Preparation of Artificial Microrna Construct Against Rice Lipase

Preparation of an artifical microRNA construct to knockout a specified lipase gene in rice

Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway, 47500 Subang Jaya, Selangor

Keywords: amiRNA, siRNA, miRNA, gene silencing, lipase, PCR

Abstract

After successfully detecting and confirming the lipase activity and the lipase gene expression of our rice sample in the previous two experiments, we would like to silence the gene of interest using miRNA-mediated silencing. To do this, we relied on the data obtained by previous work (WMD, 2005; Warthmann, et al., 2008). Also we were trying to find a suitable transformation method to transform the rice with the vectors carrying our amiRNA constructs. We found several unexpected bands in lane 5, which suggested that there may be some degradations or impurities in it. Also, we described an Agrobacterium based method to transform the plant cells.

Introduction

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.

     [pic 1]                                                                        Figure 1. The PCR scheme according to the protocol from Warthmann et al., (2008) to produce amiRNA constructs from pNW55 plasmid.

Results

[pic 2]                                                                                               Figure 2. The gel image of agarose gel electrophoresis, with lanes 1, 2, 3, 4 and 5 containing molecular standards, products of PCR tube 1, products of PCR tube 2, products of PCR tube 3 and combined products, respectively.

Track 5 contains the combined products from the 3 PCRs from lane 2, 3 and 4 using primers G4638 and G4369 (lane 4). This is proven by the presence of the ~250 bp band in lane 5, which is also present in lane 2 and lane 4 (this is most likely the 256 and 259 bp from lane 2 and 4, respectively. The ~554 bp in lane 5 is probably the fused product itself.