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Construction of artificial micro RNA with mismatch PCR to direct silencing of lipase gene

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  1. Introduction
  2. Materials and methods
  3. PCR-directed mutagenesis of template clone pNW55
  4. Verification of amplified products by agarose gel electrophoresis
  5. Discussion
  6. Conclusion

Lipase is an enzyme that hydrolyzes rice bran oil into free fatty acids, causing spoilage and restricting its usage as food ingredient. Micro RNA (miRNA) is a post-transcriptional regulator endogenously produced in plants that can be potentially altered to specifically target lipase gene, silencing it in the process. This experiment aimed to design a precursor miRNA gene with mismatch primers through fusion PCR by using the rice's own miRNA osaMIR528 as template. Three separate PCR tubes, each containing specific set of primers (primer G-4368 and primer II for reaction 1; primer I and primer IV for reaction 2; primer III and primer G-4369 for reaction 3) were prepared and sent for PCR thermo-cycling. Amplified products from three reactions were combined and subjected to fusion PCR using primer G-4368 and G-4369. Amplified products from all three reactions and fusion PCR were gel electrophoresed and UV imaged. One band was observed for reactions 1, 2, and 3 (250bp, 80bp, and 250bp respectively) and was consistent with expected data.

Two bands were observed for fusion PCR with 300bp fragment being major product, disagreeing with expected result. Deletion was suspected during PCR. As conclusion, the construction of a 554bp ami RNA precursor gene for targeted knockout of rice lipase was not successful. First three PCR reactions using designed primers managed to produce desired fragment but deletion was suspected to occur in final PCR product. Further analysis had to be done to identify possible errors that caused discrepancies with expected result.

[...] Liu H & Naismith JH 2008, efficient one-step site-directed deletion, insertion, single and multiple-site plasmid nutagenesis protocol', BMC Biotechnology, vol pp. 1-10. Martinez Patkaniowska Urlaub Luhrmann R & Tuschl T 2002, ?Single- stranded antisense siRNAs guide target RNA cleavage in RNAi', Cell, vol pp. 563-574. Ossowski S & Fitz J 2009, Web Micro RNA Designer [online]. Accesed at: [Accessed on 11th September 2014]. Ossowski Schwab R & Weigel D 2008, ?Gene silencing in plants using artificial microRNAs and other small RNAs', The Plant Journal, vol no pp. 674-690. [...]

[...] Such feature could be generated by ensuring high AC content at 5' end and high GC content at 3' end. The second requirement was at least 70% perfect matching of ami RNA to target mRNA with at least -30kcal/mol of free hybridization energy (Bernhart et al. 2006). This was the minimum free energy for stable formation of RNA heterodimers. Another mandatory criterion was no mismatch should exist at the cleavage site, maximum one mismatch in the 5' region and up to four but no more than two consecutive mismatches in the 3' part of precursor molecule. [...]

[...] Antisense RNA was a single stranded to 25 nucleotides long RNA with complementary sequence to target mRNA (Kole et al. 2012). Base pairing of antisense oligonucleotides would obstruct the ribosome binding and protein synthesizing machinery along the mRNA (Weiss et al. 1999). RNase H could also mediate degradation of target mRNA when antisense oligonucleotide hybridized by hydrolyzing the RNA/RNA or RNA/DNA heteroduplex (Kurreck 2004). In contrast to miRNA, this method was less potent and had much reduced efficiency (Martinez et al. [...]

[...] Rapid advancement in sequencing and biotechnology provide useful insights into composition and structure of rice lipase gene. Knowledge acquired sheds light on targeting lipase down to its molecular level and knocking out lipase gene is made possible. Micro RNA (miRNA) is an endogenous to 25 nucleotides long, non- translated RNA that regulates post-transcriptional events. In plants, miRNA mediates gene silencing through RNA interference (RNAi) phenomenon and a single miRNA can target a number of genes (Jones-Rhoades et al. 2006). Gene encoded for miRNA is first expressed as a long, single stranded transcript. [...]

[...] Reaction 3 was found to produce band size of 250bp, which was quite similar to the predicted 259bp. Expected band sizes for each reaction was based on theoretical assumption of accurate and efficient PCR in the 3' region of pNW5-osaMIR528 template (with primer G-4368 and primer 5' region (with primer III and primer G-4369) and middle portion (with primer I and primer IV). The brightly colored bands indicated that the bands represented major products of PCR in reaction 1 and reaction 3. [...]

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