The first functional Biobrick we have constructed is a vector with two promoters. And the two promoters actually are constructed on two strands of DNA respectively. Considering the fact that we may need a sufficient amount of dsRNA, and the high cost to extract dsRNA from engineered bacteria, we have chosen the T7 promoter whose strength is really high to obtain the dsRNA in a rather high concentration. Two T7 promoters are constructed on two different strands of DNA respectively, and transcriptional directions of the two promoters are reversed so that during the transcriptional period, the dsRNA(double strand RNA) will be synthesized. In between two promoters, there is a gene fragment which usually is the target gene of weeds or pests. To kill them effectively, the target genes we have selected are often essential thus silencing them will eventually lead to their death. Also, two terminators are constructed on reverse side to shut down the transcription so that the left part of the plasmid will not be transcript. The construction of dsRNA into the body of either plants or insects will trigger the RNAi(RNA interference).
+
we construced a vector with two promoters are constructed in one plasmid but their directions are reversed, double-stranded RNA will be produced during the transcription period.
After searching for large number of literature, we have designed a vector with much higher stability and higher efficiency in contrast to the first vector. As for this vector, only one T7 promoter and one double terminator are required. In between T7 promoter and double terminator, a sense arm, a loop and an anti-sense arm were constructed in sequence. The sense arm is the same as the sense arm in the first vector which encodes the target gene. The anti-sense arm is a gene fragment which is complementary to the sense arm. And the complementary double strands may be more easily synthesized in the help of loop which is located between them. When the hairpin RNA is successfully produced, it can trigger the RNA interference as the dsRNA does as well.
+
We found that the hairpin RNA actually possess higher stability and higher efficiency in gene silencing in living organisms including higher plants like Arabidopsis thaliana. In general, the hairpin RNA harbor similar effect with double-stranded RNA, what is different is that there is a loop inserted between the sense arm and anti-sense arm.
</p>
</p>
<figure>
<figure>
Line 59:
Line 59:
<h3 class="mdc-typography--subheading2">① Hairpin RNA with normal loop</h3>
<h3 class="mdc-typography--subheading2">① Hairpin RNA with normal loop</h3>
−
<p class="mdc-typography--body2">
−
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.
<h3 class="mdc-typography--subheading2">② Hairpin RNA with intron loop</h3>
<h3 class="mdc-typography--subheading2">② Hairpin RNA with intron loop</h3>
<p class="mdc-typography--body2">
<p class="mdc-typography--body2">
−
To achieve the best effect, we have tried another vector based on the literatures. In this plasmid, we replaced the normal loop with an intron sequence. 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 gene. And there is two possible reasons accounting for the higher stability and efficiency of this kind of construction:
+
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.
−
1. The deletion of intron from the construct by the splicesome may offer help to align the complementary arms of the hairpin in the environment which favors RNA hybridization, thus promoting to form a duplex.
+
−
2. Splicing may transiently increase the amount of hairpin RNA through retarding the hairpin’s passage from the nucleus or through creating a smaller and less nuclease- sensitive loop.
+
</p>
</p>
<p class="mdc-typography--body2">
<p class="mdc-typography--body2">
−
In Our experiment，we selected 120 base-pair-long noncoding sequence from Chloramphenicol as the normal loop.
+
In Our experiment，we selected 120 base-pair-long noncoding sequence from Chloramphenicol as normal loop.
</p>
</p>
<figure>
<figure>
Line 89:
Line 85:
<ul class="page__article__citations">
<ul class="page__article__citations">
<li class="page__article__citation__item">
<li class="page__article__citation__item">
−
[1] Total silencing by intron spliced hairpin RNAs. (2000). Nature.
+
[1] Total silencing by intronspliced hairpin RNAs.
</li>
</li>
<li class="page__article__citation__item">
<li class="page__article__citation__item">
−
[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.
+
[2] Young Sik Lee and Richard W. Carthew, Making a better RNAi vector for Drosophila: use of intron spacers
</li>
</li>
<li class="page__article__citation__item">
<li class="page__article__citation__item">
−
[3] Michael R. Green; Joseph Sambrook. (2012). Molecular Cloning: A Laboratory Manual (4th ed.).
+
[3] Molecular cloning（the 4th edition
</li>
</li>
</ul>
</ul>
Line 149:
Line 145:
</p>
</p>
<p class="mdc-typography--body2">
<p class="mdc-typography--body2">
−
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 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.
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.
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.
we construced a vector with two promoters are constructed in one plasmid but their directions are reversed, double-stranded RNA will be produced during the transcription period.
2. hpRNA construct
We found that the hairpin RNA actually possess higher stability and higher efficiency in gene silencing in living organisms including higher plants like Arabidopsis thaliana. In general, the hairpin RNA harbor similar effect with double-stranded RNA, what is different is that there is a loop inserted between the sense arm and anti-sense arm.
① Hairpin RNA with normal loop
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 intronspliced hairpin RNAs.
[2] Young Sik Lee and Richard W. Carthew, Making a better RNAi vector for Drosophila: use of intron spacers
[3] Molecular cloning（the 4th edition
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 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.
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.
