Part:BBa_K3815011

Part of the V-ATPaseB gene of Frankliniella occidentalis to synthesize dsRNA for RNAi
This is a section of the V-ATPaseB gene of Frankliniella occidentalis to synthesize dsRNA for RNAi. To product dsRNA, this sequence is inserted in the L4440 plasmid, and transformed into HT115(DE3).

purpose

vATPase-B is a gene that encodes a protein that is part of a subunit of vATPase, a type of ATP-dependent proton pump that regulates the pH of various cell organelles. Since this gene is essential for thrips, knockdown of vATPase-B results in the death of the thrips Frankliniella occidentalis.[1]

Usage and Biology

RNAi
RNAi (RNA interference) is a process in which externally introduced dsRNA suppresses the expression of genes that have complementary sequences to the dsRNA.

L4440
L4440 is a plasmid vector having two convergent T7 promoters adjacent to the multi-cloning site. By inserting a portion of the target gene sequence into the multi-cloning site of this plasmid, the target sequence is transcribed from both sides, and dsRNA can be obtained when both parts are annealed.

HT115(DE3)
HT115 (DE3) is an RNase III-deficient E. coli strain that has been modified to express T7 RNA polymerase from an IPTG-inducible promoter. It lacks dsRNA-specific RNase III, which allows it to produce high levels of specific dsRNA. These attributes allow HT115 (DE3) to be a promising strain for the preparation of large amounts of viral dsRNA in vivo.

Sequence and features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 452
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 320
  

cloning

This part was inserted in L4440. The L4440 has two t7 promoters, and this ​part are transcribed from both sides.

production and purification methods

Fig1.1.L4440 2 vATPase-B (total 7ug nucleotic acid were loaded) 3 vATPase-B (total 1ug nucleotic acid were loaded)
band of 700bp may be target RNA

We produced dsRNA and purify this [2].
The 1L LB culture was started at 37°C and IPTG (final 0.2mM) was added when OD exceeded 0.4 to induce RNA production for 4 hours. <be>E. coli was disrupted and precipitated with NaCl, and RNA contained in the supernatant was purified by phenol-chloroform treatment.

Electrophoresis in acrylamide gel was performed to confirm whether the target RNA was produced.

We could confirm RNA production.

Assay against Frankliniella occidentalis

Specifically, 200 μl of a 200 ng/μl dsRNA solution targeting vATPase-B (Milli-Q was used as a negative control) was pipetted in a PCR tube and the petiole of a green bean primary leaf was immersed. After 2 hours, three leaf fragments of 1 cm × 2 cm were cut from the soaked bean leaves and placed in three petri dishes with about 10 thrips. The mortality and leaf damage were checked after 24, 48, 72, and 96 hours.

Fig.2 RNAi assay

In particular, there was a large difference in the mean mortality rate between dsRNA-treated and negative control thrips on day 3: 8.3% and 31.3%, respectively. This result confirms the insecticidal effect of dsRNA indirectly feeding to the western flower thrips.

Reference

1 Andongma, A.A., Greig, C., Dyson, P.J., Flynn, N., and Whitten, M.M.A. (2020). Optimization of dietary RNA interference delivery to western flower thrips Frankliniella occidentalis and onion thrips Thrips tabaci. Arch. Insect Biochem. Physiol. 103, e21645.

2 Papić, L., Rivas, J., Toledo, S., and Romero, J. (2018). Double-stranded RNA production and the kinetics of recombinant Escherichia coli HT115 in fed-batch culture. Biotechnol Rep (Amst) 20, e00292.