Preparation of Artificial Microrna Construct Against Rice Lipase

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Discussion

Several PCRs were used to change the original miRNA 528 and miRNA* sequences of pNW55 vector to novel amiRNAs. As shown in Figure 2, lane 5 contains the combined products from the 3 PCR schemes described in the Materials and Methods section (Figure 1). Primers 1, 2, 3, 4, G-4368 and G-4369 used in the PCRs were generated with the help of Wiegelworld website and their sequences are complementary to the sequences in pNW55. The products of the PCRs are three amplicons; a 256-bp amplicon from the PCR using primers G-4368 and 2 (lane 2, Figure 2), a 87-bp amplicon from the PCR using primers 1 and 4 (lane 3, Figure 2) and a 259-bp amplicon from the PCR using primers G-4369 and 3 (lane 4, Figure 2). Another PCR was done using the primers G-4369 and G-4368 to yield a combined product with approximately 554 bp (Warthmann, et al., 2008), which is expected to be found in lane 5 of agarose gel. Agarose gel electrophoresis separate the DNA strands based on their size; smaller bands would travel slower from the negative to positive pole than larger bands (Sanders and Bowman, 2016). As can be seen in Figure 5, there are multiple DNA bands that are present in lane 5, not just the ~554 bp band. For example, there is a 300 bp band in lane 5 that is similar to the ones in lane 2 and 4 and this suggests that there are some amplicons that are not fused by the final PCR. Alternatively, the bands other than the 555 bp band are degraded DNA products.

The vector that can be used in the transformation of rice is osa-MIR528, similar to the one used by Warthmann, et al. (2008). To create the amiRNA constructs, three fragments in pNW55 were amplified using PCR with six primers for each amiRNA construct (Warthmann, et al., 2008). Additionally, three PCRs were performed on the pNW55 template which gave rise to 256, 87 and 259 bp amplicons, which in turn were fused to form a 554 bp amplicon, as described earlier. The 554 bp amplicon can be cloned into DNA vectors, for example pGEM-T. It can be further transferred to the multiple cloning sites (MCSs) of binary vectors (e.g. IRS154) by cutting both of them using the appropriate restriction enzymes (Warthmann, et al., 2008). To induce the expression of an inserted gene (amiRNA precursor gene in our case) in IRS154, a suitable promoter, for example the maize ubiquitin promoter can be used (Warthmann, et al., 2008). These vectors would be utilized to transform Agrobacterium tumefaciens, which can infect plant cells and transferred desired genes to them (Warthmann, et al., 2008).

The gene silencing method chosen for this experiment is miRNA-mediated silencing not siRNA-mediated silencing. One of the most defining differences between the former and the latter is that siRNAs target one type of mRNA only, which increases the chance of off-target effects happening due to downregulation on the incorrect targets (Lam, et al., 2015). On the other hand, miRNA can recognize and silence multiple target, but it has less chance to cause off-targeting (Lam, et al., 2015). Moreover, it is preferable to use miRNA rather than siRNA when working with a gene sequence that has been extensively studied (the lipase gene has been extensively studied before (Campo, et al., 2013)

Conclusion

We have managed to successfully design the amiRNA constructs from pNW55 that can target the gene responsible for lipase activity in rice bran by relying on PCR and data from Wiegelworld website. Lane contains impure products; there are most likely some amplicons that were not fused by the fusion PCR step in lane 5. Also, we have discussed the possible way of rice transformation by transferring the amiRNA construct into pGEM-T plasmid vector and to IRS154 binary vector

References

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Sanders, MF and Bowman, JL, (2016). Genetic Analysis: An Integrated Approach, 2nd Edition, Pearson, San Fransiscp, CA, USA.

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Warthmann, N, Chen, H, Ossowski, S, Weigel, D and Hervé, P, (2008). Highly Specific Gene Silencing by Artificial miRNAs in Rice, PLoS ONE, 3(3): 131-149.

WMD, (2005). AmiRNA Design Guide. Available at: http://wmd3.weigelworld.org/cgi-bin/webapp.cgi?page=Help#procedure.