Team:SZU-China/Experiment

iGEMwiki toolssearchlogin

Editing Team:SZU-China/Experiment

template

99

Experiment

Experiment
Overview
dsRNA stage

At the very beginning of our project, we wondered that whether the RNAi molecules could be used as a herbicide to kill the M. micrantha, which was so strong that almost no effective herbicides could remove it in just a few days [1]. Hence, we decided to use the traditional method to synthesize double-strand RNAs (dsRNAs) targeting several essential genes involved in respiration pathway, in order to block normal metabolism of M. micrantha and eventually kill it (Fig.1) [2]. (Click Iteration to see more) The experiments were designed as follows:

  • Setting the negative and blank controls, by spraying the leaves with irrelevant dsRNA to target genes or the dsRNA solvent (sterile water), respectively.
  • Performing qRT-PCR to check whether the transcript level of target gene was down-regulated on the treated leaves.
  • Observing the morphology of the treated leaves.

After applying dsRNA to the M. micrantha for about three weeks, we found that some part of the tested leaves turned yellow and wilted. Besides, most of the target genes were silenced one day after spraying the leaves with the dsRNA, while some of them were not. (Click Results to see more)

Fig.1 RNAi mechanism via dsRNA
Cited from http://www.51wendang.com/doc/b8ebe656d3a950a17352bb20/2
RNAi nanoparticle stage

At the dsRNA stage, we confirmed that the RNAi technology can possibly be used as the specific herbicide for M. micrantha. However, siRNAs produced from the dsRNA is less specific than short single interference RNA (siRNA) and the efficiency could not meet our requirements. We decided to synthesize RNAi nanoparticles as a vector carrying half a million copies of siRNA into the weed (Fig.2).

Fig.2 RNAi nanoparticles
Cited from Self-assembled RNA interference microsponges for efficient siRNA delivery

The RNAi nanoparticles consist of numerous hairpin-structured RNAs, which can be cleaved into countless siRNAs. We designed a closed circle sequence containing two regions that could be transcribed into a hairpin siRNA via T7 RNA polymerase. The hairpin RNA would be processed into different kinds of expected siRNAs by Dicer enzyme in vivo. Through the rolling circle transcription of the circle sequence, a lot of hairpin siRNA precursors link together and package to form a nanoball, named RNAi nanoparticle (Fig.3) [3].

Fig.3 Self-assembled RNAi nanoparticles
Cited from Self-assembled RNA interference microsponges for efficient siRNA delivery

What’s more, after discussing with many professors, we selected several siRNAs targeting different kinds of chlorophyll related genes, which might kill M. micrantha and cause obvious morphology of the treated leaves.

We also did the quality control tests on RNAi nanoparticles we synthesized through scanning electron microscope (SEM) imaging. The efficiency of generating functional siRNAs in RNAi nanoparticles seems much better than that in dsRNA and the results from qRT-PCR and morphology change were much obvious and significant. (Click Results to see more)

Hairpin siRNA stage

After CCiC, the judges pointed out that making the RNAi nanoparticles as the RNAi-based herbicide was costly and hard to be manufactured. This means that the nanoparticle-based herbicide might present small development potential and narrow market prospect. Hence, we developed the third generation of the RNAi molecules to reduce costs and make our products better applied to the market.

In the third generation, we designed a sequence containing several regions that could form hairpin siRNAs. Considering the difficulty of hairpin structure synthesis, we designed four different pairs of regions to link four kinds of hairpin siRNAs together, which could help us to produce different functional siRNAs at one time. Through E. coli proliferation, we can produce siRNAs targeting different genes in large quantities at one time. In this way, we could spray RNAi-based herbicide together to increase silencing efficiency.

Recombinant plasmid pET-28a (+) containing above mentioned regions was transformed into HT115(DE3) E. coli. The RNAi molecules transcribed in E. coli were extracted and sprayed on Mikania micrantha as an RNAi-based herbicide (Fig.4)

Fig.4 Formation of Hairpin siRNA

The RNAi molecules in the third generation were transcribed in the E. coli via IPTG induction, saving the cost on purchasing T7 RNA polymerase and T4 Polynucleotide Kinase, the most expensive biochemicals used during the synthesis of RNAi nanoparticles.

Then we did the quality control as well as the stability tests on the third generation RNAi-based herbicide. (Click Results to see more) Besides, we also carried out the Northern Blot and a G-quadruplex DNA-based, label-free and ultrasensitive strategy to detect the siRNA production in vivo [4].

Protocol
Reference

[1] Zhang, L Y , et al. "Mikania micrantha H. B. K. in China – an overview." Weed Research 44.1(2004).

[2] Robinson K E , Worrall E A , Mitter N . Double stranded RNA expression and its topical application for non-transgenic resistance to plant viruses[J]. Journal of Plant Biochemistry and Biotechnology, 2014, 23(3):231-237.

[3] Lee J B , Hong J , Bonner D K , et al. Self-assembled RNA interference microsponges for efficient siRNA delivery[J]. NATURE MATERIALS, 2012, 11(4):316-322.

[4] Yan L, Yan Y, Pei L, et al. A G-quadruplex DNA-based, Label-Free and Ultrasensitive Strategy for microRNA Detection[J]. Sci Rep, 2014, 4:7400.