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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 Diserahkan: 29 November 2015/Diterima:
10 Mac 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 RUJUKAN 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. *Pengarang untuk surat-menyurat: email: munir@ukm.edu.my
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