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

Autor:   •  October 15, 2018  •  1,837 Words (8 Pages)  •  109 Views

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According to figure 3, the gel image of PCR products shows cDNA of all four samples are less than 250bp. cDNA was amplified with two different primer pairs, the smeared bands of cDNA was amplified with primer pair 1 while the clear bands of cDNA was amplified with primer pair 2. At approximately 700bp region of the gel image, the clear band was also amplified with primer pair 2.

4. Discussion

Based on the gel image obtained (Fig 1 and Fig 2), the most visible bands were found on bran samples at approximately 50kDa. This indicates that bran contains higher amount of lipase compared to the leaf. The identification of protein lipase was analyzed by mass spectrometry. According to the Mascot Search Result, lipase was no present in any of the protein hits while the high protein hit is the Os01g0817700 protein. According to uniport, this protein codes for a phosphoglycerate mutase. The cDNA of bran leaf should be expressed in candidate gene 1, however the cDNA of bran lead is not observed from the gel image of PCR products. Errors may have contributed to this.

By comparing our results and Tiwari et al (2016) studies, primer pair for candidate gene 1 is used to amplify cDNA of LOC_Os11g43510 which codes for lipase-1, approximately 400bp. Primer pair for candidate gene 2 is used to amplified cDNA of LOC_Os01g60190 which codes for phosphoglycerate mutate, approximately 180bp.

SDS-PAGE was performed to extract lipase proteins. The gel was stained with 4-MU Butyrate to locate the location of the lipase, the presence of lipase can be detected by fluorescent bands. The bands will then further send to proteomic analysis for the identification of lipase. The other half of the gel bands was stained with Coomassie to identify the size of the lipase by comparing with the protein markers.

One of the possible limitation in this experiment is that the activity of lipase may be inactivated or disintegrate during the extraction of lipase by SDS-PAGE. High concentration of SDS inhibits the lipase activity due to its high detergent concentrations (Muga et al., 1993). If the removal of SDS was not done properly, it may not be able to restore the enzyme activity of the lipase. In addition, the rate of error for the reaction of reverse transcription is usually high due to the absent of proofreading ability of RNA (Sydow et al., 2009). Mutations such as insertions or deletions, or mismatched may have occurred.

5. Conclusion

As a conclusion, in order to prolong the shelf life of rice bran oil, the lipase activity must be reduced to increase the value of the rice crop. In this experiment, the desired lipase protein that codes for lipase activity was not extracted or may be loss due to the challenges in purifying and recovery process of lipase. The expression of candidate gene 1 which corresponds to gene LOC_Os11g43510 was not being expressed in our cDNA results. However, this method allowed us to identify lipases in rice bran. Experiment can be conducted several times to obtain the desired lipase gene take is responsible for the lipase activity.

6. Appendix

Figure 1: Gel image of SDS-PAGE of extracted proteins from bran and leaf tissues with MUF Butyrate staining. [pic 1]

[pic 2]

Figure 2: Gel image of SDS-PAGE of extracted proteins from bran and leaf tissues with Coomassive Blue Stain.

[pic 3][pic 4][pic 5][pic 6][pic 7][pic 8]

Figure 3: Gel image of PCR products on 1% agarose gel.

Table 1: Concentration of RNA and cDNA in ng/µl and A260/A280 purity ratio.

RNA

cDNA

Bran

Leaf

Bran

Leaf

Concentration (ng/µl)

5.50

32.7

198

75.5

A260/A280 ratio

1.8

2.0

1.7

1.7

Reference:

Jeon, CJ & Agarwal, K 1996, ‘Fidelity of RNA polymerase II transcription controlled by elongation factor TFIIS’, Proceedings of the National Academy of Sciences, vol. 93, pp. 13677-13682.

Lacerda, DBCL, Junior, MSS, Bassinello, PZ, Caliari, M & Castro, MVL 2013, ‘The kinetics of lipase activity and hydrolytic rancidity of raw, parboiled, and extruded rice bran during storage’, Food Science and Technology, vol. 33.

Muga, A, Arrondo, JL, Bellon, T, Sancho, J & Bernabeu C 1993, ‘Structural and functional studies on the interaction of sodium dodecyl sulfate with beta-galactosidase’, Archives of Biochemistry and Biophysics, vol. 300, pp. 451-457.

Ramezanzadeh, FM, Rao, RM, Windhauser, M, Prinyawiwatkul, W, Tulley, R & Marshall, WE 1999, ‘Prevention of Hydrolytic Rancidity in Rice Bran during Storage’, Journal of Agriculture Food Chem, vol. 47, pp. 3050-3052

Seetharamaiah, GS & Chandrasekhara, N 1989, ‘Studies on hypocholesterolemic activity of rice bran oil’, Atherosclerosis, vol. 78, pp. 219-223.

Sydow, JF & Cramer, P 2009, ‘RNA polymerase fidelity and transcriptional proofreading’, Current Opinion in Structural Biology, vol. 19, pp. 732-739.

Tiwari, GP, Chiang, MY, Silva, JRD, Song, BK, Lau, YL & Rahmen S 2016, ‘Lipase genes expressed in rice bran: LOC_Os11g43510 encodes a novel rice lipase’, Journal of Cereal Science, vol. 71, pp. 43-52.

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