7 RNA fragment was inserted into plasmid pUC19 and pUC18, recom- polymerase. The pT7-6 vector does not contain ribosome bind- binant plasmids pSC1-1 and pSC1-2 was obtained respectively, ing site ATG codon upstream of polylinker. Escherichia coli BL21 which were transformed into E. coli DH5A , JM109 and HB101. The (DE3), which contains the gene for T7 RNA polymerase on the E. transformants were cultured, and the expressed gene products coli chromosome under the control of the lac promoter, containing were identified by using antiserum. The gene encoding insecti- pT7-6-IP can express the insecticidal protein gene with IPTG. The cidal protein was not expressed in E. coli, nor induced by IPTG. western blot showed that the expressed protein can reacted with Then we cloned pSC1-1 EcoRI-BamHI fragment into pT7-6, insecticidal protein antiserum. These evidences indicated that the and transfered into E. coli BL21 (DE3). The lysate of E. coli BL21 gene cloned should be the coding region of insecticidal protein. (DE3) with pT7-6-IP was analyzed using SDS-page and Western Cloned the insecticidal protein gene has significance in many blot. Compared with the contained pT7-6 E. coli total protein, aspects, including clarifying the function and structure of the one additional 26 kDa band was found on SDS-page, and it insecticidal protein, wide application in developing new microbial had positive signal on western blot. The result showed that the insecticide and gene-engineered plants with insect-resistance, expression protein had same character as natural insecticidal and the basic study on the inheritance of insect pathogen protein. microbes. Acknowledgements DISCUSSION This work was supported by grants from The National Key Technology R&D Program of China 2007BAC18B03. 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Cloning of the gene encoding an insecticidal protein inPseudomonas pseudoalcaligenes

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Springer Journals
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Copyright © 2009 by University of Milan and Springer
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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Abstract

Annals of Microbiology, 59 (1) 45-50 (2009) Cloning of the gene encoding an insecticidal protein in Pseudomonas pseudoalcaligenes Jie ZHANG, Jian ZHAO, Daxu LI, Sujun LIU, Li LI, Qun SUN, Ming HUANG, Zhirong YANG* Life Sciences School, Key laboratory for Bio-resources and Eco-environment of Chinese Education Ministry, Sichuan University, Chengdu, Sichuan, P.R. China, 610064 Received 31 October 2008 / Accepted 26 January 2009 Abstract - 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 DH5A was transformed using a partial Sau3AI digest of P. pseudoalcali- genes chromosomal DNA ligated with pUC19 vector cut by BamHI. The resulting genomic library was screened using an oligodeoxy- nucleotide 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 kb BamHI-KpnI fragment of the smallest clone (pSC1) contained the N terminal sequence of the insecti- cidal 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. A BamHI-KpnI fragment was cloned into pT7-6. A novel protein was expressed in E. coli BL21 (DE3) and its western blot similar to natural toxin. Key words: Pseudomonas pseudoalcaligenes; insecticidal protein; locust; gene cloning. INTRODUCTION for safety test of microbial insecticide of U.S. Environmental Protection Agency, a bacterial locusticide containing P. pseudo- alcaligenes and Bacillus thuringiensis had been tested in our A pathogen was isolated from natural dead yellow-spined bam- laboratory by standard testing procedure on vertebrates, such boo locust (Ceracris kiangsu Tsai, Orthoptera: Locustidae) in as rats, mice, rabbits, chickens, pigeons, quails, pigs, fish, etc. Geleshan farm of Chongqin. In Liu et al. (1995) its pathogenic- The results showed that the insecticide had neither toxicity nor ity was confirmed by the Koch’s postulates. It was identified as pathogenicity to the test animals. The bacterial locusticide had Pseudomonas pseudoalcaligenes according to its physiological the certificate for pesticide temporary registration of the Ministry and biochemical properties as well as the G+C content of DNA of Agriculture of China since 1997. (63.73 mol %). The results of preliminary bioassay showed As already shown (Zhang et al., 1997, 1998) an extracellular that the pathogen can infect the grasshoppers and C. kiangsu. insecticidal protein was discovered in P. pseudoalcaligenes. The Pseudomonas pseudoalcalingenes was found effective against insecticidal protein was purified by gel-filtration chromatogra- the major species of grassland locusts in Qinghai and Sichuan, phy on Sephadex G-100 and anion-exchange chromatography China. The fatality rate to Aulacobothrus sinensis was 87.3% 10 on DEAE-Sephadex A-50 from the suspension of P. pseudoal- when bred with corn leaf, which dipped with 10 CFU/ml of P. caligenes culture. Certain biophysical and biochemical properties pseudoalcalingenes 5 d. The fatality rates showed significant were also studied. The molecular weight of the subunit of the differences between different species of locust; the fatality rate insecticidal protein is about 26 kDa and pI is 5.16. Amino acid to Locusta migratoria mardlensis was 48% and the third instar composition analysis showed that it was an acidic protein. The locust was the most sensitive to the strain. The LC was 3.27 x 7 insecticidal protein is sensitive to heat, which has inactivity in 2 10 CFU/ml for third instar C. kiangsu, 5 d. The strain could also minutes at 80 °C, is stable at pH 6.6-10. It is sensitive to pepsin infect Gyrnaephora ruoergends, Lencarda separata and Maladera and insensitive to trypase. It does not cause haemolysis. orientails to some extent (Yang et al., 1996a, 1996b). The structure of the insecticidal protein and mechanism of According to the Ministry of Agriculture of China’s standards controlling locusts was discovered in P. pseudoalcaligenes in for safety test of insecticide, and referring to the requirements recent study. Laser Raman spectra analysis indicates that the molecule of the insecticidal protein has 58% B -sheet, 8% coil and has trans-gauche-trans configuration of C-C-S-S-C-C linkage * Corresponding Author. Phone: 86-028-85460487; (Luo et al., 2000). The majority of tyrosyl residues are exposed E-mail: bioyang@163.com 46 J. ZHANG et al. and a few are buried in the protein. The insecticidal protein acts Electrophoretic separation and detection of proteins. SDS- on the foregut, midgut, hindgut, Malpighian tubes and fat body, PAGE was performed in a vertical gel slab (Ausubel et al. 1998) and kills locusts as an intact molecule. After 24 to 48 h, it was by discontinuous buffer system on determination of insecticidal observed that all these tissues and cells showed pathological protein. The 12% separating gel and the 4% stacking gel were used. The gel slabs were 1.5 mm x 14 cm x 14 cm. The clearly changes in various degrees, and so did the host organelles, such visible bands of protein were obtained on the gel after fixation, as cytoblast, mitochondria, endoplasmic reticulum and ribo- staining and discoloration. By compared with the standard pro- some. Particularly, the changes of the mitochondria are much teins of specific molecular mass, the 26 kDa band was deter- more serious than those of others (Zhao et al., 2002). Under the mined, cut and kept. electron microscope it could be observed that many mitochondria The colorimetric test was performed by the method of expanded, partial cristae broke and blurred. Detection of oxygen Bradford (1976) to determine the protein content, with bovine measurement system shows the efficiency of oxidative phos- albumin as the standard protein. phorylation and the ATP synthesis decreases, while the activity of mitochondrial ATPase is almost not affected. That means the Determination of amino acid composition and N-terminal energy utilization of locusts is in gear, but the shortage of the sequencing. After the 1 mg purified insecticidal protein was supplying of energy results in their death. hydrolyzed in 6 M HCl 24 h, the Hitachi 835-50 high speed auto- Cloning insecticidal protein gene is essential to the con- matic amino acid analyzer was used to analysis the amino acid struction of transgenic grass or the optimization of structure composition. in subsequent structure-activity studies. In this study we used The Beckman LF3000 automatic protein sequencing machine the purified insecticidal protein to obtain rabbit antiserum, and was used for N-terminal amino acid sequencing (Matsudaira, sequenced the N-terminal amino acids, synthesized the cor- 1987). responding oligonucleotide probe as methods by Ding and Yang (2001). A genomic library of P. pseudoalcaligenes was construct- Preparation and characterization of antibody to insecticid- ed and the gene encoding the insecticidal protein was cloned and al protein from Pseudomonas pseudoalcaligenes. Antigen sequenced. was obtained from the 26 kDa protein band after repeated freezing and melting, homogenization and complete emulsifica- tion. Injection at the first time was performed on 10 locations MATERIALS AND METHODS on rabbit’s body, with each injected by 0.1 ml (about 600 μg/ml insecticidal protein) antigen. The first booster was on the fourth Bacterial strains, culture conditions, plasmid. The strain P. week, the second and third booster after 2 weeks in turn. Blood pseudoalcaligenes was isolated from a dead locust (C. kiangsu) was collected from the ear-vein of the rabbit, and stood for 4 h in 1991 and had been preserved in our lab. The enhanced before stayed overnight at 4 °C. We removed blood clot, collected beef-extract peptone medium (0.35%beef extract, 1%peptone, blood serum after centrifugation and determined the antibody 0.8%NaCl, 0.35%fructose; pH 7.2), LB medium containing 100 titer of blood serum. μg/ml ampicillin, SOB medium, and SOC medium were used for The indirect ELISA with protein G-alkaline phosphatase con- culture. Plasmids pUC19 and pT7-6 were utilized. jugate was applied to detect antiserum from rabbit (Mayer and Walker, 1980). Coated plate with 100 μl antigen solutions (50 The separation and purification of insecticidal protein. mM Na CO -NaHCO buffer pH9.6 with 0.05% NaN , 10 μg/ml 2 3 3 3 Pseudomonas pseudoalcaligenes was inoculated on the enhanced prude insecticidal protein) before incubated overnight at room beef-extract peptone medium and shaking-cultivated at 35 °C for temperature. Blocked residual binding capacity of plate with 48 h. The supernatant was collected after centrifugation at 6000 TTBS buffer, added 100 μl rabbit antiserum diluted in TTBS buffer x g for 20 min followed by ultrafiltration on the Millipore ultra- to each of the coated wells, incubated 3 h at room temperature. filtration system. The collected proteins solution of 10-40 kDa Added 100 μl goat anti-rabbit IgG-ap diluted in TTBS buffer with were dialyzed and condensed with PEG20000 powder to assay 1% BSA, and incubated 3 h at room temperature. The sample insecticidal activity, obtain the crude insecticidal protein. was detected with absorbance at 405 nm. The DEAE-cellulose-32 column chromatography was used to Western blot was applied to identify the specificity of anti- purify the protein. The impure protein was washed by elution serum (Simpson, 2004). SDS-PAGE-separated proteins were buffer, followed by equilibrium buffer containing NaCl (0-0.6 M) in blotted onto nitrocellulose. The TTBS block buffer and BCIP/NBT linear gradient. The collected fractions were subjected to analysis visualization solution were used. of insecticidal activity. The eluants of insecticidal activity were combined and concentrated after dialysis. Preparation and harvesting of genomic DNA fragments The concentrated solutions were separable by Sephadex after restriction enzyme digestion. The P. pseudoalcaligenes G-100 gel filtration. We collected the filtrated fractions and then genomic DNA was extracted by CTAB protocol (Ausubel et al., measured the purity by assaying the absorbance at 280 nm and 1998). Genomic DNA solution of 500 μl was divided into several the insecticidal activity. The fractions with insecticidal activity small parts and Sau3AI serially diluted was added respectively were combined and subjected to dialysis before concentrated for partial digestion. Digestion condition was optimized to make (Schagger and Von Jagow, 1987). the length of DNA fragments between 2-10 kb. The 2-10 kb DNA was recovered using low melting temperature agarose. Force microdrop feeding insect bioassay. Biological activ- ity of the protein was assayed by delivering 30 μl drops of Ligation of DNA fragment to plasmid. Bulk and small amount each protein dilution into mouth of third instar C. kiangsu of plasmid was extracted by the alkaline lysis method. The plas- with a microsyringe drive. The mortality of locust in 5 d was mid pUCl9 of 2696 bp with Amp and lacZa sites, digested with accounted to determine the insecticidal activity of the sample BamHI, was treated with calf intestine alkaline phosphatase (De Leon and Ibarra, 1995). (CIAP). Ann. Microbiol., 59 (1), 45-50 (2009) 47 Dephosphorylated linear plasmid pUCl9 of 0.1 μg was mixed Preparation and identification of the antibody to insecti- with 0.3 μg foreign DNA fragments and incubated to complete cidal protein from Pseudomonas pseudoalcaligenes the ligation reaction. The recombinant DNA was transferred to E. Immunodiffusion assay was used to identify the rabbit antiserum. coli DH5A competent cell (Lnoue et al., 1990). No precipitation lines were found in the reaction between preim- mune serum with crude insecticidal protein, it suggested that Radiolabeling of probe. According to the results of N-terminal there was no nonspecific reaction between the rabbit serum and sequence, designed an oligonucleotide probe. The oligonucle- the crude insecticidal protein. Antibody was detected from the otide sequence was end-labeled with [G - P] adenosine triphos- antiserum which was obtained after the second booster injection phate (Amersham Pharmacia, Piscataway, NJ, USA) using T4 and diluted 4 and 16 times. The precipitation lines appeared in polynucleotide kinase (Promega), incubated 45 min at 37 °C, crude insecticidal protein reacting with rabbit antiserum obtained DNA fragments was end-labeled with [A - P] cytosine triphos- after the third booster injection and even diluted 64 times, and phate using klenow fragment (Sambrook et al., 2002), incubated this suggested there be a high titer of the rabbit antiserum. 4 h at room temperature. The titer of rabbit antiserum after the third booster injection was tested by ELISA, Coated plate with 100 μl antigen solution In situ hybridization. The in situ lysis membrane of E. coli was with 10 μg/ml prude insecticidal protein, according to concentra- prepared at first. DNA was transferred from agarose gel onto tion change of protein, protein adsorption quantity was about nylon membrane, hybridized with the radiolabeled oligonucle- 0.32 μg each well. It was found that the maximum dilution factor otide probe in TMAC hybridization solution. After the intensity of the antiserum which appeared positive reaction was 12800. of radioactivity of background had detected the X ray film was To identify the specificity of the antiserum, the purified total exposed 6 d in -20 °C. bacterial protein, the total protein of supernatant from bacterial culture and crude insecticidal protein 10-40 kDa were subjected Expression of insecticidal protein in Escherichia coli. on electrophoresis, and then transferred onto pyroxylin filter Digested pSC1-1 with EcoRI and BamHI, recovered the 1.4 kb membrane with tank transfer systems, followed by the reaction fragment which contain insecticidal protein, and cloned into with the primary antibody and goat anti-rabbit IgG-AP in turn. EcoRI-BamHI-cleaved pT7-6, constructed pT7-6-IP, transferred The result showed that the antiserum can only reacted with into E. coli BL21 (DE3). When the culture reached the mid- insecticidal protein, suggested that the antiserum have high exponential phase, 1.0 mM IPTG was added to induce gene specificity (Fig. 1). expression. Cultivation was continued for an additional 2 h. Lysed bacteria with 1X SDS loading buffer, the supernatant were ana- Cloning of the gene encoding the insecticidal protein from lyzed on for SDS-page and Western blot. Pseudomonas pseudoalcaligenes The pUC19 fragment with dephosphorylated BamHI ends was ligated to 2-10 kb Sau3AI partial digested P. pseudoalcaligenes RESULTS genomic DNA fragments that were recovered from agarose slices using low-melting-point agarose gel. Then we transferred the Separation and purification of the Pseudomonas pseudoalcali- recombinant plasmid into E. coli DH5A , and about 10000 recom- genes insecticidal protein and the N-terminal sequencing binant colonies were obtained, which were genomic library of P. The insecticidal activity was assayed, the supernatant showed pseudoalcaligenes. toxic effect to grasshopper, while the pellet did not. The super- After screening the recombination colonies by oligonucle- natant lost its toxic effect completely after treatment by final otide probe, we obtained 5 positive clones. All positive clones concentration 100 μg/ml protease K at 37 °C for 4 h. That sug- had insert fragment, the longest fragment was about 10 kb, and gests that the insecticidal protein exist in the supernatant of bacterial culture. During the isolation and purification of the insecticidal protein M 1 2 3 1 2 3 from the bacterial culture, crude insecticidal protein of 10-40 kDa kDa was derived from the supernatant of bacterial culture by Millipore 97.6 ultrafiltration. The purified insecticidal protein was derived by DEAE-cellulose-32 column chromatography, and Sephadex 66.2 G-100 gel filtration. Only one single band was detected by SDS- 43.0 PAGE. The molecular weight of the subunit was approximate 26 kDa by calculation according to its mobility on SDS-PAGE, while the relative molecular weight calculated by Sephadex G-100 gel 31.0 filtration was 28 kDa. This proved that the insecticidal protein from P. pseudoalcaligenes was composed by one peptide chain. 20.1 The amino acid composition of the insecticidal protein was assayed. 14.4 Purified insecticidal protein powder was dotted on the PVDF membrane after being dissolved in the appropriate reagent. After fixating, it was sequenced using Beckman LF3000 auto- A B matic protein sequencing machine. The results suggested that its N-terminal sequence was Gly-Val-Trp-Gln-His-Gln-Ser-His-Ala- FIG. 1 - Identification of the specificity of antiserum by Western Ala, so we designed an 17 mer oligonucleotide probe based on blot analysis. A: SDS-PAGE, B: Western blot. Lane M: this sequence (5’-GGNGTNTGGCARCAYCA-3’, N means any basic protein molecular marker, lane 1: the total bacterial radical, Y means pyrimidine, N means purine). protein; lane 2: the total protein of supernatant from bacterial culture; lane 3: the crude insecticidal protein. 48 J. ZHANG et al. M 1 2 3 4 5 6 7 1 2 3 4 5 6 7 kb 21.23 5.15 4.27 3.63 2.03 1.58 1.38 A B FIG. 2 - A: double restriction endonuclease digested pSC1, B: hybridization with oligodeoxynucleotide probe. Lane M: L DNA/ EcoRI+HindIII, lane 1: EcoRI-BamHI, lane 2: SphI-BamHI, lane 3: SalI-BamHI, lane 4: SmaI-BamHI, lane 5: XbaI-BamHI, lane 6: KpnI-BamHI, lane 7: HindIII-BamHI. There are seven hybridization positive signals with oligonucleotide probe. The corresponding fragments were about 3.2 or 3.6 kb EcoRI-BamHI fragment, about 4.2 kb SphI-BamHI fragment, about 2.4 kb SalI-BamHI fragment, about 7.2 kb SmaI- BamHI fragment, about 4.0 or 4.5 kb XbaI-BamHI fragment, about 1.4 kb KpnI-BamHI fragment, about 2.8 kb HindIII-BamHI fragment. the shortest fragment was about 6.5 kb. All positive clones were TGA (GenBank accession no. DQ790644). In downstream of the digested with EcoRI completely. Hybridization with 17 mer oligo- terminate code, no other terminate code or reverse repeated nucleotide probe showed all of them had 5 kb positive hybridiza- sequence was found. The deduced amino acid sequence of tion signal fragment. this gene should contain Gly-Val-Trp-Gln-His-Gln-Ser-His-Ala- So the shortest recombinant plasmid pSC1 was used for fur- Ala (from 23 to 32 amino acids) which is same as N-terminal ther identification. Analysis with restriction endonuclease, only sequence of insecticidal protein. That indicated the ORF of 1.4 one recognition site by BamHI was found in plasmid pSC1, and kb fragment should contain insecticidal protein coding region the recognition site was in insert fragment. The plasmid pSC1, (Fig. 3). double digested with BamHI and other restriction enzyme, was There were 22 amino acid residues between initiation codon separated in agarose gel and transferred to nylon membrane. ATG of the ORF corresponding amino acid and N-terminal There are seven positive hybridization signals with oligonucle- sequence of insecticidal protein, for insecticidal protein was otide probe. Hybridization with oligonucleotide probe showed extracellular enzyme, so it should be a signal peptide. Ala Leu that the 1.4 kb BamHI-KpnI fragment was the smallest fragment, Ala (20 to 22 amino acids) should be cleavage site of signal which contained the gene encoding insecticidal protein (Fig. 2). peptide (Oliver, 1985). Most signal peptides have a tripartite structure: a positively Sequencing of the gene encoding the insecticidal protein charged N-domain, containing lysine and/or arginine residues; The BamHI-KpnI fragment was subcloned into pUC19, we named a hydrophobic H-domain; and a C-domain which specifies the recombinant plasmid pSC1-1. Sequencing the 1.4 kb frag- the cleavage site for specific signal peptidase (SPase). The ment by the ABI 310 Genetic Analyzer, we found that there N-terminal peptide of insecticidal protein only had one basic was an open reading frame of 828 bp, which coded the protein amino acids-Arg. The signal peptide had a C-domain, an atypical consist of 276 amino acid residues and had a terminate code of N-domain and lack of H-domain. FIG. 3 - Sequencing of the gene encoding the insecticidal protein. Ann. Microbiol., 59 (1), 45-50 (2009) 49 The deduced mature protein was 254 residues. According to Sequencing and the structural analysis of the gene encoding the sequence of the insecticidal protein gene, we deduced that insecticidal protein may help us unveil the structural charac- the molecular mass of the mature protein was 26.7 kDa and its teristics and the active domains of insecticidal protein, clarify isoelectric point was 5.57. This was consistent with the data of the regulation of gene expression, and construct high efficient purified protein. gene-engineered strains. Based on the N-terminal sequence of During the sequence analysis of the insecticidal protein insecticidal protein, we designed the oligonucleotide probe which gene, it was found that the codon usage had preference to G and was used to screen the gene library of P. pseudoalcaligenes. C, with the frequency of the third base of codon being G or C The coding region of insecticidal protein from P. pseudoalcali- reaching 71%. The amount of base in the coding chain contains genes has 828 bp that codes 276 amino acid residues. It was was high G and C of approximate 62.6%, and this coincided speculated that 22 amino acid residues of N terminus was for a with the high content of G and C in P. pseudoalcaligenes (Henry signal peptide. The signal peptide of insecticidal protein was dif- and Bessieres, 1984; Dumora et al., 1997; Bever and Iglewski, ferent from Secretory (Sec-type) signal peptides, Twin-arginine 1998). The 5’-end region of insecticidal protein gene had Shine- signal peptides are cleaved by one of the various type I SPases Dalgarno sequence AGGAAC, similarly to that of ribosome bind- of B. subtilis; Lipoprotein signal peptides, which are cleaved by ing site of E. coli (Stormo and Schuneuder, 1982). There was the lipoprotein-specific (type II) SPase; Type IV prepilin signal no sequence similar to -10 regions (TATAAT) or -35 regions peptides, which, in B. subtilis, are cleaved by the prepilin-spe- (TTGACA) in the promoter of E. coli were found. cific SPase ComC (Tjalsma et al., 2000). The sequence features By nucleotide-nucleotide BLAST (blastn) on NCBI, there resembled signal peptides of bacteriocins and pheromone which was no highly identical nucleic acid sequence with that of are exported by ATP-binding cassette (ABC) transporters. The insecticidal protein from P. pseudoalcaligenes, suggesting that signal peptides cannot be predicted by the regular algorithms for there was lack of identity between insecticidal protein from signal peptide prediction, such as SignalP, as it consist only of P. pseudoalcaligenes and other bacterial toxin. Furthermore, N- and C-domains and completely lack a hydrophobic H-domain compared with genes of insecticidal crystal protein of B. thur- (Nielsen et al.,1997). ingiensis, exotoxin A of P. aeruginosa, botulinum toxin A and B The gene coding insecticidal protein from P. pseudoal- of Clostridium botulinum, and diphtheria toxin, the identity was caligenes lacked significant identity with other bacterial toxins. lower than 15%. Therefore, insecticidal protein from P. pseudoalcaligenes was By NCBI conserved domain searching, 46.5% of the amino a novel insecticidal toxic protein discovered firstly in our lab. acid sequence of insecticidal protein was aligned with COG1804 Sequencing the cloned gene coding insecticidal protein revealed (CaiB, Predicted acyl-CoA transferases/carnitine dehydratase that it had the same N-terminal coding region as the natural [Energy production and conversion]), 42.6% of the sequence insecticidal protein. This region could code a mature protein of was aligned with KOG3957 (Predicted L-carnitine dehydratase/ 26.7 kDa whose molecular weight and amino acid composition alpha-methylacyl-CoA racemase [Lipid transport and metabo- was similar to that of the natural insecticidal protein. lism]), 58.9% of the sequence was aligned with pfam02515 The insecticidal protein gene was not expressed in E. coli, (CoA_transf_3, CoA-transferase family III). nor induced by IPTG. The results above might indicate that the promoter of insecticidal protein gene could not be recognized Expression of insecticidial protein gene in Escherichia by E. coli, or there were some fragments lost. Furthermore, the coli cloned fragments could not be expressed even with the help of The rabbit antiserum screening from the 5 positive clones in situ the lac promoter. lysis was negative neither induced or not by IPTG. The 1.4 kb The pT7-6 used to express genes under control of T7 RNA fragment was inserted into plasmid pUC19 and pUC18, recom- polymerase. The pT7-6 vector does not contain ribosome bind- binant plasmids pSC1-1 and pSC1-2 was obtained respectively, ing site ATG codon upstream of polylinker. Escherichia coli BL21 which were transformed into E. coli DH5A , JM109 and HB101. The (DE3), which contains the gene for T7 RNA polymerase on the E. transformants were cultured, and the expressed gene products coli chromosome under the control of the lac promoter, containing were identified by using antiserum. The gene encoding insecti- pT7-6-IP can express the insecticidal protein gene with IPTG. The cidal protein was not expressed in E. coli, nor induced by IPTG. western blot showed that the expressed protein can reacted with Then we cloned pSC1-1 EcoRI-BamHI fragment into pT7-6, insecticidal protein antiserum. These evidences indicated that the and transfered into E. coli BL21 (DE3). The lysate of E. coli BL21 gene cloned should be the coding region of insecticidal protein. (DE3) with pT7-6-IP was analyzed using SDS-page and Western Cloned the insecticidal protein gene has significance in many blot. Compared with the contained pT7-6 E. coli total protein, aspects, including clarifying the function and structure of the one additional 26 kDa band was found on SDS-page, and it insecticidal protein, wide application in developing new microbial had positive signal on western blot. The result showed that the insecticide and gene-engineered plants with insect-resistance, expression protein had same character as natural insecticidal and the basic study on the inheritance of insect pathogen protein. microbes. Acknowledgements DISCUSSION This work was supported by grants from The National Key Technology R&D Program of China 2007BAC18B03. 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Journal

Annals of Microbiology – Springer Journals

Published: Nov 24, 2009

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