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 Ecr is an ideal RNAi target site to kill aphids with a broad spectrum destruction effect.
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 Ecr is an ideal RNAi target site to kill aphids with a broad spectrum destruction effect.
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.
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</p>
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<h1 class="mdc-typography--headline">What is Bioclay?</h1>
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<p class="mdc-typography--body2">
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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.
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</p>
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<p class="mdc-typography--body2">
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LDH nanosheets are set of inorganic layered materials. The general formulation of it is shown below:
In the formulation, most divalent and trivalent metal ions can be utilized(M<sup>2+</sup> and M<sup>3+</sup>) and any forms of anions(A<sup>-</sup>) with x=0.2-0.4 and n=0.5-1.0.
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.
+
</p>
+
+
<h1 class="mdc-typography--headline">Sustained release of dsRNA</h1>
+
<p class="mdc-typography--body2">
+
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 CO<sub>2</sub> in the atmosphere and humidity, the reactions below have elaborated the mechanism of the degradation of Bioclay:
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.
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.
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.
At first, we construced a vector with two reverse promoters in one plasmid to produce double-strain RNA (dsRNA). This is the traditional way to produce dsRNA.
2. hpRNA construct
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 hairpin RNA (hpRNA) with a loop between sense and antisense form of target sequence. It can be produced using only one promotor.
① Hairpin RNA with normal loop
At first, we used loop120 (a 120bp sequence selected from Chl resistant gene.) as our loop. We selected the sequence for it has a high gibbs free energy (low tendency) to form hairpin.
② Hairpin RNA with intron loop
As the intron sequence will be sliced during the pre-mRNA processing and the stability is much higher than the previous two methods according to a relevant paper, it will be a better option for us to silence the target sequene.
In Our experiment，we selected 120 base-pair-long noncoding sequence from Chloramphenicol as normal loop.
Strain select
We use the M-JM109LacY (dicer deficit type, can’t degrade dsRNA), as a manufactory to produce dsRNA.
Reference
[1] Total silencing by intron spliced hairpin RNAs. (2000). Nature.
[2] Young Sik Lee and Richard W. Carthew, Lee, Y. S., & Carthew, R. W. (2003). Making a better RNAi vector for Drosophila: Use of intron spacers. Methods, 30(4), 322–329.
[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.
But we met an huge obstacle : select vital gene fragments with high specificity in purpose of decreasing RNAi off-taget rate. But the truth is existing genome data of weeds is deficient right now and there are rare gene notes for sequencing species. Also,many malignant weeds are homologous from the victim crops they do harm to, the gene discrepancies is too less.
We felt hopeless after doing a lot of work then we paid attention to an easier one—Arabidopsis , which has clear genetic background. It is an ideal candidate for tandem RNAi verification.
Pest
In consideration，we chose aphids as experiment materials. First, It is a kind of worldwide pests, creating great impact in agriculture fields. Second, studies towards on aphids are relatively mature. And the materials is easy to get form nature.
Target gene
Arabidopsis
We find a target gene—trxz, whose deficiency will result in complete albino phenotype , it’s very easy to observe.
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 could demonstrate that TRX z constitutes a subunit of the 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 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:
\[ \mathrm{[(M^{2+}_{(1-x)}M^{3+}(OH)_2)^{x+} \times (A^{m-}_{x/m} \times nH_2O)^{x-}]} \]
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:
\[ CO_2+H_2O \longrightarrow H_2CO_3 \logrightarrow H^+ + HCO_3^- \]
\[ Mg_3Al(OH)_8 - A^- + 5H^+ \logrightarrow 3Mg^{2+} + Al'(OH)'_3 +A^- +5H_2O \]
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.
