Lipase; artificial micro RNA; fusion PCR; targeted gene knockout; endogenous micro RNA; RNA interference; post-transcriptional regulation
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: wmd3.weigelworld.org. [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. [...]
[...] Materials and methods PCR-directed mutagenesis of template clone pNW55. Three PCR sets were prepared. To three separate PCR tubes, 1µL of template clone pNW55 was added to 19µL of pre-mix PCR recipe, composing of 14.4 µL of deionized water, 2µL of 10X PCR buffer µL of deoxynucleoside triphosphate (dNTP) µL of Pfu polymerase, 1µL of forward primers and 1µL of reverse primers. For PCR reaction the forward primer was primer 4368 and reverse primer was primer II. For PCR reaction the forward primer was primer I and reverse primer was primer IV. [...]
[...] Further analysis had to be done to identify possible errors that caused discrepancies with expected result. Introduction Lipase (EC ) is an enzyme that hydrolyzes ester bonds in lipids, generating glycerol and free fatty acids (FFT) (Lee et al. 2003). This catalytic activity plays important role in sustaining embryo development during seed germination by mobilizing stored lipids in endosperms to generate energy (Vijayakumar & Gowda 2012). With rice acting as staple food in most part of the world, millions tons of rice bran, a byproduct of rice milling process, are produced annually. [...]
[...] Jones-Rhoades MW, Bartel DP & Bartel B 2006, ‘MicroRNAs and their regulatory roles in plants', Annual Review of Plant Biology, vol pp. 19-53. Kole Krainer AR & Altman S 2012, therapeutics: beyond RNA interference and antisense oligonucleotides', Nature Review, vol no pp. 125-140. Kurreck J 2004, ‘Antisense and RNA interference approaches to target validation in pain research', Current Opinion in Drug Discovery & Development, vol no pp. 179-187. Lacerda DBCL, Junior MSS, Bassinello PZ, Caliari M & Castro MVL 2013, kinetics of lipase activity and hydrolytic rancidity of raw, parboiled, and extruded rice bran during storage', Food Science Technology, vol no pp. [...]
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