Preparation of an Artificial Microrna Construct to Knockout a Specified Lipase Gene

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miRNA (amiRNA) sequence suitable for targeting the lipase gene. Initial miRNA sequences of the template are replaced by amiRNA sequences, this was done by PCR using specifically designed Primers (I + IV). Respective PCR product displays an 87 bp band on track 3 – this sequence consists of amiRNA which will eventually be processed into miRNA specific to our targeted lipase gene.

PCR with G-4368 + Primer II and Primer III + G-4369 was also performed to obtain flanking RNA sequences which house multiple cloning sites (MCS) – required for subsequent cloning. As annotated on Figure 1., these 2 fragments are 256 bp (track 2) and 259 bp (track 4), respectively. Subsequently, a mix of the aforementioned modification PCR products are fused to give a single DNA fragment to be cloned - Primers G-4368 + G-4369 was used. As observed in track 5, there is a 554 bp band which consists of the whole amiRNA construct with MCS. As this approach requires 4 PCR set up reactions, it is suggested be time-consuming as unsuccessful reactions can introduce errors (Liang, He, Li, & Yu, 2012).

Faint bands (annotated in orange and yellow) were also observed on the gel. The faint band of ~500 (orange box) bp may be due to fusion of the two flanking RNA sequences (256 bp and 259 bp). On the other hand, bands in the yellow box may be due to the presence of aforementioned PCR products which have not fused.

Constructed amiRNA is then subsequently inserted into T-DNA of a Ti-plasmid i.e. pGreen plasmid. pGreen plasmid is engineered to have an extensive MCS which would be compatible with our artificial miRNA vector, pNW55 (Hellens, Edwards, Leyland, Bean, & Mullineaux, 2000). Ti-plamid can be cloned into a bacterial host such as Agrobacterium tumefaciens or Agrobacterium rhizogenes. When integrating amiRNA into a Ti-plamid, selection markers such as antibiotic resistance may be added to identify Agrobacteium cells carrying the Ti-plasmid. However, there is a growing concern regarding the development of antibiotic resistance among harmful microorganisms. Alternatively, blue-white screening can be used – vector should have an MCS in lacZ gene fragment so that successfully transformed Agrobacteium cells carrying recombinant Ti-plasmids will produce white colonies and can be visually observed. Additionally, using a smaller vector increases the chance of transformation.

Vector itself should also contain additional marker/reporter genes to distinguish successfully transformed plant cells from other uninfected plant cells. An antibiotic resistance marker can be used as bacterial resistance i.e. phenotype of Spectinomycin and Kanamycin-resistant plants can be easily distinguished from each other. Promoter and terminator sequences are essential to induce and repress expression of genes i.e. nos gene.

To repress lipase genes in rice plant, T-DNA housing the amiRNA construct has to be infected into said rice plant cells – recombinant T-DNA is incorporated into plant’s genomic DNA. Agrobacterium infection may be induced by immersion of cultured embryogenic rice calli in Agrobacterium culture solution (containing antibiotic). Due to the presence of antibiotic, transformed plant cells with antibiotic resistance genes (in T-DNA) will be able to grow. Also, only healthy calli are selected to be grown into complete rice plant (Sahoo, Tripathi, Pareek, Sopory, & Singla-Pareek, 2011).

Conclusion

In all, the aims of this experiment has been met, though Agrobacterium infection step has not been conducted yet – we successfully designed an amiRNA construct with the ability to target our specified lipase gene. This demonstrates the effect of amiRNA as a gene silencing tool and also possible applications in modulating desirable traits in rice plants.