In the previous two experiments, we have successfully detected the lipase gene and its activity from the rice line sample, as well as analyzing and confirming the gene expression. The next step of creating a rice line with reduced lipase activity would be silencing the gene of interest. In this experiment, the approach used to silence the gene of interest is using artificial microRNAs (amiRNAs). amiRNAs can be used in the reverse genetics to directly silence the gene of interest and allow us to analyze the effects of such silencing on the organism phenotype (Warthmann, et al, 2008). We are trying to knock out and reduce the expression of the gene responsible for gene activity in rice bran with the help of bioinformatics data from Wiegelworld. Data from websites like Wiegelworld help us to design primer sequences that are to be used in the PCR to produce amiRNA constructs (Warthmann et al, 2008).
Several PCRs can be performed to produce precursor gene of the amiRNAs that would be used to silence the gene of interest, specifically the gene responsible for the lipase activity in rice bran. The PCR schemes were performed using primers G4638, 1, 2, 3, 4, 5 and G4369, as described in the materials and methods section. In this experiment, the precursor of the rice miRNA Osa 528 would be utilized to produce another, almost identical precursor. This precursor gene is to be transferred to the genome of the rice using appropriate promoter to make the organism synthesize the precursor RNA (and hence, the miRNAs) that would silence the lipase gene.
In plant, microRNAs are endogenous single stranded small RNAs that are created by the processing of double-stranded RNAs (dsRNAs) encoded by the microRNA genes (Sanders and Bowman, 2016; WMD, 2005). microRNAs associate with RNA strands that are complementary to them and induce their degradation or inhibit their translation into polypeptides (Sanders and Bowman, 2016). It is widely applied in the field of transcriptomics and proteomics to study the function of the specific genes and the effects of downregulation on those genes (Verdonk and Sullivan, 2012).
The objectives of the experiments are to design amiRNA constructs that specifically target the gene responsible for lipase activity in rice bran with the help of information from Wiegelworld and research done by Warthmann and colleagues (2008). We also aim to discuss possible transformation techniques which are to be used to transfer the precursor gene to the plant cells.
Materials and Methods
The amiRNA constructs that target our gene of interest were designed with the help of Wiegelword website (http://wmd3.weigelworld.org) and research done by Warthmann and colleagues (2008), as described during the practical session.
Three tubes with the same template DNA were set, containing primers 2 and G-4368 for tube 1, primers 1 and 4 for tube 2 and primers 3 n d G-4369 for tube 4. Several polymerase chain reactions (PCRs) were performed on the tubes, as described in Figure 1 below. The conditions at which the PCRs were performed are of 95°C for 2 minutes; 34 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds; 72°C for 7 minutes for the first round and 95°C for 2 minutes; 34 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for1 minutes; 72°C for 7 minutes for the PCR fusion step.
Upon the completion of the PCRs, 2 µL of the content of tube 1, 2 and 3 were mixed with 2 µL of sample buffer and inserted into lane 2, 3 and 4 of 1.5% agarose gel, respectively. 1 µL of the DNA ladder and another µL of sample buffer were mixed and added into lane 1. A mixture with unknown content was added into lane 5. The gel was then run and visualized under UV light.
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)
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