Team:SZU-China/Measurement
Measurement
SZU-China 2019 iGEM team aimed to produce an RNAi-based herbicide for M. micrantha (Micrancide)through RNA interference (RNAi) technology. During the whole process, we developed two new and innovative approaches to measure the efficiency of Micrancide at both morphological and genetic changes.
During the research on plant epigenetics, it is hard to detect the changes in the apparent morphology of plants quantitatively. However, it would be easy to analyze and demonstrate the plant changing degree if we could quantify the change. Hence, SZU-China 2019 team developed a Matlab script (Etiolation_rate_script.m) for M. micrantha to measure the etiolation rate of leaves.
Since leaves are different, we need to redefine the green every time. Hence, the script will scan the picture to calculate the area of leaves. Then, click the green area, and the script will pick the pixel of the circle area of 10% of the leaves area with the clicking point as the center and calculate the average of the green. Then it will define this pixel as the standard of green.
We define the x% of standard pixel as the critical value. The pixel recognized by the script below this value is regarded as the browning area, while the one above this value is defined healthy area. The x is variable according to the etiolation degree.
According to the principle mentioned above, the script will recognize and use red color to mark the browning area automatically and then calculate the etiolation rate.
Put the leaf photo to be processed into the directory of the script, and copy the name of the picture to a specific location of the script
After clicking to run, the program will pop up a window of a leaf. Then, you need to click on the green area of the leaf and the program will automatically calculate the etiolation rate of the leaf and mark it.
We used this script to get the etiolation rate of the tested M. micrantha leaves and predicted how long the tested leaves would die. We sprayed Micrancide on four leaves and took the photos of them for ten days. Then, we matted the image of leaves from the photos and ran the script.Here are one of the results.
According to the results, we carried out statistical analysis on the average etiolation rate of each group on the tenth day and got the etiolation rate.
Then we built the model to obtain the etiolation rate equation and predicted that M. micrantha would die after 13.4 days (when the etiolation rate is more than 80%, we think this leaf is dead). Click Model to see more.
And the experimental results showed that after 15 days, the tested M. micrantha were dead completely, which meant that the Matlab script and the model we built were persuasive and reliable to some extent.
As we all know, RNAi molecules are short and easy to degrade by RNase. Hence, it is difficult to detect siRNA inside the leaves in vitro. However, we had to detect whether there was exogenous siRNA introduced into the tested M. micrantha. We have tried to carry out Northern Blot strictly avoiding RNase but still failed (Fig.1).
Fig.1 The result of Northern Blot (Nothing could be seen)
Then, we found a new method to test it, which was a G-quadruplex DNA-based, label-free and ultrasensitive strategy for microRNA detection [1].
A cDNA strand, which is completely complementary to the target miRNA and partly complementary pairing with G-rich DNA, was designed first. Then this cDNA can be competed off from the cDNA/G-rich DNA duplex to form a cDNA/RNA heteroduplex and release the G-rich oligonucleotides when the target-siRNA was introduced. Recent research progress has demonstrated that G-quadruplex DNA, a specific type of G-rich nucleic acid sequence, can be remarkably recognized by thioflavin T (ThT) with high selectivity, unlike the triplex, duplex or single-stranded forms of DNA. The fluorescence intensity of ThT exhibits a considerable increase upon binding to G-quadruplex DNA, which can be utilized as a signal reporter. The conformation of released G-rich oligonucleotides would change into G-quadruplex DNA with the presence of 2.0 mM K+. Then, the ThT remarkably recognizes and selectively binds to the G-quadruplex DNA, resulting in a significant enhancement in the fluorescence signal (Fig.2).
Fig.2 Schematic representation of the direct detection of miRNAs
The concrete experiments are detailed in the Experiment (Fig.3).
Fig.3 siRNA Detection
This year, we first introduced this strategy to test the function of toxin Tse2 and finally verified that Tse2 could work, and this testing method was useful and reliable. For more information, please visit BBa_K314200. The Toxin Tse2 can arrest the growth of prokaryotic and eukaryotic cells when expressed intracellularly, so the Toxin(-) group would grow better than the Toxin(+) group. And from the results of the Trieste iGEM team 2012, we can see the trend of E. coli growth (Fig.4).
Fig.4 Toxin Tse characterization from Trieste iGEM team 2012
Compared with the results from the Trieste iGEM team 2012, the toxin Tse2 could work, and this label-free method to test hairpin siRNA had good repeatability and high reliability (Fig.5).
Fig.5 The Fluorescence Emission of different treated E. coli samples
Then, we applied this method to detect siRNA introduced into leaves and got the results (Fig.6).
Fig.6 siRNA detection of different days using the new methods
We were doubted whether these results were reliable, so we also did the RT-qPCR to test the endogenous target gene expression. Then we compared the results (Fig. 7).
Fig.7 The siRNA detection results vs. the RT-qPCR results
From the figure, we can see that target genes were significantly silenced after treated for 2 days and recovered after about 4 days according to the RT-qPCR results, while the siRNA in vivo increased dramatically after treated for 1 day and then decreased gradually according to the new method results, from which we can conclude that with the increase of siRNA in vivo, the gene expression was down-regulated as expected and the decrease of siRNA led to the recovery of target gene expression. Hence, we can reach the conclusion that this new method is reliable and can reflect the change of siRNA in vivo to some extent.
These two measuring methods were useful and reliable to a certain extent according to the results of the experiment. The first method can be applied to plant epigenetic experiment and quantify the apparent changes of the plants by adjusting the parameters. The second one can be applied to the experiment related to microRNA and other experiments that need to carry out Northern Bolt, which is much more convenient and time-saving than NB.
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[4] Sun N, Li D, Hou J, et al. A propeller‐like small molecule as a novel G‐quadruplex DNA binder: The study of fluorescent sensing property and preferential interactions with human telo21 structure[J]. Chemical Biology & Drug Design, 2019, 93.
