Determination of Lipase Protein and the Study of Gene Expression in Bran and Leaf Tissue of Rice Plant (oryza Sativa)

Determination of lipase protein and the study of gene expression in bran and leaf tissue of Rice Plant (Oryza sativa).

Cassandra Liew Chi Yen, Ngeow Sue Sie, Wong Chong Voon, Ashwathi Nambiar

Student ID: 26608057, Monash University Malaysia, Jalan Lagoon Selatan, Bandar Sunway, 47500 Subang Jaya, Selangor, Malaysia.

______________________________________________________________________

ABSTRACT

Rice (Oryza sativa) highly consumed by majority of the population around the world. The value of the rice crop can be improved by producing Rice Bran Oil (RBO). Grains and bran are the by-product of rice milling process. The Bran contains oil that can be potentially be used as cooking oil. However, the shelf life of RBO is hard to maintain due to its lipase activity that will degrade the lipids, triacyl glycerol (TAG) into free fatty acids. In this experiment, we tried to identify the lipase proteins in the growing rice plant by performing SDS-PAGE to separate plant proteins based on their size and determine which proteins are involve in lipase activity. We have also study the expression of RNA for two candidate genes and compare the expression patterns with the proteins detected by SDS-PAGE. The RNA is being converted to cDNA by reverse transcription reaction and identify gene that is responsible for the lipase activity by PCR.

KEY WORDS: Triacylglycerol lipase, Rice bran, Sodium dodecyl sulphate polyacrylamide gel electrophoresis

1. Introduction

The presence of plant lipases in rice bran leads to rapid degradation of triacyl glycerol (TAG) into free fatty acids and glycerol which contributed to the low stability of rice bran oil. The free fatty acids reduces the pH level which gives off a foul smell due to its acidity (Malekian 2000).

Rice bran oil ranked high among most of the vegetable oils due to the balanced profile of their major fatty acids such as palmitic, oleic and linoleic acids (Larissa et al., 2017).  According to Seetharamaiah and Chandraekhara (1989) research, linoleic acid has the best outcome in producing reductions in cholesterol levels. However, the major issue here is the low stability of RBO due to the activity of lipase that will degrade triglyceride into free fatty acids decreasing the shelf life and result in unpleasant, foul smell that is not suitable for consumption (Ramezanzadeh et al.,1999). To prevent this, gene expression of lipase must be reduced so that the degradation process will slow down and reduce rancidity (Lacerda et al., 2013). Therefore, the lipase protein involved in breaking down of tryglyceride is being studied to prevent the degradation of the trigylcerides.

In this experiment, we identify lipase protein which is responsible for the lipase activity and study the gene expression in rice. Extraction of lipase protein from rice plant was performed and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins, then followed by assaying the lipase activity. Further, expression of the RNA was tested to determine whether the patterns of the expression are well-matched with lipase detected at the protein level.

2. Materials and methods

2.1 Extraction of Rice Protein

30mg of rice tissue (leaf and bran) were ground into fine powder using Liquid Nitrogen and transferred into Eppendorf tube. 100µl of Extraction buffer (62.5 Mm Tris HCl (ph7.4), 10% glycerol, 0.1% SDS, 2 Mm EDTA, 100 mM DDT) was added. Sample tubes were vortexed for 2 mins and incubated for 10 minutes on ice. The tubes were centrifuged at 12000 rpm for 10 minutes, supernatant was collected. 20µl from the collected supernatant was added into a new Eppendorf tube with 20µl of sample buffer and placed on a heating block (100°C) for 5 minutes. The tubes were then centrifuged for 15 seconds and 20µl of each sample were loaded onto the polyacrylamide gel and run at 150V at for 40 minutes.

2.2 SDS-PAGE with extracted protein

After electrophoresis, the gel was immersed in 50ml of 25% Isopropanol for 5 minutes in a gel rocker. The gel is washed with 50ml of distilled water for 5 minutes in the gel rocker. Further, the gel was rinsed with 50 Mn Na-Phosphate buffer (pH 7.0) for three times. 40ml of the 4-MU Butyrate was added and allowed it to stain for one minute. The gel was observed under UV light and visible bands were cut and sent for proteomic analysis.

2.3 Gel staining with Coomassie stain

The other half of the gel was placed in 100 ml distilled water and microwaved for 90 seconds. The water was decanted and 50ml of Gelcode Blue Safe Protein was added to the gel and microwaved for 90 seconds. After microwaving, the gel was allowed to agitate gently on a shake for 5 minutes. The staining agent was decanted and the gel was destained with 100 ml ultrapure water in a microwave for 90 seconds. The gel was removed from the microwave and allowed it to agitate on the shake for another 5 minutes. The water is decanted. The gel was observed under UV light and visible lipase band is cut and placed in Eppendorf tube for protein sequence analysis by mass spec.

2.4 Extraction of RNA, preparation of cDNA and amplification of lipase genes

Rice tissue (leaf and bran) were ground and lysed. The lysate was homogenized with QIAshredder. RNA in rice tissue was extracted based on RNeasy Plant Mini Procedure Protocol (RNeasy Mini Handbook 2012). Preparation of cDNA was performed by Reverse Transcription Reaction. PCR was carried out for two candidate genes for lipase using primers of the two genes. PCR products were loaded onto the agarose gel for the separation of cDNA. The gel was observed under UV light and photograph of the gel was taken (BTH3820 Practical Manual 2017). The DNA ladder used was Thermo Scientific GeneRuler 1kb DNA ladder.

3. Results

Based on MUF Butyrate staining (figure 1), lipase band was more visible in Bran (Grain) samples with approximately 50kDA length according to the protein markers. As for Coomassie staining (figure 2), the length of lipase was visible at approximately 50kDa as well. Based on the mass spec analysis, Os01g0817700 protein has the highest protein hit, however lipase did not appear to be in the top hit results.

The concentration of RNA extracted from Bran is 5.5ng/µl with a 260/280 purity ratio of 1.8, while the RNA concentration of Leaf is 32.7 ng/µl with a 260/280 purity ratio of 2.0. The cDNA concentration of Bran obtained is 198 ng/µl with a 260/280 purity ratio of 1.7 and cDNA of leaf is 75.5 ng/µl with a 260/280 purity ratio of 1.7.