2. Material and Methods
2.1 DBM strain
The DBM strain ‘Fuzhou lab’ were reared on radish seedlings without exposure to insecticides for 5 years, spanning at least 100 generations (You et al. 2013).
2.2 Preparation of brush border membrane vesicles (BBMV)
Midgut BBMVs were prepared as described previously (Wolfersberger et al. 1987). Fifth-instar larvae were immobilized on ice and dissected in cold dissection buffer (17 mM Tris-HCl, pH 7.5, 5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 300 mM mannitol, 1 mM phenylmethane sulfonyl fluoride (PMSF)) to isolate the midgut epithelium. Midgut epithelial tissue was homogenized in an equal volume of ice-cold 24 mM MgCl2, then incubated on ice for 15 min, followed by centrifugation at 25,006 g at 4°C for 15 min to collect the supernatant. The centrifuged pellet was resuspended in ice-cold dissection buffer in 0.5 volume of the initial homogenate and then the BBMV extraction procedure was repeated as described above. The supernatants collected from the two extractions were combined and BBMVs were precipitated by centrifugation at 30,000 g at 4°C for 1 h and stored at −80°C. Protein concentration was measured using the BCA Protein Assay Kit (Rockford, USA) according to the manufacturer’s instructions.
2.3 On-membrane capture
Ten micrograms of each Bt protein was separated in a 10% gel by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In parallel, same amount of Bt proteins was separated by another 10% gel. Then, proteins were transferred to nitrocellulose membrane using an Amersham Semi-Dry Transfer Unit (Freiburg, Germany). Proteins one NC membrane were denatured by incubating the membrane in denaturing and renaturing buffer (100 mM NaCl, 20 mM Tris (pH 7.6), 0.5 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 0.1% Tween-20, and 1 mM dithiothreitol (DTT)) containing 6 M guanidine-HCl for 30 min at room temperature (Wu et al. 2007). The membrane was then washed with denaturing and renaturing buffer containing 3 M guanidine-HCl for 30 min at room temperature, then washed with denaturing and renaturing buffer containing 0.1 M and no guanidine-HCl for 1 h at 4°C. The membrane was blocked with Pierce protein-free buffer (Rockford, USA) for 1 h at room temperature. Membranes were incubated with 30 µg total BBMV proteins (final concentration 10 µg/mL) in protein-binding buffer (100 mM NaCl, 20 mM Tris (pH 7.6), 0.5 mM EDTA, 10% glycerol, 0.1% Tween-20, and 1 mM DTT) overnight at 4°C. The Bt proteins on the NC membrane were positioned by aligning to Bt proteins on the other NC membrane that visualized with Ponceau S, then were cut for trypsin digestion.
On-membrane digestion was carried out as described by Luque-Garcia et al. (Luque-Garcia et al. 2008). Nitrocellulose bands were washed at least six times with Milli-Q water (Merck Millipore, Shanghai, China), then incubated in trypsin solution (12.5 ng/µl prepared in 50 mM NH4HCO3 buffer (pH 8)) at 37°C overnight. After digestion, samples were dried in a vacuum, dissolved in acetone (90 µl acetone/4 mm2 nitrocellulose), vortexed, and incubated for 30 min at room temperature. Acetone containing dissolved nitrocellulose was carefully removed and precipitated peptides were air-dried. Peptides were resuspended by adding 20 µl of 2% acetonitrile in 0.1% formic acid. All solutions were sonicated for 10 min before mass spectrometry analysis.
2.4 Q Exactive LC MS/MS analysis
In the analysis of complex mixtures, peptides of similar mass often co-elute; therefore, resolution is key in mass spectrometry (Michalski et al. 2011). Shotgun proteomics using the Q-Exactive instruments (Thermo Fisher Scientific, USA) is usually performed at 17,500 resolution at m/z 200 with a transient length of 60 ms. The higher resolution in MS/MS spectra helps to assign fragments of large precursors. Data were analyzed in MaxQuant using the integrated Mascot search engine (Michalski et al. 2011). The total number of MS scans exceeded 5000, and the total number of MS/MS scans exceeded 16 000. The average number of isotope patterns detected was close to 35,000, a very high number considering that the gradient was not particularly long, presumably because of the short MS and MS/MS cycle time of 1 s.
2.5 Far western blot
Far western blot was performed as previously described (Wu et al. 2007). Briefly, Bt proteins (Cry1Ac, Cry1Ab, and Cry1Bd) were separated on a 12% SDS-PAGE gel, then transferred to polyvinylidene difluoride (PVDF) membrane. Protein denaturing and renaturing on the membrane was performed exactly as per the protein co-blotting procedure described above. The membrane was then blocked with 5% milk in phosphate-buffered saline (PBS) for 1 h at room temperature. The membrane was then incubated with 5 µg purified His-GSS1 or His-GSS2 proteins (final concentration 1 µg/mL) in protein-binding buffer overnight at 4°C. Membranes were probed with anti-His primary antibodies, then washed with PBST (1% Tween-20 in PBS buffer), incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, and exposed to X-ray films after reacting with electrochemiluminescence (ECL) substrates.
2.6 Purification of GSS1and GSS2 proteins
Primers used for cloning in this study are listed inSupplemental Table 1 . GSS1 and GSS2 were cloned into the pET28A vector using BamHI and HindIII sites, and over-expressed in the BL21 (DE3) Escherichia coli strain.
Bacterial cells were harvested by centrifuging at 3000 g for 15 min at 4°C. Cells were washed with bacterial cell lysis buffer to remove residual culture medium. Washed cells were harvested by repeating centrifugation at 3000 g for 15 min at 4°C. After decanting the supernatant, the wet pellet was weighed, and E. coli cells resuspended in about 3 mL of lysis buffer per gram of cell pellet. The suspension was stirred for 30 min at 4°C, lysozyme was added to a concentration of 0.1% (w/v ), then the mixture was incubated for 30 min at 4°C while shaking gently. The suspension was centrifuged at 23,000 g for 30 min at 4°C and the supernatant discarded. The pellet was dissolved in inclusion body binding buffer (20 mM Tris-HCL (pH 7.9), 5 mM imidazole, 0.5 M NaCl, 8 M urea). The cleared inclusion bodies solution was applied to a column containing Ni-Agarose beads equilibrated with 3 × 5 mL ultrapure water and 3 × 5 mL protein binding buffer. The column was washed with 15 bed volumes of inclusion body binding buffer before eluting polyhistidine-tagged proteins with 5–10 bed volumes of inclusion body elution buffer (20 mM Tris-HCL (pH 7.9), 500 mM imidazole, 0.5 M NaCl, 8 M urea). Protein purity was typically >90% as determined by SDS-PAGE and Coomassie blue staining. Protein concentrations were measured using the BCA Protein Assay Kit according to the manufacturer’s instructions.
2.7 His-tag pull-down
The Pierce His Tag Protein Interaction Pull-Down Kit (catalog number 21277) was used to detect the binding of GSS1 and GSS2 with Bt proteins. Solubilization of proteins (His-GSS1 and His-GSS2) from inclusion bodies (a requirement of this kit) was carried out according to the method developed by Simpson (55). Cells were lysed as described in “Cloning and purification of GSS1and GSS2”. The cell lysate was centrifuged at 23,000 g for 30 min at 4°C before decanting the supernatant and measuring the wet mass of the pellet. The pellet was resuspended in 10 volumes of lysate washing buffer and the suspension stirred for 1 h at room temperature. The mixture was again centrifuged at 23,000 gfor 30 min at 4°C, the supernatant decanted and the pellet recovered. Wash steps were repeated three more times. The pellet was then dissolved in 9 volumes of solubilization buffer C per gram wet weight of inclusion body pellet, and the mixture incubated for 1 h at room temperature. Nine volumes of renaturation buffer C were added slowly to the solubilized pellet and the mixture incubated for 2 - 4 h at 25°C. Two milliliters of Ni-Agarose beads were added to renaturation buffer C and incubated overnight on a magnetic stirrer at 4°C. Protein–bead complexes were collected by pressing renaturation buffer C through a column with a 0.25 nm filter. Polyhistidine-tagged proteins were eluted with 5–10 bed volumes of elution buffer (20 mM Tris-HCL (pH 7.9), 500 mM imidazole, 0.5 M NaCl). Imidazole was removed from purified His-GSS1, His-GSS2 and Bt proteins (Cry1Ac, Cry1Ab and Cry1Bd) by dialysis against Tris-buffered saline buffer (25 mM Tris-HCl, 0.15 M NaCl (pH 7.2)).
A His-tag protein was added to spin columns containing equilibrated HisPur Cobalt Resin (Thermo Scientific, Rockford, USA) and incubated at 4°C for at least 30 min on a rotating platform with a gentle rocking motion. Spin columns were centrifuged at 1250 g for 30 s to 1 min to remove solution. Beads were washed 5 times with 400 μL of wash solution. Up to 150 μg of prepared Bt proteins was added to columns, which were then incubated overnight at 4°C. Beads were washed 5 times with washing buffer to remove non-specifically bound proteins. Proteins were eluted by adding 250 μL of elution buffer (1 mL of 290 mM imidazole elution buffer made with 70 μL of 4 M imidazole stock solution to 930 μL of wash solution) to the spin column. Spin columns were incubated for 5 min on a rotating platform with gentle rocking motion, before centrifuging at 1250 g for 30 s to 1 min. Proteins were analyzed by SDS-PAGE and visualized by silver stain.
2.8 Plasmids and plant transformation
Plasmids for double-stranded RNA (dsRNA) expression were constructed as previously described (56). The pBSK intron vector was a pBluescript II SK vector (Stratagene) containing a 120-nucleotide intron of theArabidopsis thaliana RTM1 gene between the NotI and XbaI sites. Sense and antisense target fragments with restriction enzyme sites at both ends were obtained by PCR amplifying DBM cDNA clones with primer pairs (Supplemental Table 1 ). The two PCR fragments were inserted at inverted repeats into the corresponding sites of the pBSK intron vector. The dsRNA construct generated was then used to replace GUS in pBI121 to generate the Pro 35S::dsRNA construct. The final RNAi vector was introduced into Agrobacterium tumefaciens strain GV3101. Transgenic Arabidopsis plants were generated using the floral dip method, screened on half-strength Murashige and Skoog (MS) agar medium containing 30 µg/mL kanamycin.
Analysis of dsRNA expression levels were analyzed using T2 homozygous plants. Freshly hatched DBM larvae were fed with leaves of thedsGFP and dsGSS1 lines for 7 d, respectively. Expressions of GSS1 and GSS2 were detected by real-time PCR (primers listed in Supplemental Table 1 ).
2.9 Quantitative reverse transcription PCR (RT-qPCR)
RT-qPCR was performed using an Eppendorf Mastercycler ep realplex, using gene-specific and allele-specific primers to detect expression patterns. Each reaction was performed in a 20 μL volume containing 10 μL SYBR Green (Fermentas), 0.4 μL Rox Reference Dye II, 1 μL of each primer (10 mM), 1 μL of sample cDNA, and 7.6 μL UltraPure distilled water (Invitrogen). The PCR program used was: 95°C for 10 s, 40 cycles at 95°C for 20 s, 60°C for 30 s. Relative quantification was calculated using the comparative 2-△△CT method (Schmittgen and Livak 2008). All data were normalized to the level of RP49 from the same sample.
2.10 Silkworm transformation
Transformation plasmids were constructed based on the initial piggyBac vectors pBac[3xp3-DsRed, IE1-EGFP]. The 3xp3 promoter was removed by cutting with NotI and AgeI, and was replaced with the HR5 enhancer followed by the IE1 promoter to generate the pBac[HR5IE1-DsRed, IE1-EGFP] plasmid based on homologous recombination using the ClonExpressTM II One Step Cloning Kit (Vazyme Biotech Co., Ltd.). The open reading frames (ORFs) of GSS1 and GSS2 were replaced with EGFP to generate the silkworm transformation plasmids, pBac[IE1-DsRed, IE1-GSS1] and pBac[IE1-DsRed, IE1-GSS2]. Primers are listed in Supplemental Table 1 .
Silkworm microinjection was performed as described previously (Tan et al. 2013). The transformation plasmid was microinjected into preblastoderm G0 embryos (Nistari strain), which were then incubated at 25°C in a humidified chamber for 10–12 d until larval hatching. Larvae were reared on fresh mulberry leaves or an artificial diet (Nihonnosanko) under standard conditions. Putative transgenic adult G0 were mated with each other, and G1 progeny were scored for the presence of red fluorescence using fluorescence microscopy (Nikon AZ100). Positive G1 progeny were mated with wild-type moths to generate hybrid silkworms expressing one copy of dsRed and a target gene. Hybrid silkworms with red fluorescence were fed with Cry1Bd-treated leaves as described below.
2.11 Bt toxin treatment
To test the tolerance of RNAi-silenced DBM larvae, two leaves from 4-week-old A. thaliana plants with genotypes dsGFP anddsGSS1 were laid on a moistened filter paper in a 15 mm petri dish. Freshly hatched DBM larvae were placed on leaves of each genotype to feed for 7 d. On the eighth day, larvae were transferred to fresh leaves coated with either a diluted suspension of Bt toxin in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 8.0), or HEPES buffer as a control. Mortality was recorded after 24 h and LC50 values were calculated by probit analysis based on the dose determined to be high enough to kill 100% of larvae.
To test the tolerance of transgenic silkworms, four squares of mulberry leaf (4 × 4 cm), coated with 50 μL of a diluted suspension of Cry1Bd in HEPES buffer (pH 8.0) were fed to 10 second instar larvae for 24 h. The dose high enough to kill 100% of susceptible larvae was determined. Probit analysis was carried out using SPSS software (version 12.0) to determine LC50.