Difference between revisions of "Team:Lanzhou/Tandem RNAi"

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Our human practices focused on one single purpose—better design, in the spirit of caring and changing the world.
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The total work contains 6 aspects: public engagement, education, consulting experts, conference communications, questionnaires and designing a Bio-pesticide handbook. Also, we have gained a lot of valuable information about the society, the advice and feedback which offered a positive effect on our project and optimized our design in every aspect, from experiments, device to modelling.
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<p class="mdc-typography--subheading2">At the same time, with social responsibility, we also contributed a lot to the society, whatever in education or popularization of science because we want to make the world a better place through our efforts. </p>
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<p class="mdc-typography--subheading2">Besides, due to our hardship this year, we did a special work to reflect the situation and problems of iGEM teams in China. We sincerely hope our experiences could not only inspire the future iGEM teams to go through their hard time but give some directions to solve possible difficulties they would meet.</p>
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<p class="mdc-typography--subheading2">We really appreciated all the support we have received from the society and relevant communities in our HP work.</p>
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<h1 class="mdc-typography--headline">RNAi Mechanism</h1>
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RNA interference first discovered as an ancient antiviral system in plants, when long dsRNA come into cells, they will be rapidly cut by dicer or dicer-like enzyme (double-stranded RNA specific endonucleases of Rnase III family), forming 21 to 28-nt RNA duplexes with the characterize of 2-nucleotide 3’overhangs and 5’phosphates, which are called siRNAs (small interfering RNA). SiRNAs conform a ribonucleoprotein complex RISC (RNA-induced silencing complex).
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RISC includes slicer and Argonaute protein with Rnase H-like domain, which first mediates the unwinding of siRNA duplex. The passenger strand is degraded by Argonaute, then the guide RNA binds to the target mRNA in a sequence specific manner. This binding mediates target mRNA cleaved by slicer, meanwhile the fragments of mRNA will be recognized as the aberrant and destroyed by cells, inducing gene silence in a post transcriptional level ( PTGS ,Post Transcriptinal Gene Silence).
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<h1 class="mdc-card__title mdc-card__title--large">Engagement</h1>
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RNAi mechanism is the basis of our project.
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<figcaption>Figure1. In Arabidopsis, four Dicer-like (DCL) proteins (DCL1 to DCL4) have been identified. Three of which are involved in processing dsRNAs that come from different sources. DCL1 is required for the production of siRNAs derived from long dsRNAs, including viruses and transgenic hairpins used in experimental RNA interference.</figcaption>
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Tandem RNAi
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<h1 class="mdc-card__title mdc-card__title--large" style="color: black;">Conference</h1>
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We noticed that many weeds are perennial plants, and they always chosen by pests as natural shelters to survive the brutal winter. This phenomenon inspired us to wipe out weeds and pests together by a method—tandem RNAi, which means triggering RNA interference in at least two organisms at the same time. In our project, we magnified RNAi effect by selecting vital genes of target organisms, whose silence will result in phenotype change or death. And we used dsRNA irrigation to realize the tandem RNA interference.
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In the latest research, Hunter et al. confirmed that citrus trees can absorb dsRNA through roots and phloem injections (Hunter et al. 2012). Besides, It has been proved when Arabidopsis roots were soaked in dsMob1A (Mob1A gene is required for organ growth and reproduction), the root lengths and numbers were significantly suppressed and plants could not bolt or flower. These results directed a possible way to deliver dsRNA into plants through dsRNA irrigation .  
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Meanwhile, we noticed dsRNA spraying has been verified successfully in disease and mandibulate insects control, but it can’t affect the piercing-sucking pests which live on phloem sap, plant stems or root system. For these sap-sucking insects, dsRNA needs to be delivered through the phloem sap. And this delivery could be realized by dsRNA irrigation. Demonstrated by Araujo that the dsRNA flowing through the plant vascular system could be taken up by psyllids that feed on the phloem. As a result, the insects’ mortality increased when the dsRNA of an arginine kinase was absorbed by the host tree. (Araujo et al. 2006; Hunter et al. 2012).
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Taken together, we made an assumption: when designed dsRNA contains both vital genes information of target plant and insect, dsRNA irrigation-mediated RNAi are able to affect insects and their host plants together.
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<h1 class="mdc-card__title mdc-card__title--large" style="color: black;">Questionnaire</h1>
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Based on this deduction, we constructed our project—Bio-pesticides
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<figcaption>Figure2.Tandem RNAi </figcaption>
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Maybe you would still doubt the power and efficiency of our tandem-RNAi-based bio-pesticides. Here we give u a cardiotonic.
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<h1 class="mdc-card__title mdc-card__title--large">Handbook</h1>
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<h2 class="mdc-typography--title"><font size="5">&bull;</font>Amplification of the RNAi system</h2>
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It was estimated that only two molecules of dsRNA per cell were able to induce RNAi In C. elegans. In another report, injection of dsRNA into the intestine of a C. elegans hermaphrodite generated RNAi, which could be stably inherited to the F2 generation. These two findings led to the proposal that RNAi signals could be systemic and amplifiable in nature.
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The amplification effect should be possible with the following mechanisms
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1. RISC reuse<br>
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RISC can be reused in the last step of RNA silencing pathway and It has been reported that RISC can degrade more than 30 mRNA substrates. Which amplifies and prolongs the silencing effect.
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2.  Generation of siRNA<br>
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A single starting dsRNA molecule are able to generate multiple siRNA molecules (about 45 siRNAs/Kb), it’s a kind of amount magnification, and even without the supply of new dsRNA, the silencing effect could still be maintained for a long time.
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3.  RDRP mechanism<br>
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In addition, RNAi amplification rely on an RNA-dependent RNA polymerase (RdRP) system, the role of RDRP can be explained by"random degradation PCR model": RdRP could use target mRNA as templates to synthesize new dsRNA in the guide of single stand siRNA (as primer), then dicer cut new dsRNAs and generate amount of secondary siRNAs and into the next round of cycle reaction. After multiple cycles, the signal has been maginified, meanwhile there has been enough RISC complexes to finish silencing. In addition, cleaved mRNA fragments can be regarded as aberrant RNA, contributing to dsRNA denovo synthesis( without primer)mediated.by RDRP.
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RDRP were demonstrated in nematodes (EGO1, RRF1, RRF3), chondromyces (QDE1) and fission yeast, but there is no direct evidence that RdRP enzyme was found to be active in other organisms. Otherwise, the work of Lipardi demonstrated that RdRP activity was found in the embryo pyrolysis of drosophila, but no RdRP homologous sequence was found in fruit flies and human genomes.
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On the whole, the amplification system guarantee the basic strength and duration of RNAi.
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<figcaption>Figure 3.  RNAi amplification mechanism</figcaption>
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<h2 class="mdc-typography--title"><font size="5">&bull;</font>Movement of RNA silencing signal in plant</h2>
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In plants, RNA silencing produces cell non-autonomous signal molecules that can move over short or long distances through vascular system, leading to the sequence specific silencing of a target gene in a well defined area of cells or throughout the entire plant.
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Molecular transport in plants can occur either symplastically through the channels connecting the adjacent cells termed plasmodesmata, or apoplastically through a transfer process acrossing the intercellular spaces, cell membrane and the cell wall.
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In short range silencing spread, the silencing molecules (21nt siRNAs, miRNAs, tasiRNAs or other type) most probably follow the symplastic route. The silencing spread is limited to 10–15 cells beyond the production site and with no need of an amplification mechanism. Silencing molecules are then transported with high velocity through the phloem pathway from photosynthetic sources (i.e. leaves) to sucrose sinks (i.e. roots and growing points).
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The long distance or systemic movement of the silencing signal takes place over days. This spread is mediated by PTGS (post transcriptional gene silencing )or TGS (transcriptional gene silencing), which conforms to the mobile 21 nt siRNAs that stem from exogenous dsRNA.
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<figcaption>Figure 4.  RNA silencing signal movement mechanism</figcaption>
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The mechanism ofs silencing signal movement in plants make sure that when dsRNA is absorbed by roots, it will trigger systemic RNAi and silence target gene throughout the entire plant.
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<h1 class="mdc-typography--headline">Final performance</h1>
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Our RNAi-based bio-pesticides, unlike chemical pesticides and transgenic crops containing Bacillus thuringiensis (Bt) toxins, are able to target a specific organism, thereby offering pests and weeds species-specific management.
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In experiments, designed dsRNA contains both target plant and pest key vital genes information and via dsRNA irrigation plants, realize the “Two birds, one stone” . See more details in construction.
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<figcaption>Figure 5. Tandem dsRNA</figcaption>
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<figcaption>Figure 6. Tandem hpRNA</figcaption>
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DsRNA irrigation strategies can be improved by mixing the RNAs with chemical reagents or nano materials (like bioclay) to stabilize the RNAs and thus increase the strength and duration of plant protection. Overall, this new generation of tandem-RNAi-based pesticides looks promising to meet our world's increasing demands for increasing safety and quality of crop yields to feed the growing population.
 
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<h1 class="mdc-typography--headline">Reference</h1>
 
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[1] Min, Zhang1†, Yuwen, Zhou2†, Hui, Wang1, Huw, Dylan, Jones3, Qiang, Gao2, Dahai, Wang1, Youzhi, Ma1*, and, Lanqin, Xia1*. Identifying potential RNAi targets in grain aphid (Sitobion avenae F.) based on transcriptome profiling of its alimentary canal after feeding on wheat plants[J]. BMC Genomics, 2013, 14(560): 2-15
 
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[2] Ting, Yan, †, Hongmei, Chen, †, Yongwei, Sun, Xiudao, Yu, and, Lanqin, Xia, *. RNA Interference of the Ecdysone Receptor Genes EcR and USP in Grain Aphid (Sitobion avenae F.) Affects Its Survival and Fecundity upon Feeding on Wheat Plants[J]. Int. J. Mol.Sci., 2016, 17(2098): 1-14
 
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[3] Yao T P, Forman B M, Jiang Z, et al. Functional ecdysone receptor is the product of EcR and Ultraspiracle genes[J]. Nature, 1993, 366(6454): 476-479.
 
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[4] Coleman, A. D. et al. “Persistence and Transgenerational Effect of Plant-Mediated RNAi in Aphids.” Journal of Experimental Botany 66.2 (2015): 541–548. PMC. Web. 1 Nov. 2017.
 
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[5] Buhtz A, Springer F, Chappell L, Baulcombe DC, Kehr J (2008) Identification and characterization of small RNAs from the phloem of Brassica napus. Plant J 53: 739–749
 
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[6] Dunoyer P, Himber C, Voinnet O (2005) DICER-LIKE 4 is required for RNA interference and produces the 21-nucleotide small interfering RNA component of the plant cell-to-cell silencing signal. Nat Genet 37: 1356–1360
 
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[7] Dunoyer P, Himber C, Ruiz-Ferrer V, Alioua A, Voinnet O (2007)Intra- and intercellular RNA interference in Arabidopsis thalianarequires components of the microRNA and heterochromaticsilencing pathways. Nat Genet 39: 848–856
 
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[8] Schwach F, Doonan JH, Baulcombe DC (2010) The Arabidopsis RNA-directed DNA methylation argonautes functionally diverge based on their expression and interaction with target loci. Plant Cell 22: 321–334
 
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[9] Schwach F, Vaistij FE, Jones L, Baulcombe DC (2005) An RNAdependent RNA-polymerase prevents meristem invasion by Potato virus X and is required for the activity but not the production of a systemic silencing signal. Plant Physiol 138: 1842–1852
 
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[10]Voinnet O, Vain P, Angell S, Baulcombe DC (1998) Systemic spreadof sequence-specific transgene RNA degradation is initiatedby localised introduction of ectopic promoterless DNA. Cell 95:177–187
 
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[11] Zilberman D, Cao X, Jacobsen SE (2003) ARGONAUTE4 control of locus specific siRNA accumulation and DNA and histone methylation. Science 299: 716–719
 
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[12] Coleman, A. D. et al. “Persistence and Transgenerational Effect of Plant-Mediated RNAi in Aphids.” Journal of Experimental Botany 66.2 (2015): 541–548. PMC. Web. 1 Nov. 2017.
 
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{{Lanzhou/Footer}}
 
{{Lanzhou/Footer}}

Revision as of 03:57, 2 November 2017

Lanzhou

Lanzhou2017

Our human practices focused on one single purpose—better design, in the spirit of caring and changing the world.

The total work contains 6 aspects: public engagement, education, consulting experts, conference communications, questionnaires and designing a Bio-pesticide handbook. Also, we have gained a lot of valuable information about the society, the advice and feedback which offered a positive effect on our project and optimized our design in every aspect, from experiments, device to modelling.

At the same time, with social responsibility, we also contributed a lot to the society, whatever in education or popularization of science because we want to make the world a better place through our efforts.

Besides, due to our hardship this year, we did a special work to reflect the situation and problems of iGEM teams in China. We sincerely hope our experiences could not only inspire the future iGEM teams to go through their hard time but give some directions to solve possible difficulties they would meet.

We really appreciated all the support we have received from the society and relevant communities in our HP work.