Engineered bacteria construction
Prediction of Effective Fragments of CHS1
The deduced amino acid sequences of SlCHS1 and SlCHS2 were analyzed by using DASTAR software. The open reading frame (ORF) was predicted according to the ORF finder tool (https://www.ncbi.nlm.nih.gov/orffinfer/). The molecular weight (MW) and isogenic point (pI) of SlCHS1 and SlCHS2 were calculated using ExPASy (http://web.expasy.org/compute_pi). The signal peptides of SlCHS1 and SlCHS2 were predicted using SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP). The functional domain was predicted by using SMART software (http://smart.embl-heidelberg.de/). The membrane-spanning domain was predicted by TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). The phylogenetic tree was constructed with MEGA 7.0 software using the neighbor-joining method with 1000-fold bootstrap resampling. Protein sequences from different insect species were obtained from GenBank (http://www.ncbi.nlm.nih.gov/) and used in the phylogenetic analysis.
Figure 1 Multiple sequence alignment of the conserved catalytic domain of the chitin synthases (CHSs) from three insect species. CHSs are from Spodoptera litura (Sl), Spodoptera exigua (Se) and Helicoverpa armigera (Ha). Seven characteristic motifs (M 1–7) for insect chitin synthases are indicated with red boxes.
Construction of bacteria expression vectors
The bacteria expression vectors were constructed based on pET28a. Three vectors were designed for expressing amiR-CHS1, VLP ( HIV TAT-conjugated coat protein (CP) dimer), and VLP plus amiR-CHS1 ( VLP-amiR-CHS1, Figure 3) in Escherichia coli.
Figure 2 Vector design for expressing amiRCHS1, VLP and VLP-amiRCHS1 in bacteria. pac: the site where CP recognizes RNA and assembles it; TAT, transmembrane peptide. The above vectors are all modified from pET28a.
Detection of RNA Expression
Three bacteria expression vectors were expressed in E.coli BL21(DE3) that lacks RNase III activity (BL21Δrnc). RNA in different strains before and after IPTG induction (overnight) was extracted and detected with formaldehyde denaturing gel. Northern blot showed the expression amiR-CHS1 in BL21Δrnc that expresses amiR-CHS1 and VLP-amiR-CHS1.
Figure 3 Detection of amiRNA accumulation in BL21ΔRNC. After IPTG induction, amiRNA expression was detected in the strains expressing VLP, amiR-CHS1 and VLP-amiR-CHS1 (138 nt). Samples of 10 µg total cellular RNA were loaded in each lane of the RNA blot. The Gelview-stained gel before blotting is shown below the blot as a loading control.
Detection of Protein Expression
Proteins were extracted from different BL21Δrnc strains before and after IPTG induction (overnight). Successful expression of VLP was determined by SDS-PAGE in bacteria expressing VLP and VLP-amiR-CHS1.
In summery, both protein and amiRNA could efficiently express in BL21Δrnc.
Figure 4 Detection of protein expression level in BL21ΔRNC. After IPTG induction, the expression of 2×CP (28 kDa) was detected in the VLP and VLP-amiR-CHS1-expressing strains. Sample load, 10 μg.
Insect feeding and bioassays
VLP improved RNAi efficiency in S. litura
Bioassay was conducted via feeding S. litura larvae with an artificial diet supplemented with the engineered bacteria. Only the VLP-amiR-CHS1 markerdly reduced the larval weight gain7 days after feeding (DAF).
Figure 5 Bioassay of S. litura larvae 7 days after feeding (DAF). (A) Representative examples of S. litura larvae. (B) Larval weight of S. litura. Data are means ± SE (n ≥ 30).
Relative expression of SlCHS1
As expected, the expression of target gene was significantly reduced in larvae fed with VLP-amiR-CHS1-expressed bacteria compared with other groups.
Figure 6 Analysis of CHS1 expression in S. litura after 7 d of feeding by qRT-PCR. Data are means ± SEM (n ≥6). Different lowercase letters above the columns indicate significant differences (P < 0.05, one-way ANOVA with Tukey’s multiple range test).
Detection the stability of the amiRNA
To test whether the MS2 VLP could reduce the degradation of amiRNA by nuclerase in the midgut of S. litura, RNA from different BL21Δrnc strains was extracted and digested with intestinal fluid. The results showed that naked amiR-CHS1 was quickly degraded after 5 min of incubation and completely degraded after 10 min. By contrast, the amiCHS1 encapsulated by the MS2 VLP could be detected even after 10 minutes of incubation. These results indicate that the MS2 VLP could protect amiRNA from degradation in the intestinal fluid of S. litura.
Figure 7 Stability test of S. litura's intestinal fluid against amiRNAs. Northern blotting of amiR-CHS1 in engineered E. coli extract after incubation with intestinal fluid (15-fold dilution) of tobacco cutworms at room temperature. Naked amiR-CHS1 is strongly degraded after 5 minutes and completely degraded after 10 minutes. In contrast, amR-CHS1 filled with VLP is relatively stable. Sample load, 5 μg.
Silencing CHS1 caused developmental defect of S. litura
Bioassay of S. litura was performed till the molting stage. The pupation and molting of S. litura larvae were significantly inhibited by feeding the VLP-amiR-CHS1.
Figure 8 Effects on pupation and molting of S. litura after silencing SlCHS1. (A,B) Phenotype of S. litura 25 and 32 DAF.
Compared with CK ,VLP and amiR-CHS1, VLP-amiR-CHS1 was more effective in affecting the pupation rate and deformity rate of S. litura.
Figure 9 Representation (%) of developmental and mortality rates of S. litura larvae until molting stage after feeding on engineered bacteria.
Production of transplastomic tobacco plants
Plastid transformation vector construction
These results indicated that the expression of amiRNA in bacteria could effectively interfere with the growth and development of S. litura. Subsequently, PLASTID PESTICIDES™ constructed plastid transformation vectors pMJ11 and pMJ12 expressing amiRNAs.
Figure 10 Tobacco plastid transformation vector. The selectable marker gene aadA is driven by the psbA promoter (CrPpsbA) and the 3'UTR of the rbcL gene (CrTrbcL) of Chlamydomonas reinhardtii. GaccD: tobacco accD promoter; Prrn: tobacco rRNA operon promoter; TrrnB: rrnB terminator of E. coli.
Biolistic transformation
Sterile tobacco leaves were bombarded with plastid plasmids using a 1100 psi rupture disk at target distance of 9 cm. Following the biolistic bombardment, the leaf samples were diced into 5 × 5 mm and were placed on regeneration media including 500 mg/L spectinomycin.
Authentic transplastomic clones carrying an aadA gene are resistant to both spectinomycin and streptomycin, whereas spontaneous spectinomycin-resistant mutants are resistant only to spectinomycin. While resistance is manifested as formation of green calli with regenerating shoots, sensitivity is indicated by formation of scanty white callus in the leaves. Resistance to streptomycin and spectinomycin indicates the presence of selectable aadA gene. However, double selection delays shoot formation. Hence, we scored the presence of aadA gene by resistance to streptomycin plus spectinomycin, but screen homoplastic transplastomic plants on spectinomycin.
Table 1. Summary of plastid transformation experiments conducted in tobacco.
Figure 11 Screening of leaves transformed with dual antibiotics. (A) Three groups of tobacco leaves transformed with the vector Nt-amiRCHS1 (B) Four groups of tobacco leaves transformed with the vector Nt-VLP-amiRCHS1.
Homoplasy confirmation of transplastomic plants by Southern blot
Figure 12 Generation of transplastic tobacco plants expressing MS2 VLPs for delivery of amiRCHS1 against S. litura. (A) Physical map of the targeted region in the tobacco plastid genome (ptDNA) and maps of the transformed region of the tobacco plastid genome in the Nt-VLP, Nt-amiRCHS1, and Nt-VLP-amiRCHS1 transplastic lines. The SphI restriction sites used for restriction fragment length polymorphism (RFLP) analysis of transplastomic lines are indicated, and the sizes of the restriction fragments detected in Southern blot analyses are given. (B) Southern blot analysis of plastid transformation of tobacco line. SphI-digested DNA samples were separated by electrophoresis in a 1% agarose gel and hybridized with the DIG-labeled probes shown in (A). The absence of the 4.9 kb hybridization signal of the wild-type tobacco plastid genome indicated that all transplastic lines reached homoplasmy.
Figure 13 Stable inheritance of plastid transgenes and wild-type-like phenotypes of transplastomic tobacco plants. (A) Seed assays to confirm homoplasmy of transplastomic tobacco plants. Seeds obtained from wild-type (Nt-wt) and transplastomic plants were germinated on synthetic medium containing spectinomycin (500 mg L−1). Resistance of seedlings to the antibiotic and lack of segregation confirm the homoplasmic state of the transplastomic lines. Phenotypes of transplastomic tobacco lines grown in soil for 3 (B) and 6 (C) weeks. Scale bars: 1 cm in (A) and 10 cm in (B,C).
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