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

Abstract

MicroRNAs (miRNAs) are small RNA molecules which have the ability to coordinate the expression of genes in eukaryotes. During plant development and growth, miRNAs down-regulates/represses specific genes which are not required under certain circumstances. RISC complexes are associated with these 21-nucleotide mRNA sequences, directing cleavage of mRNA with complementary sequences. Artifical microRNA (amiRNA) corresponding to an identified lipase gene can be designed and constructed to replace initial miRNA duplex sequences. Here, we outline the protocols required to knockout a specified lipase gene in rice tissue. amiRNA sequences are designed using bioinformatics tools i.e. WMD3. amiRNA technology is a robust and dynamic tool to study specific gene functions and their respective metabolic pathways.

Introduction

Rice cannot be stored for long period of time because lipase enzyme present within the bran layer oxidizes lipids into free fatty acids. As a result, rice turns rancid with an unpleasant odor. Eradicating this issue would improve population wellbeing in general, as people would be encouraged to incorporate bran oil products into their diet. Products of rice bran oil are rich in various vitamins and are potent in lowering cholesterol levels (Nantiyakul et al., 2012).

In this study, we employed amiRNA technology to knockout/repress lipase activity within rice tissues. amiRNA pathway is often preferred when targeting gene expression compared to conventional small interfering RNA approaches (siRNA). This is because we are able to track amiRNA-expressing cells by looking at their correlated markers. Also, less effort is required to express multiple amiRNAs compared to siRNAs. Overlapping PCR is used to construct the amiRNA by site directed mutagenesis on the template miRNA. OsMIR528 is used as a template to generate specifically designed miRNA which is able to target our lipase gene of interest – the duplex sequences are replaced by the amiRNA sequences designed using WMD3 tool. Short primer sequences are also designed to replace miRNA sequences with amiRNA sequences.

Consequently, functional mature amiRNA are produced with this experimental protocol as they are processed like natural miRNA molecules. miRNA can down-regulate the expression of specific lipase gene by cleaving RNA or by inhibiting translation (Jones-Rhoades, Bartel, & Bartel, 2006).

Materials and Methods

Primers used to construct amiRNA sequences were designed using WMD3 – Web microDNA designer. Materials and methods described by Weigelworld and Norman Wartmann et al. (2008) were followed closely. Products of 4 PCR reactions were run on 1% agarose gel.

Results

PCR products of Tube 1, 2 and 3 were run on track 2, 3 and 4, respectively. Primers used are G-4368 + Primer II, Primer I + Primer IV and Primer III + G-4369. As a result, 256 bp, 87 bp and 259 bp bands are produced.

On track 5, there is a 554 bp band. Additionally, faint bands of ~500 bp and ~250 bp were also observed (labelled in orange and yellow).

Figure 1. Annotated gel electrophoresis results of PCR reactions with G-4368 + Primer II, Primer I + Primer IV, Primer III + G-4369 and G-4368 + G-4369 for lanes 2 to 5, respectively.

Discussion

The aim of this experiment is to induce silencing/repression of a specified lipase gene in rice via the miRNA pathway. First, an artificial miRNA needs to be designed to target the lipase gene we are interested in. OsMIR528 is used as a template – WMD3 is used to produce an artificial