The first functional Biobrick we constructed is a vector with two reverse T7 promoters. Double-strain RNA (dsRNA) will be synthesis during transcription process. This is the tranditional way to produce dsRNA. We advanced this method by adding double terminators outside T7 promotors. In addition, We standardized the method by adding biobrick preffix and suffix in it. Eventually, we got BBa_K2377016 (pDP), a standard plasmid backbone for dsRNA production.
To generate dsRNA expression vectors, we can simply put target sequences into the plasmid using RFC10 standard. This allows us to do high throughput target selection during a short period of time.
2. hpRNA construction
Traditional way to generate RNAi gene silencer using two reverse T7 promotors has a relative low efficiency due to the interference of two RNA polymerases during transcription process. To address this problem, we designed hpRNA with a loop between sense and antisense form of target sequence. hpRNA can be generated using only one promotor. In addition, it's more stable than dsRNA.
① Hairpin RNA with normal loop
② Hairpin RNA with intron loop
At first, we used loop120 (a 120bp sequence selected from Chl resistant gene.) as our loop. Afterwards, we saw from papers that the intron sequence in hpRNA can be spliced by spliceosome in plant. This process raises the efficiency of RNAi in plant by an unknown mechanism. Therefore, intron could be a better option of loop.
Bacteria strain selection
We use the M-JM109LacY (RNaseIII deficit type, uncapable to degrade dsRNA/hpRNA), as a manufactory to produce dsRNA/hpRNA.
Reference
[1] NA, S., SP, S., MB, W., PA, S., AG, G., and PM, W. - Total silencing by intron-spliced hairpin RNAs, D - 0410462, T - ppublish.
[2] YS, L., and RW, C. - Making a better RNAi vector for Drosophila: use of intron spacers, D - 9426302, T - ppublish.
[3] Michael R. Green; Joseph Sambrook. (2012). Molecular Cloning: A Laboratory Manual (4th ed.).
Target organism
Plant
At first, we aimed at setaria virids, a kind of maliganant weed resistant to pesticide glyphosate.
We met huge obstacles : First, it is hard to select vital gene fragments with high specificity due to the deficient genome data of weeds and low discrepancies between malignant weeds and victim crops . This may increase the off-target rate of our strategy. On the other hand, the growth cycle of setaria virids is too long.
We found it infeasible after doing a lot of work on setaria virids, so we turned our attention to a more feasible target—Arabidopsis , which has clear genetic background and shorter growth cycle. It is an ideal candidate for tandem RNAi verification.
Pest
We chose aphids as our pest target for it is a worldwide pest, creating great impact in agriculture fields and it is easy to capture from nature. In addition, large amount of studies on Aphids gave us a lot of ripe experiences.
Target gene
Arabidopsis
We chose a target gene—trxz. Trxz deficiency results in complete albino phenotype , making it easy to be observed.
TRX z is a branch of plastidial thioredoxin (TRX), lying between x- and y-type TRXs thus was named TRX z, which is a likely candidate for mediating thiol-modifications in chloroplasts.
Recently, several independent studies demonstrate that TRX z constitutes a subunit of plastid-encoded RNA polymerase (PEP). Meanwhile the generation of the full PEP complement is an essential early step during chloroplast bio-genesis and its disturbance cannot be reversed if a certain time point in the program has been passed.
The Arabidopsis knockout mutant lines of TRX z exhibit a complete albino phenotype and was inhibited in chloroplast development.
What’s more, we noticed that a analysis of promoter-GUS indicated that TRX z is expressed in young tissues with decreasing expression during leaf maturation.
So our verification need to be at the young stage of the otherwise the phenotype maybe not so obvious.
Aphid
Theoretically, any genes necessary for insects survival, growth, development, reproduction or feeding can be used as RNAi targets(Vilcinskas 2011). The ecdysone receptor (ECR) is a such kind of gene, which could up-regulate the vital genes expression through the interaction with ultra-stable protein (USP) under the existence of ecdysteroid hormone.
Therefore, Ecr gene silencing can effectively increasing the insects mortality and reducing the reproduction rate. Studies have shown when fed aphids with 7.5 ng / ul dsRNA Ecr, the mortality rate up to 70% after 8 days.
According to the literature, the similarity of Ecr gene sequence between pea aphid and grain aphid was 98.4% and with more than 90% similarity in other kind of aphids, so we chose ECR as the target sequence of RNAi. So Ecr is an Ideal RNAi target site to kill aphids with a broad spectrum destruction effect.
Background
Through papers we knew that dsRNA can sustain for more than 2 weeks in soil (Jeffrey G. Scott, et al, 2013), but which is still too short to guarantee enough RNAi efficiency for weeding. Fortunately, we found a kind of special material—bio-clay, which could storage dsRNA as shelters.
What is Bioclay?
Bioclay is a complex combining dsRNA(double-stranded RNA) and LDH(layered double hydroxide) which is reported to provide sustained protection for the dsRNA from the nuclease activity and a effective carrier of negatively charged molecules such as dsRNA. Additionally, it also facilitates sustained release of dsRNA on the leaf surface. As the construction of dsRNA and mechanism of RNAi are elaborated in other parts of wiki, here, the characteristics of LDH will be introduced.
LDH nanosheets are set of inorganic layered materials. The general formulation of it is shown below:
\[ \mathrm{[(M^{2+}_{(1-x)}M^{3+}(OH)_2)^{x+} \cdot (A^{m-}_{x/m} \times nH_2O)^{x-}]} \]
In the formulation, most divalent and trivalent metal ions can be utilized(M2+ and M3+) and any forms of anions(A-) with x=0.2-0.4 and n=0.5-1.0.
Figure 1. Schematic illustrations of LDH
dsRNA delivery mechanism
It is basically known to all that negatively charged molecules such as dsRNA cannot easily transcend cellular membranes because the cellular membranes are also negatively charge. However, if these anionic molecules are incorporated within the inter layers of LDH, the negatively charged molecules will not be exposed to the negatively charge membranes. The positively charged platelets intercalate the dsRNA, thus masking its strong negative charge. Aside from this, the high delivery efficiency is also benefited from the innate ability of LDH to escape from endosomes which is considered a major barrier to effective delivery. Through LDH-mediated endosomal buffering, rupture of endosomal vesicles and release of intact therapeutics into the cytoplasm, the delivery efficiency is substantially enhanced.
Sustained release of dsRNA
It has already been proved that the stability of dsRNA will be enhance for a long period under environmental conditions. The sustained release of dsRNA is facilitated through the formation of carbonic acid on the leaf surface from CO2 in the atmosphere and humidity, the reactions below have elaborated the mechanism of the degradation of Bioclay:
\[ CO_2+H_2O \longrightarrow H_2CO_3 \longrightarrow H^+ + HCO_3^- \]
\[ Mg_3Al(OH)_8 - A^- + 5H^+ \longrightarrow 3Mg^{2+} + Al'(OH)'_3 +A^- +5H_2O \]
where A- could be bound dsRNA. Through the previous work done by other labs, they have proved that the amount of aluminum and magnesium of Bioclay will decrease 28% and 22% respectively. And the dsRNA will not be degraded completely in 30 days. While if there is no LDH, the dsRNA will be degraded completely in 20 days.
dsRNA uptake
Despite of two reports of dsRNA uptake through roots and one through flower buds, the uptake of dsRNA can also be achieved through the supply on the leaf surface through some sprays onto the leaves. Besides, there is also the possibility of systemic movement of dsRNA and its RNA breakdown products from mature sprayed leaves to new leaves when they form at the shoot apex.
Future Application
Due to the substantial advantage brought by the Bioclay, we have a strong desire to introduce it into the market along with our bio-pesticides. The appealing characteristics including protect dsRNA from degradation, sustained release of dsRNA and the dsRNA delivery into cells will expand the application of Bioclay into many fields. The future work will mainly focused on lowering the price of Bioclay and the optimizing characteristics of LDH.
Reference
[1] Gasser, M. S. (2009). Inorganic layered double hydroxides as ascorbic acid (vitamin C) delivery system--intercalation and their controlled release properties. Colloids Surf B Biointerfaces, 73(1), 103-109. doi: 10.1016/j.colsurfb.2009.05.005
[2] Ladewig, K., Niebert, M., Xu, Z. P., Gray, P. P., & Lu, G. Q. (2010). Efficient siRNA delivery to mammalian cells using layered double hydroxide nanoparticles. Biomaterials, 31(7), 1821-1829. doi: 10.1016/j.biomaterials.2009.10.058
Mitter, N., Worrall, E. A., Robinson, K. E., Li, P., Jain, R. G., Taochy, C., . . . Xu, Z. P. (2017). Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses. Nat Plants, 3, 16207. doi: 10.1038/nplants.2016.20
Future Work
Owning to the time and technology limitations, part of our project need to be achieved in the future. At present, most of genomes of weeds have not been sequenced, so we had to temporarily substitute Arabidopsis for real weeds as the basic plant target to verify whether our idea works. We believe that one day when the sequencing and other related technologies become more advanced, overcoming current restrictions, our Bio-pesticides could be a real sword towards all the threats would do harm to crops, not only weeds, pests but also fungi and virus induced diseases, etc.
In application, we added cracking-agent artificially to make engineering bacteria lysis and release dsRNA product. In the future, we hope to optimize this step by using a suicide system linked to quorum sensing. When the cells population or target production concentration is up to a specific threshold as we want, bacterium will be triggered self-lysis spontaneously, which will be more convenient for users.
Consideration on how to let plants absorb dsRNA, Apart from dsRNA irrigation, we designed a device to make plants easily take up dsRNA. In the future, we intend to express cellulase and pectinase along with dsRNA spraying, to let them generate minor wounds on the plant leaves, therefore, offer an access to deliver dsRNA into plant cells. This is just an assumption, many details need to be settled down in the future.
Hope that all our efforts has been the basis of changing the future world a better place.
