With more than 160,000 species, Lepidoptera is recognized as the second most bio-diverse group of insects after Coleoptera. It includes many of the most devastating pests of forests, crops, and stored products as well as some  insects important to humans, such as the silkworm, Bombyx mori. The silkworm is a lepidpptran of economic value highly domesticated by man (Neils et al., 2007; Chitvan et al., 2009).

Sericulture has several thousand years of history in China. A sericulturist always faces the challenging of  rearing silkworms free of bacterial infection which results in growth retardation and death. The infected larvae cause a severe symptom of diapause. The diapausing larvae are typically infected by the common soil bacterial, Bacillus thuringiensis (Ohba, 1996). The annual economic losses caused by Bacillus thuringiensis infection in the Hangjiahu regions of Zhejiang Province, known as the home of silk in China, are incalculable.

The spore-forming bacterium Bacillus thuringiensis synthesizes parasporal crystal toxin during sporulation. Bt toxin usually works in the insect midgut, where Bt protoxins are activated by gut protease to produce activated toxins, activated toxin, which bind to specific receptors to confer toxicity. Large numbers of studies show that the reactions between Bt toxins and insect gut are determined by many gene products expressed in the insect gut. These include proteins and enzymes involved in Bt protoxin activities, toxin bind to receptors and toxin degradation products. These results imply that there are interacting systems for Bt toxin functions existing in the insect midgut. Changes in these systems might cause particular Bt toxin specificity and efficacy, and could affect Bt toxic lethal action to a variety of insects (Crickmore et al., 1998; Knowles, 1994).

Silkworm was the first lepidopteran insect to have its complete genome sequenced and has become the model insect species for Lepidoptera research (Kazuei, et al., 2004). Mining highly toxic Bt strains from the midgut of diseased silkworm larvae and identifying the Bt toxin functional gene has significant importance for studying insect resistances to Bt toxin.  

In this study, we collected 100 diapausing silkworm larvae from different farmers in the Hangjiahu region in Zhejiang Province. Sodium acetate & temperature separation was used to isolate Bacillus isolates from the midgut tissue and slime of collected samples. The Bacillus thuringiensis strains was further characterized by using staining, crystal shape observation, SDS-PAGE, plasmid profile and bioassay to make clear the genotype and Bt toxin genes.

Results
Isolation of Bacillus strains and Bacillus thuringiensis isolates
We dissected 100 diapausing silkworm larvae collected from the Hangjiahu of Zhejiang Province and used midgut tissue and slime to isolate the Bacillus strain using sodium acetate & temperature separation. From 218 bacillus strains harvested, six isolates were further identified to be Bacillus thuringiensis based on parasporal crystal formation observed under the oil lens optical microscope and scanning electronic microscope. Larvacidal assays were carried out using crude Bt proteins and the results indicated that Bt strain W015-1 was highly toxic to the lepidopteran Plutella xylostella (data not shown), It was deposited in the HITAR Bacillus Collections with Accession No 20050509W015. 

Parasporal inclusion morphology of W015-1 isolate 
Strain W015-1 was grown in BP solid medium at 30℃ for 3 days until the parasporal crystals were observed through oil lens light microscopy, and then the crystals were examined by scanning electron microscopy (SEM). It was clear that the parasporal crystals from Bt W015-1 were typically bipyramidal in shape (Figure 1). SDS-PAGE analysis indicated that the intact parasporal crystal of W015-1 has a dominant polypeptide of about 130 kD after grown for 20 hours during the sporulation stage (Figure 2). The crystal proteins of W015-1 were processed into about 80 kD fragments with 1 μmol/L trypsin treatment (Figure not shown).

Figure 1 Scanning electron micrograph of the spores and crystal proteins from Bt W015-1 (6.6 mm×15 k)

Figure 2 SDS-PAGE profile of parasporal inclusion proteins of  Bt W015-1

Plasmid profiles of W015-1
Bacillus thuringiensis commonly harbors a varied number of  large plasmids with different molecular mass. Most of the cry genes are located on these large plasmids (Carlson et al., 1996). Therefore, plasmid size and number are usually considered as tools to identify the strain characteristics (Procar et al., Vilas-Boas, 2004). In this study, the Pulsed-Field Gel Electrophoresis (PFGE) was employed to compare plasmid profiles of Bt W015-1 and the reference strains Bti, HD1 and HD73. The plasmid profiles of Bt W015-1 in size and number are the same as HD71,  but significantly different from Bti AND508 and HD1 (Figure 3).

Figure 3 PFGE banding patterns of large plasmids from Bt strain W015-1

Identification of cry-type toxin genes
The polymerase chain reaction-fragment length polymorphism (PCR-RFLP) was used to identif the cry genotype of W015-1 (All tested universal primers not listed in this paper). The primer pairs of K5un2/K3un2 and K5un3/K3un3 produced PCR fragments in size of 1.6 kb and 1.4 kb, respectively (Figure 4A). Both PCR amplicons were digested with Pstâ… and XbaI, Pstâ… and EcoRâ… , respectively (Figure 4B). The 1.6 kb fragments digested by Pstâ… and XbaI are 820 bp, 550 bp and 320 bp in size which are obviously larger than those of expecting sizes (801 bp, 518 bp and 322 bp). Similarly, the 1.4 kb fragments digested by Pstâ… and EcoRâ…  are 800 bp, 470 bp and 280 bp in size which are a little larger than those of expecting sizes (726 bp, 434 bp, 244 bp and 59 bp). It is clear that the 59 bp digested fragment was lacking in the strain Bt W015-1. Although the PCR amplicon of W015-1 is the same size as that of HD73, the RFLP patterns of the strains are different in size and number. The results indicated that Bt W015-1 had a different cry genotype than that of standard strain HD73. Purified PCR amplicons were ligated into pMD18-T vector to construct the recombinant plasmid pMDK2 and pMDK3 for sequencing (Beijing Genome Institute, Beijing, China). The sequence analysis revealed that deduced amino acid residues of the amplicons generated by the above primer was highly similar to cry 1Ac1, which implied that the W015-1 strain contains a cry1Ac type gene. 

Figure 4 Identification of cry1A genotype of Bt strain W015-1 and HD73 based on PCR-RFLP

Cloning of cry1Ac gene
In order to obtain the full length sequence of Cry1Ac from W015-1, inverted PCR was used to amplify the sequences through primer pair  cryI5/cryI3 designed by primer software based on the sequences of plasmids pMDK2 and pMDK3. 

The restriction endonucleases NedⅠ, SalⅠ,BglⅡ and BamH Ⅰ were used to completely digest the plasmids of W015-1, and then inactivated the restriction enzymes were inactivated in a 65℃ water bath for 15 min, followed by the addition of T4 DNA ligase to randomly connect the digested fragments. The inverse PCR produced the 1.6 kb fragment in size (Figure 5) based on the randomly NedⅠdigestion fragment as template.. This product was ligated into the vector pMD18-T to construct the recombinant plasmids pMDIS for sequencing. The sequences of recombinant pMDK2, pMD1K3 and pMDIS were assembled into a spliced DNA sequence of 3 772 bp, which contained a 3 534 bp open reading frame (ORF). The primer pairs of E1A5/E1A3 were designed based on the assembled sequence to amplify the full length sequence of the ORF about 3.5 kb (Figure 6). This further confirmed that the cry 1Ac gene existed in the strain W015-1.

Figure 5 Full length cry1Ac gene amplified by inverted PCR

Figure 6 Full sequence of cry1Ac22 gene amplified by PCR

The sequence of this gene deposited in the GenBank with accession number EU282379 was designated as cry1Ac22 based on the nomenclature system proposed by Crickmore et al (1998). The coding sequence of cry1Ac22 with 3 534 bp in length encodes a putatively weak acidic polypeptide of 1 178 amino acid residues with estimated molecular weight of 133 kD and iso-electric point of 5.04 , which includes 30.90% hydrophilic amino acids, 32.64% hydrophobic amino acids, 13.83% acidic amino acids and 11.43% basic amino acids. The deduced amino acid sequence of cry1Ac22 has 99% similarity to cry1Ac1, whereas three sites of 233 (T / R), 448 (M / I) and 1158 (K / E) are differences existing between cry1Ac22 and cry1Ac1. Multiple sequence alignment and conserved domain of cry1Ac22 were generated by using clustalW and proDom programs (http://www.ebi.ac.uk/Tools/clustalw2/index.html; http://prodom.prabi.fr/prodom/current/html/form.php), The results indicated that the Cry1Ac22 toxic protein has three protein domains and five conserved blocks that are typical features of Cry 1A proteins (Figure 7). Three-dimensional structure prediction revealed that Cry1Ac22, whose three-dimensional structure was identified by X-Ray analysis, is highly similar to cry1Aa and cry3Aa (http://swissmodel.expasy.org/workspace/index.php?func=modelling_simple1). 

Figure 7 Predicted 3-D crystal structure of Cry1Ac22 protein

Expression of cry1Ac22 gene and bioassay analysis
The cry1Ac22 was ligated into pQE30 to construct recombinant plasmid pQE30-cry1Ac22. The expression of cry 1Ac22 was induced by IPTG in the strain E. coli M15. SDS-PAGE analysis indicated that the band of 133 kD inclusion protein was present in the whole cell lysate with IPTG induction and no band was present without IPTG induction (Figure 8). The inclusion of cry1Ac22 can be hydrolysed to a trypsin-activated form with a molecular weight of about 80 kD with the 1 μmol/L trypsin treatment (Figure 8). Bioassay of a trypsin-activated form of cry1Ac22 was carried out and the results showed that expressed Cry1Ac22 protein exhibited high toxicity to second instar larvae of Plutella xylostella (LC50: 4.135 ×10⁸ cfu/mL; 95% FL: 3.368~5.122 ×10⁸ cfu/mL).

Figure 8 SDS-PAGE analysis of cry1Ac22 gene expressed in Escherichia coli M15 cells

Methods and Materials
Collection and anatomy of Silkworm
One hundred diseased and dead silkworm larvare with brown or black diapausing symptoms were collected from farmers in the Hangjiahu region of Zhejiang Province and placed in individual the 15 mL tubes. The larvare were dissected to obtain the midgut tissue and slime for isolating bacteria.

Isolation of Bacillus and Bacillus thuringiensis
Bacillus isolates  were obtained with the use of high temperature sodium acetate according to Xie et al (2009) and Hossain et al (1997) . The midgut tissue and slime were completely dissolved in 20 mL BPA medium incubated for 4~5 hours at 30°C with shaking at 220 r/min, and then moved to a water bath at 75℃ for 15 min, 1 mL of dissolved solution was proportionally diluted in 9 mL sterilzed water to a final dilution ratio of 10^-2 and 10^-3 for plating. The isolates were grown on NB solid medium plates for three days (beef extract 5 g, peptone10 g, NaCL34 g, distilled water 1 000 mL, pH value 7.0~7.4, 1.5% (w/v) agar, at 121℃ for 20 min autoclave sterilization). Single colonies were   re-plated and then used to observe the morphology and parasporal crystal through oil lens optical microscopey and scanning electronic microscopey. The isolates were stored in 50% glycerin solution at low temperature refrigeration. 

Strains, plasmids and growth conditions
The strains and plasmids used in this study are listed in Table1. B. thuringiensis W015-1 was isolated from the diapausing silkworm larvae. B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis were used as reference strains. pQE30 and pMD 18T were the vectors for cloning and expression of cry gene. Bt strains were grown in BP medium (Lecadet et al., 1980) and G-Tris medium (Aronson and Thompson, 1971) at 30℃. All E. coli strains were grown at 37℃ in Luria-Bertani (LB) medium and Terrific Broth (TB) medium (12 g Bacto-tryptone, 24 g yeast extract, 4 mL glycerol ddH₂O to 900 mL). All culture medium should be autoclaved at 121℃ for 20 min before using. For solid media 1% agar was added . Ampicillin (100 μg/mL) or Kanamycin (12.5 μg/mL) were added to culture media as required.

Table 1 Strains and plasmids used in this study

Observation of parasporal crystal by Scanning Electron Microscope (SEM)
Bt strains were grown in BP medium at 30℃ for 3 days until sporulation was complete as examined by light microscope with an oil-immersion lens. The spores and crystals were collected by centrifugation at 4℃ at 12 000 g for 10 min, and the precipitate was washed three times with ice-cold sterilized double-distilled water. The spore-crystal suspensions were placed on aluminum mount and fixed in 1% OsO₄ after the samples were air-dried overnight. The samples were then coated with gold in an IB-5 ion coater (HITACHI, Japan),The SEM observation was conducted on a HITACHI S-3400N (HITACHI, Japan) at a voltage 15 kv following instructions for the devise (Zhang et al., 2009).

Plasmids profiles of Bt strains
Bt W015-1 wase grown to the final OD₆₀₀ value of 2.0 at 30℃ in LB medium with shaking at 220 r/min. The strain cells were pelleted by centrifugation at 10 000 g for 5 min and re-suspended in GTE buffer [2.5 mmol/L Glucose, 25 mmol/L Tris–Cl,10 mmol/L EDTA (pH 8)]. Lysozyme was added to the suspension at a final concentration of 10 mg/mL and incubated 1 hour at 37℃ for enzymatic lysis of cell wall. Lysozyme (1.0% SDS; 0.8 mol/L NaOH) was added at 2 ×volumes and mixed gently by inverting the suspension 6 to 8 times. A half volume of 3 mol/L sodium acetate was added and incubated on ice bath for more than 4 h. After centrifugation at 4℃ at 12 000 g for 15 min, the supernatant was extracted with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) and then the plasmid DNA was precipitated from the aqueous phase with 2 volumes of cold 96% ethanol.

CHEF Mapper^® XA Pulsed Field Electrophoresis System (Bio-Rad, USA) was employed to detect the plasmid profiles of Bt W015-1 in accordance with the manufacture’s instruction. Parameters were set for automatical separation of a plasmid DNA with size range of 15 kb to 500 kb (Voltage: 6 V/cm, Running time: 20 hours, Included angle: 120℃, Initial Switch Interval time:1.19 sec, Final Switch Interval time= 44.69 sec). The plasmids DNA was subjected to electrophoresis in 1% low-melting-point agarose(Amresco, USA) at 14℃ with 0.5×TBE buffer (45mmol/L Tris•Cl,1mmol/L EDTA). The plasmids DNA was then stained in ethidium bromide (5 μg/mL) after electrophoresis for 30 min , followed by destaining in double-distilled water (at 4℃) for 2 hrs. Water was changed  three times in the course of decoloring and plasmids were then observed under ultraviolet light.

SDS-PAGE analysis of Bt strains
Bt W015-1 was inoculated in 5 mL of liquid LB medium and grown overnight at 30℃ with shaking at 200 r/min. At 12 hrs post inoculation, the mixture was transferred into eppendorf tubes with 200 mL BP medium by 1% volume ratio to continue culturd at 30℃ with shaking at 200 r/min.  A 1 mL spore-crystal mixture was harvested every two hours for SDS-PAGE analysis after centrifugaing at 4℃ at 12,000 g for 5 min and re-suspending the pellet in 100 L sterilized water after removing the supernatant. After 25 L NaOH (0.5 mol/L)  was added, samples were placed at room temperature for 10 min, and 63 L 3 × loading sample buffer (3.63 g Tris, 0.3 g bromophenol blue, 6 g SDS, 30 mL glycerol, 15 mL β-mercaptoethanol dissolved in 100 mL double-distilled water and pH 6.8) was added to the suspension. The supernatant was loaded onto 7.5% gel immediately for SDS-PAGE analysis. Laemmli’s electrophoresis procedure were followed up Laemmli’s method in this study (Laemmli, 1970).

Identification of  cry genotype by PCR-RFLP
DNA templates for PCR were prepared following Yu’s method ( Yu, 2006). Thirty four primer pairs were used to identify the cry gene (Xie el al., 2009), including the primer pairs of K5un2 / K3un2 and K5un3 / K3un3 for screening the Bt strain cry type genes (Table 2) (Kuo and Chak, 1996b). 50 μL PCR reaction volume sample contains 5 μL10×PCR buffer(promega, USA), 0.5 μL (4 U/μL)Taq polymerase (promega, USA), 1 μL dNTP mixture (to 250 μmmol/L final concentration), 1 μL each primer, 1 μL (50~100 ng) template DNA, after adding 40.5 μL ddH₂O to 50 μL. The PCR was performed in a PTC-200 Thermo Cycler (MJ Research, USA) with the procedures as follows: pre-denaturation at 94℃ for 5 min, then followed by 30 cycles (94℃ 1 min, 53℃ 1 min and 72℃3 min), and finished at 72℃for 10 min. PCR products were examined by 1% agarose gel electrophoresis and purified using the TIANgen Midi Purification Kit (Tiangen, Beijing, China). PCR-RFLP analysis  followed the procedures of Kuo and Chak (1996a). Table 3 shows the PCR-RFLP banding patterns of cry1Ac1 revealed by using 2% agarose gel electrophoresis (Kuo and Chak, 1996a).

Table 2 Primers for genes identification/cloning or expression

Table 3 PCR-RFLP analysis of the cry1Ac1

Cloning of cry-type gene
The PCR products amplified by primer pairs K5un2/K3un2 and K5un3/K3un3 were ligated into the Easy Vector pMD18T to construct recombinant plasimds pMDK2 and pMDK3, then sequenced by Automatic DNA Sequencer (ABI-3730XL, Huda, Beijing). According to the sequences, primer pairs of cry1I5/cry1I3 were designed for the inverted PCR amplification. Plasmid DNA of Bt W015-1 (1.0 to 2.0 μg) was digested completely by Nedâ… , Salâ… ,Bglâ…¡and BamHâ… in a 37℃ water bath for 3 hours, respectively, prior to inactivating with restrictive endonuclease in a 65℃ water bath for 15 min, and then added T4 ligase was then added to connect the digested fragments at 4℃ overnight (over 12 hours). The inverted PCR was performed using connected products as the template, and the inverted PCR products were inserted into the pMD18T vector to construct a recombinant plasmid named pMDIS for sequencing. Finally,  three sequences were assembledfrom the recombinants of pMDK2, pMDK3 and pMDIS to make the full length gene sequence (Triglia et al., 1988).

Expression of cry1Ac22 and Bioassay
The primer pair E1A5/E1A3 was designed based on the full sequence of cry1Ac22, in which the BamH â…  and Sal â…  restrictive endonuclease sites were introduced into the 5’ end of the forward and reverse primers, respectively. The sequence confirmed gene was ligated into the prokaryotic expressing vector pEQ30 to make the recombinant vector pEQ30-cry1Ac22. The recombinant plasmid was further transformed into Escherichia coli M15 and then were incubated in TB medium adding 12.5 μg/mL Kanamycin, at 37℃ for about 1.5 h until the value of OD₆₀₀ reached about 0.5, IPTG was added to the mixture at the final concentration of 1 μmol/L for inducing Cry1A22 for 4~10 hours at 30℃ The expression cells were collected by centrifugation at 12 000 g at 4℃, for 10 min and completely suspended in an equal volume of TE. Lysozyme was then added  to digest at the final concentration of 20mg/mL and incubated at 37℃ while shaking at 200 r/min for 30 min. The cells and proteins were re-collected by centrifugation, the pellet was washed with an equal volume of 1 mol/L NaCl three times and then broken up by ultrasonic treatment (Model VC-130, Sonics and Materials Inc, USA) for 20 min. The concentration of expressed Cry1Ac22 protein was measured by the Lowry assay with a standard marker protein of bovine serum albumin (BSA) (Lowry et al., 1951). The expressed proteins were disolved in 50 mmol/L Na₂CO₃ (pH 10.0) with 1 mol/L typsin  added  to digest during incubation at 37℃ for 1 hour. SDS-PAGE procedures were the same as that mentioned above.

To bioassay, each leaf 8cm diameter disk of Chinese cabbage leaves were immersed in different concentrations of typsin treated proteins containing 0.02% Triton X-100, for 10 sec and air dried naturally at room temperature for 2 hour. Each leaf disks was placed in an individual Petri dish (10 cm diameter) lined with moistened filter paper, and 10 second-instar larvae of Plutella xylostella were introduced into each Petri dish. The experiment wasreplicated six times and buffer and sterilized water were used for reference and blank. Larval assays were performed at 26°C and 65% relative humidity for 96 hrs with a photoperiod of 14 hrs light/10 hrs dark . The LC₅₀ with the 95% confidence intervals, were estimated by SPSS software for windows (SPSS Inc, Chicago, USA) (Sayyed et al., 2001).

Conclusions
In this paper, we isolated and characterized Bacillus thuringiensis W015-1, from the diapausing silkworm larvae,It showed high insecticidal activity against the lepidopteran P. xylostella. Bt W015-1 synthesizes a bipyramidal crystal with a molecular weight of 130 kDduring sporulation. The plasmid profiles of Bt W015-1 are similarity to the reference strain HD73 whereas there were dissimilarities  in size and number to HD1 and Bti. The cry genotype of W015-1 has obvious differences in the restricted enzyme sites with that of model strain HD73. 

We cloned the cry1Ac22 gene that has a length of 3,537 bps encoding 1,178 amino acid residues. Cry 1Ac22 has high amino acid sequence identity to Cry1Ac1 with the differences existing in the sites of 233(T/R), 448(M/I) and 1158(K/E). The Cry1Ac22 inclusion protein with a molecular weight of 133 kD, was expressed in E. coli induced by ITPG , The trypsin-activated form of the recombinant protein was found to have high insecticidal activity against larvae of Plutella xylostella, compared to that of model strain HD73.

With respect to the reference strains, W015-1 has different plasmid profiles, cry genotypes and crystal proteins. Thus, it is believed that Bt W015-1 could be used as a potential biopesticide alternative to Btk.

Authors’ contributions
LX, WFZ and ZML conducted all the research works for this paper. YGC was involved in collecting the diseased silkworm larvae. LX and WFZ jointly completed the data analysis and manuscript preparation. YZL participated in experimental management and reviewed the manuscript. XJF coordinated the project and was fully involved in the experimental design, data analysis and manuscript preparation. All authors read and approved the final manuscript.

Acknowledgements
Authors would like to thank Madam Ruying Zhao from the Jiaxing Bureau of Agriculture in Zhejiang Province for her helps in collecting the diseased silkworm larvae. We greatly appreciate Dr. Phil Grau, Sr. Entomologist of SynTech Research, for reading and revising the manuscript. We also thank two anonymous reviewers for their strict criticism on this paper. This work was initiated by theChina National Bt Collection Initiative  project and partly supported by the National 863 Program of China (Project No. 2004AA2111112). 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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