Production of functional double-stranded RNA using a prokaryotic expression system in Escherichia coli

Abstract

RNA interference (RNAi) is a nucleic acid metabolism system utilized for the post-translational regulation of endogenous genes or for defense against exogenous RNA or transposable elements. Double-stranded RNA (dsRNA)-mediated RNAi shows broad application prospects to improve existing plant traits and combat invading pathogens or pests. To improve dsRNA transcriptional efficiency using a prokaryotic expression system, Trxz gene, an essential gene for the early development of chloroplasts in Arabidopsis thaliana, was chosen for a functional study. Two types of recombinant expression vectors, pDP-Trxz and phP-Trxz-N/L, were constructed to generate dsTrxz, the dsRNA which specifically induces Trxz gene silencing. Gel electrophoresis tests showed that phP vectors performed better and produced more dsRNA than the pDP vector under the same conditions. Purification of dsTrxz by enzymatic digestion indicated that highly purified dsRNA can be obtained through the use of DNase enzymatic hydrolysis assay. To confirm the knockdown effect of the dsRNA, a root immersion assay was performed, and we found that the root immersion culture could continue to affect the growth and development of A. thaliana. This included inhibiting the development of new leaves, causing weak plant development, leaf whitening, and other symptoms. This indicated that in vitro expressed dsRNA can be absorbed through Arabidopsis roots and can continue to trigger Trxz gene silencing. To delay dsRNA degradation and extend the effectiveness of RNAi, nanomaterial layered double hydroxide (LDH)-mediated BioClay was performed. We found that LDH-mediated BioClay alleviates the degree of dsRNA degradation, which provides a new idea for the storage and transportation of dsRNA.

Keywords: LDH; RNAi; dsRNA; hpRNA; immersion of roots.

Figure 1

Double promoter recombination vector pDP‐Trxz (a) and hairpin transcriptional vector phP‐Trxz‐L (b) and phP‐Trxz‐N (c) construct. The arrows indicate the direction of transcription. All plasmids were based on pUC19 cloning vectors. T7: T7 promoter. Ter, terminator

Figure 2

Comparison of recombination vector transcriptional efficiency. (a) Lanes 1 and 2: Transcript dsTrxz produced by pDP‐Trxz recombinant vector in the independent repeated trials; Lane 3: Transcript hpTrxz‐L produced phP‐Trxz‐L recombinant vector; Lane 4: Transcript hpTrxz‐N produced phP‐Trxz‐N recombinant vector; Lane 5: negative control vector pDP expressed in M‐JM109‐LacY. (b) Quantitative analysis of gel bands demonstrated that dsTrxz transcript produced by phP vector in lanes 3 and 4 is significantly higher than pDP vector in lanes 1 and 2. The bands intensity was evaluated with ImageJ. **p < 0.01 (one‐way ANOVA). (c) The Escherichia coli transfected with recombinant plasmid was collected for total RNA extraction. Total nucleotides concentration was analyzed on an Epoch Spectrophotometer system (BioTek) and the dsRNA concentrations were calculated via experimental groups and the control group

Figure 3

Enzymatic purification of dsRNA crude extracts. Gel electrophoresis evaluated the dsRNA crude extracts from M‐JM109‐LacY, Trxz‐N, and Trxz‐L treated with (+) or without (−) DNase for 15 min. Neg1, extracts with control phP vector in M‐JM109‐LacY. Neg2, extracts of M‐JM109‐LacY

Figure 4

Immersion of roots in hpTrxz‐N inhibits Arabidopsis development. (a) The same batch of Arabidopsis seedlings grown on MS medium for 7 days after germination was divided into six subgroups. Each subgroup was treated with different concentrations of dsTrxz (hpTrxz‐N) with a concentration gradient of 0, 0.1, 1, 10, 100, and 1,000 ng/L. The average leaf whitening rate (LWR) was observed after treatment for 7 days. (b) Arabidopsis was cultured in liquid medium containing 10 ng/L hpTrxz‐N. After 4 weeks of growth, the difference in morphology between the Arabidopsis in the experimental and control groups was measured, including LWR, leaf number, and maximum leaf mean length. *p < 0.05, **p < 0.01 (one‐way ANOVA)

Figure 5

BioClay delays dsRNA degradation. (a) The purified dsTrxz samples were separately dissolved in LDH (with a particle size of 172 nm) and double distilled water (ddH₂O) to a final concentration of 20 ng/μl. All samples were left to stand at room temperature and collected on days 0, 5, 10, 17, and 21 for agarose gel electrophoresis. (b) Quantitative analysis of dsRNA concentration demonstrated that dsTrxz dissolved in LDH (BioClay) delays dsRNA degradation, more so than in ddH₂O

Figure A1

Molecular structure of layered double hydroxide