Difference between revisions of "Part:BBa K1954002:Design"

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pGAP-Vitellogenin-GFP device

Design Notes

Whitefly (Bemisia tabaci) is globally considered as a polyphagous agricultural pest that causes severe direct and indirect damage to many crops by feeding and transmitting viruses to plants[1].

Vitellogenin plays a vital role in oocytes and embryo development in insects and Vitellogenin gene is responsible for the reduction of oxidative stress in honeybees. We cloned this gene into yeast to test if it reduces oxidative stress in eukaryotes [2]. The recombinant yeast Pichia pastoris has been described in [3]. Oxidative stress in eukaryotes is casually linked to the production of carbonyl derivatives. By measuring carbonyl derivatives concentration, oxidative stress can be quantified [4]. By using RNAi to create yeast without the Vitellogenin gene and comparing oxidative stress with the wild type cells, can show whether Vitellogenin is suitable to reduce oxidative stress in human cells.

RNAi in insects: dsRNA is cleaved by a RNase III, often called Dicer, into 21-25 nt-long short interfering RNA duplexes (siRNA). These siRNAs are incorporated into the RNA-induced silencing complex (RISC). After discarding the passenger strand, the RISC complex binds to the target mRNA, cutting it and thereby hindering translation [5].

The protein Vitellogenin has previously been expressed in the methyl-trophic yeast Pichia pastoris which has shown to impact cellular production of essential amino acids and long-chain polyunsaturated fatty acids. This effect is consistent with the putative role of Vitellogenin in the health of metazoans, a group which includes wasps (Encarsia formosa) and Silverleaf whitefly (Bemisia tabaci). Targeting Vitellogenin for increased production in certain wasps, or decreased production in Silverleaf whitefly, could both be future routes to controlling the whitefly as a pest.

In this project we attempted to characterize kinetics of antisense-based down-regulation of recombinant Vitellogenin expression in Pichia pastoris. A genetic construct that complies with the BioBrick standard and enables both Vitellogenin expression and inducible Vitellogenin down-regulation by action of anti-sense RNA was assembled.

After cloning vitellogenin gene into the plasmid, the promoter pAOX1 is sensitive to methanol. In the absence of methanol, pGAP is active and the fusion protein GFP-Vitellogenin is synthesized. The cell becomes green due to GFP. In the presence of methanol, pAOX1 becomes active and starts to synthesize anti-sense mRNA, which binds onto the mRNA of the fusion protein. This forms double stranded mRNA, which will be degraded by the cell. The green color of the cell due to GFP disappears.

The experiment investigates the kinetics of anti-sense mRNA and may help Syngenta design the final gene construct in the bio-control of white fly.

References

[1] Upadhyay S, Singh H, Dixit S, Mendu V, Verma P. Molecular characterization of vitellogenin and vetellogenin receptor of Bemisia tabaci. PLOS ONE(2016): 10.1371

[2] Seehuus S, Norberg K, Gimsa U, Krekling T, Amdam G. Reproductive protein protects functionally sterile honey bee workers from oxidative stress. PNAS (2006): 103, 962-967

[3] Ding J, Lim E, Li H, Kumar J, Lee S, Lam T. Expression of Recombinant Vitellogenin in the yeast Pichia pastoris. Biotechnology and Bioengineering (2004): 85

[4] Nystrom T. Role of oxidative carbonylation in protein quality control and senescence. The EMBO Journal (2005): 24

[5] Huvenne H, Smagghe G. Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: a Review. Journal of Insect Physiology (2010): 56, 227-235