ACCELERATING AGENG?

What if we are able to make an organism reduce their healthy lifespan as a way of killing them? Will this have useful real world applications? Find out how Biosynthage are working on using RNAi to increase oxidative stress as a way of pest control

VITELLOGENIN

The main topic of UCL Biosynthage is aging and we have covered various areas of research such as: SOD gene therapy for lungs, oxidative-stress induced lycopene synthesis and pH-induced bacteriocin production in dentistry. We have a deep interest in helping old people suffering from oxidative stress and plan to cover more areas of science relevant to it, which has made us apply for the Syngenta grant.

Syngenta, a Swiss agriculture company, sponsors iGEM 2016 and plans to offer 4 iGEM teams and over 300 university teams $5000 grant in RNA-based biocontrols corporate innovation to explore how to best demonstrate the value of new tools for the future of agriculture and food production. UCL Biosynthage has luckily been awarded with this grant.

Background information

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 (vg): plays vital role in oocytes and embryo development in insects and vitellogenin gene is responsible for the reduction of oxidative stress in honeybees. We are planning to clone 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 vitellogenin gene and comparing oxidative stress with the wild type, it shows whether vitellogenin is suitable to reduce oxidative stress in human cells.

The protein vitellogenin has previously been expressed in the methyl-trophic yeast, Pichia pastoris, and 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.

RNAi definition 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 will bind to a homolog mRNA, cutting it and thereby hindering translation [5].

RNAi for the whitefly (B.tabaci) bio-control via oral route: Regarding to the objective of the Sygenta proposal, biocontrol must be applied in the wild by feeding or targeting the specific pest (B. tabaci) that lives on crops. Thus, injecting RNAi into the body of the pest becomes inapplicable. According to [6], the artificial diet (with/without dsRNA/siRNAs) was filter-sterilized (0.22 μm). A 100 μl diet was sandwiched between the two layers of UV-sterilized parafilm and stretched on the inner surface of the tube cap. The entire process was carried out aseptically. The caps of insects containing tubes were replaced with the diet containing caps and tubes were kept upright. The diet was replaced on alternate days to minimize the possibilities of degradation in test samples and prevent microbial contamination.

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

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.

Unexpectedly, the gene company from which we has purchased the gene has reported issues about the toxicity of the bacteria producing those genes. We are very surprised as it has been proved in previous research that it works well in the genetic recombinant yeast cell P. pastoris. We are still waiting for the gene to conduct our experiment.

References

• 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
• 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
• 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
• Nystrom T. Role of oxidative carbonylation in protein quality control and senescence. The EMBO journal(2005): 24
• 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