Team:SZU-China/Iteration
Iteration
RNA interference (RNAi) technology was all the rage at a time, and the biologists almost scrambled to study the mechanism and the application of it. Then, as the technique of CRISPR gene editing got increasingly popular, the RNAi was not as hot as it used to be. We barely found the papers applying the RNAi molecules to the plants as the herbicide. Hence, we were doubted that whether it could be used as an herbicide to kill the M. micrantha, which was so strong that there were almost no effective herbicides that could remove it in just a few days. Therefore, we decided to synthesize the most traditional RNAi molecule, double-strand RNAs (dsRNA), as the vector to carry the exogenous siRNA into the plants. We designed the dsRNA targeted on different kinds of respiration related genes, to block M. micrantha normal metabolism, trying to make it dead.
We carried out the Real-time fluorescence quantitative PCR to test the gene expression of the tested M. micrantha. We set the control and the experimental groups. The gene expression level shows that dsRNA can silence the target genes (Fig.1). These gene sequences encode citrate synthase-related proteins. We could see that on the first day, the expressions of gene were downregulated, that is the relative gene expression was under 100%.
Fig. 1 The Relative Target Gene Expression after using dsRNA
Besides, after about three weeks, from the apparent morphology of the leaves, we could confirm that dsRNA did go through the leaves and worked inside the M. micrantha (Fig.2). The upper line was blank group, and the lower line was experimental group. The other operating conditions of the two groups were the same. The control group were applied the solvent of dsRNA (the sterile water), and the experimental group were sprayed the dsRNA mixture. We could clearly see that the experimental leaves were turning brown and wilting.
Fig.2 the morphology of the leaves after testing for three weeks
However, the efficiency of dsRNA was quite low and less specific, and we had to spray for many times to get the wilting results. Therefore, we held a brainstorm with our PIs and advisors to improve our methods.
At the dsRNA stage, we could confirm that the RNAi technology would possibly be used as the specific herbicide for M. micrantha. However, since the dsRNA is so long that it is not as specific as short interference RNA (siRNA) and the efficiency could not meet our requirements, we decided to use a new way to synthesize RNAi nanoparticles as the vector to carry half a million copies of siRNA into the weed, after the brainstorm. However, there were no professors in our school knew how to synthesize the RNAi nanoparticles, so we had to explore it by ourselves and meanwhile, interview some experts that might give us some related suggestions. After discussing with the experts and trying hard, we finally synthesized the RNAi nanoparticles. (Click Results to see more results) Here is the scanning electron microscope (SEM) pictures of the RNAi nanoparticles we synthesized (Fig.3).
Fig.3 The SEM pictures of the RNAi nanoparticles
What’s more, after discussing with many professors, we selected the siRNAs that could target different kinds of chlorophyll related gene, which could kill the M. micrantha as well as helping us better observe the apparent morphology of the tested leaves. The qRT-PCR results are as follows (Fig.4). We could see that all the target genes were silenced three days after treatment, while the negative control nearly had no change, which showed that the siRNA has high specificity inside M. micrantha.
Fig. 4 The Relative Target Gene Expression after Using RNAi nanoparticles
Meanwhile, we traced the morphology of tested leaves for 10 days and found black spots on the leaves and some of them had bleached margin (Fig.5). The first line was the photo of the blades before treating, and the second line was the photo taken after 10 days, and they were just sprayed RNAi nanoparticles once. The results were much better than that of dsRNA.
Fig.5 The Morphology of the Leaves after Spraying RNAi Nanoparticles for 10 Days
However, after CCiC, the judges pointed out that making the RNAi nanoparticles in vitro as the RNAi-based herbicide was costly and hard to be manufactured, which meant that the herbicide presented small development potential and narrow market prospect. Hence, we developed the third generation of RNAi molecules to reduce costs and make our products better applied to the market.
The RNAi molecules in the third generation were transcribed inside the E coli induced by IPTG, which reduced the cost of purchasing T7 RNA polymerase and T4 Polynucleotide Kinase, the most expensive biochemicals used during the synthesis of RNAi nanoparticles. Then we did the quality tests on the third generation mentioned above and tested the stability of this kind of RNAi-based herbicide.
From the results of qRT-PCR, we could see that all the genes were silenced two days after treatment, while the negative control remained unchanged (Fig.6). These gene sequences targeted on the chlorophyll related gene. Besides, we could clearly see that the gene silence results were more stable than that of RNAi nanoparticles.
Fig. 6 The Relative Target Gene Expression after Using hairpin siRNA
Meanwhile, we had a collaboration with Jiangnan-China team and tried their biosurfactant. We set four tested groups, sterile-water-sprayed, surfactant-sprayed (to test whether it would do harm to the plants), surfactant-RNAi hairpin siRNA-sprayed and water- RNAi hairpin siRNA-sprayed, respectively. Then, we traced the morphology of tested leaves for 10 days and found the leaves were wilting and turning black (Fig.7). The photos were taken after 10 days, and they were sprayed hairpin siRNA every day. The results were much more significant than before.
Fig.7 The Morphology of the Leaves after Spraying Hairpin siRNA every day for 10 Days
Considering the Micrancide is RNAi-based herbicide, which is easy to be degraded by RNase, the container of it should be RNase-free before the RNAi molecules pumping inside. After discussing with some experts, we decided to design the vial made of glass as the container, which could be placed under a high temperature of 250℃ for 3 hours to remove the RNase. Apart from that, it is not easy to oxidize and can be stored for a long time. However, after we interviewed the farmers, they told us that glass was too heavy to carry and they preferred something light, convenient and unbreakable.
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High temperature resistant
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Not easy to oxidize
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Can be stored for a long time
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Heavy
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Fragile
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Inconvenient
Then, we found the degradable plastic with high temperature resistant. It still could not be put under a high temperature of 250℃ but can go high temperature and high-pressure sterilization using 0.1% DEPC to remove the RNase. The second generation is much lighter, more portable and easier to transport. Moreover, the bottle will be sealed with a pressure-sensitive gasket to keep the Micrancide away from the degradation.
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High temperature resistant (but not that high)
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Degradable
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Light, convenient and unbreakable
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Easy to oxidize
