Sains Malaysiana 45(10)(2016): 1443–1451

 

Profil Pengekspresan Gen Mengekod delta 6 dan delta 12-Asid Lemak Desaturase Cunninghamella bainieri semasa Biosintesis Asid Gamma-Linolenik

(Expression Profiles of delta 6 and delta 12-Fatty Acid Desaturase Encoding Genes Cunninghamella bainieri during Gamma-Linoleneic Acid Biosynthesis)

NOOR ADILA ABDUL KARIM, ZAMRI ZAINAL, AIDIL ABDUL HAMID, FARAH DIBA ABU BAKAR & ABDUL MUNIR ABDUL MURAD*

 

Pusat Pengajian Biosains dan Bioteknologi, Fakulti Sains dan Teknologi, Universiti Kebangsaaan Malaysia, 43600 Bangi, Selangor Darul Ehsan, Malaysia

 

Received: 29 November 2015/Accepted: 10 March 2016

 

ABSTRAK

Delta 6-asid lemak desaturase dan delta 12-asid lemak desaturase merupakan enzim yang diperlukan bagi langkah desaturasi semasa proses biosintesis asid gamma-linolenik (GLA) oleh kulat oleaginus. Objektif kajian ini ialah untuk menganalisis profil pengekspresan gen mengekod enzim delta 6-asid lemak desaturase (des6) dan delta 12-asid lemak desaturase (des12) kulat oleaginus Cunninghamella bainieri semasa penghasilan GLA. Jujukan gen separa bersaiz 1372 pb bagi des6 dan 1008 pb bagi des12 telah dipencil daripada C. bainieri. Analisis pengekspresan gen menggunakan kaedah tindak balas berantai polimerase kuantitatif masa sebenar (RT-qPCR) menunjukkan perubahan kadar pengekspresan des6 adalah lebih tinggi berbanding kadar pengekspresan des12 semasa penghasilan GLA. Pengekspresan des6 adalah tertinggi selepas 24 jam dikultur dalam medium penghasilan GLA. Namun, kadar pengekspresannya menurun hingga jam ke-96 pertumbuhan tetapi meningkat semula pada jam ke-120. Bagi des12, kadar pengekspresannya adalah lebih sekata dengan pengekspresan tertinggi dikesan pada jam ke-120. Analisis penghasilan GLA menunjukkan jumlah GLA dalam sel berkolerasi dengan kadar pengekspresan des6. Hasil kajian mencadangkan bahawa aras pengekspresan des6 adalah penting dalam menentukan aras GLA dalam C. bainieri.

 

Kata kunci: Asid gamma-linolenik; Cunninghamella bainieri; delta 6-asid lemak desaturase; delta 12-asid lemak desaturase; kulat oleaginus

 

ABSTRACT

Delta 6-fatty acid desaturase and delta12-fatty acid desaturase are enzymes required for the desaturation steps during gamma-linolenic acid (GLA) biosynthesis in oleaginous fungi. The objectives of this study were to analyse the expression profiles of genes which encode for the delta 6-fatty acid desaturase (des6) and delta 12-fatty acid desaturase (des12) from an oleaginous fungus, Cunninghamella bainieri, during GLA production. Partial gene sequences with the size of 1372 bp for des6 and 1008 bp for des12, were isolated from C. Bainieri. Gene expression analyses via real-time quantitative PCR (RT-qPCR) indicate that the changes in the expression profile of des6 is higher during GLA production as compared to the expression profile of des12. The expression of des6 is the highest after 24 h of growth in the GLA-inducing medium. However, its expression showed a decreasing pattern until 96 h of growth but increased at 120 h. The expression profile of des12 is more consistent with the highest expression levels detected at 120 h of growth. GLA production analysis showed that the amount of GLA in the cell is correlated with the expression of des6. The results of this study suggested that the expression of des6 plays an important role in determining GLA production in C. bainieri.

 

Keywords: Cunninghamella bainieri; delta 6-fatty acid desaturase; delta 12-fatty acid desaturase; gamma linoleneic  acid; oleaginous fungi

 

REFERENCES

Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J. & Wittwer, C.T. 2009. The MIQE Guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry 55(4): 611-622.

Certik, M. & Shimizu, S. 1999. Biosynthesis and regulation of microbial polyunsaturated fatty acid production. Journal of Bioscience and Bioengineering 87(1): 1-14.

Chan, P.L., Rose, R.J., Murad, A.M.A., Zainal, Z., Low, E.L., Ooi, L.C., Ooi, S.E., Yahya, S. & Singh, R. 2014. Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture. PLOS One 9(6): e99774.

Conti, E., Stredansky, M., Stredanska, S. & Zanetti, F. 2001. γ-Linolenic acid production by solid-state fermentation of Mucorales strains on cereals. Bioresource Technology 76: 283-286.

Dyal, S. & Narine, S.S. 2005. Implications for the use of Mortierella fungi in the industrial production of essential fatty acids. Food Research International 38(4): 445-467.

Folch, J., Lees, M. & Stanley, G.H.S. 1957. A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226: 497-509.

Gunstone, F.D. 1992. Gamma linolenic acid-occurrence and physical and chemical properties. Progress Lipid Research 31: 145-161.

Hansson, L. & Dostalek, M. 1988. Effect of culture conditions on mycelial growth and production of γ-linolenic acid by the fungus Mortierella ramanniana. Applied Microbiology and Biotechnology 28: 240-246.

Jang, H.D., Lin, Y.Y. & Yang, S.S. 2005. Effect of culture media and conditions on polyunsaturated fatty acids production by Mortierella alpina. Bioresource Technology 96: 1633-1644.

Kendrick, A. & Ratledge, C. 1992. Desaturation of polyunsaturated fatty acids in Mucor circinelloides and the involvement of a novel membrane-bound malic enzyme. European Journal of Biochemistry 209: 667-673.

Khandake, R.M., Ranjekar, P.K. & Harsulkar, A.M. 2009. Cloning of a novel omega-6-desaturase from Flax (Linium usitatissimum L.) and its functional analysis in Saccharomyces cerevisiae. Molecular Biotechnology 42: 168-174.

Khunyoshyeng, S., Cheevadhanarak, S., Rachdawong, S. & Tanticharoen, M. 2002. Differential expression of desaturase and changes in fatty acid composition during sporangiospores germination and developement in Mucor rouxii. Fungal Genetics and Biology 37: 13-21.

Manikan, V., Kalil, M.S., Omar, O., Kader, A.J.A. & Hamid, A.A. 2013. Effects of Mg2+, Fe3+, Mn2+ and Cu2+ ions on lipid accumulation by Cunninghamella bainieri 2A1. Sains Malaysiana 43(3): 443-449.

Muhid, F., Kader, A.J.A., Yusoff, W.M.W., Omar, O. & Hamid, A.A. 2012. Pembangunan inokulum untuk penghasilan lipid dan asid gamma linolenik (GLA) oleh Cunninghamella bainieri 2A1. Sains Malaysiana 41(4): 431-437.

Murad, A.M.A., Karim, N.A.A., Hashim, N.H.F., Adnan, A.M., Zainal, Z., Hamid, A.A. & Bakar, F.D.A. 2011. Identification and characterisation of an oleaginous fungus producing high g-linoleneic acid. The Internet Journal of Microbiology 9(2): 1-4.

Napier, J.A., Hey, S.J., Lacey, D.J. & Shewry, P.R. 1998. Identification of a Caenorhabditis elegans Δ6-fatty acid desaturase by heterologous expression in Saccharomyces cerevisiae. Biochemical Journal 330: 611-614.

Noor Adila, A.K., Farah Diba, A.B., Zamri Z., Wan Mohtar, W.Y., Aidil, A.H., Mahadi, N.M. & Murad, A.M.A. 2007. Comparison of methods for isolating high quality DNA and RNA from an oleaginous fungus Cunninghamella bainieri strain 2a1. Malaysian Journal of Microbiology 3(1): 7-13.

Pfaffl, M.W. 2001. A new mathematical model for relative quantification in real time RT-PCR. Nucleic Acids Research 29: 2002-2007.

Qiu, X., Hong, H.P., Datla, N., MacKenzie, S.L., Taylor, D.C. & Thomas, T.L. 2002. Expression of borage Δ6-desaturase in Saccharomyces cerevisiae and oilseed crops. Canadian Journal of Botany 80: 42-49.

Ratledge, C. & Wynn, J.P. 2002. The biochemistry and molecular biology of lipid accumulation in oleaginous microorganism. Advances in Applied Microbiology 51: 1-50.

Sakai, H. & Kajiwara, S. 2005. Cloning and functional characterization of aΔ12 fatty acid desaturase gene from the basidiomycetes Lentinula edodes. Molecular General Genomics 273: 336-341.

Sakuradani, E. & Shimizu, S. 2003. Gene cloning and functional analysis of a second Δ6-fatty acid desaturase from an arachidonic acid-producing Mortierella fungus. Biosciences Biotechnology and Biochemistry 67(4): 704-711.

Sakuradani, E., Kobayashi, M. & Shimizu, S. 1999. Identification of a Δ12-fatty desaturase from an arachidonic acid-producing Mortierella fungus by heterologous expression in the yeast Saccharomyces cerevisiae and the fungus Aspergillus oryzae. European Journal Biochemistry 261: 812-820.

Sayanova, O., Beaudoin, F., Libisch, B., Shewry, P. & Napier, J. 2000. Mutagenesis of the borage Δ6 fatty acid desaturase. Biochemical Society Transaction 28: 636-638.

Sayanova, O., Smith, M.A., Lapinskas, P., Stobart, A.K., Dobson, G., Christie, W.W., Shewry, P.R. & Napier, J.A. 1997. Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain results in the accumulation of high levels of Δ6-desaturated fatty acids in transgenic tobacco. Proceedings of the National Academy of Sciences, USA 94: 4211-4216.

Watanabe, K., Oura, T., Sakai, H. & Kajiwara, S. 2004. Yeast Δ12-fatty desaturase: Gene cloning, expression and function. Biosciences Biotechnology Biochemical 68(3): 721-727.

Wei, D., Li, M., Zhang, X., Ren, Y. & Xing, L. 2004. Identification and characterization of a novel Δ12 fatty acid desaturase gene from Rhizopus arrhizus. FEBS Letters 573: 45-50.

 

 

*Corresponding author; email: munir@ukm.edu.my

 

 

 

 

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