Sains Malaysiana 39(6)(2010):
927–933
Transformasi
Gen Metalotionin (eiMT1) daripada Eleusine indica ke
dalam Pokok Tembakau, Nicotiana tabacum Berperantaraan Agrobacterium
tumefaciens
(Transformation
of Metallothionein Gene (eiMT1) from Eleusine indica into
Tobacco, Nicotiana tabacum by Agrobacterium tumefaciens)
Nik Marzuki Sidik*, Roslina Mat Yazid, Che Radziah Che Mohd. Zain
& Ismanizan Ismail
Pusat
Pengajian Biosains dan Bioteknologi, Fakulti Sains dan Teknologi
43600
Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia
Diserahkan:
16 Jun 2009
/ Diterima: 5 Mei 2010
ABSTRAK
Metalotionin
(MT)
merupakan protein pengikat logam berberat molekul rendah dan kaya dengan
sistein yang hadir dalam pelbagai jenis organisma termasuklah bakteria, kulat, tumbuhan
dan haiwan. MT tumbuhan dipercayai mengambil bahagian dalam metabolisme
dan penyahtoksikan logam dengan cara pengkelatan ion-ion logam berat. Fungsinya
yang unik ini telah mendorong minat untuk memencilkan gen daripada rumput
sambau, Eleusine indica. DNA pelengkap
(cDNA) eiMT1
telah diklonkan ke dalam vektor binari pBI121 untuk ditransformasikan ke dalam
pokok tembakau melalui perantaraan Agrobacterium tumefaciens.
Penyaringan pokok tembakau transgenik dengan PCR dilakukan
menggunakan 3 pasang pencetus yang direka khas iaitu pasangan CMTF dan CMTR, 35SF dan PMTR, dan
pasangan pencetus khusus-gen MTFS2 dan MTRS2.
Ketiga-tiga pasangan pencetus ini berjaya menghasilkan saiz serpihan DNA jangkaan
iaitu masing-masing 270 pb, 1.1 kb dan 170 pb. Penjujukan terhadap serpihan
bersaiz 170 pb dan analisis jujukan menunjukkan persamaan 100 % dengan eiMT1.
Kajian pengekspresan gen melalui pendekatan transkripsi berbalik-PCR membuktikan
bahawa transgen eiMT1 telah berjaya diekspreskan dalam 11 daripada 19 pokok
transgenik yang dikaji.
Kata
kunci: eiMT1; Eleusine indica; metalotionin; transformasi;
tembakau
ABSTRACT
Metallothioneins
(MTs)
are low molecular weight, cysteine-rich metal-binding proteins found in a wide
range of organisms including bacteria, fungi, plant and animal. Plant MTs
are thought to play a role in metal metabolism and detoxification via chelating
of the metal ions. This unique feature results in the interest to
isolate gene from goosegrass, Eleusine indica. eiMT1 cDNA was
cloned into pBI121 binary vector for the transformation into tobacco by Agrobacterium
tumefaciens. Screening of transgenic plants was conducted using three
specially designed primers, CMTF and CMTR and 35SF and PMTR, and a
pair of gene specific primers, MTFS2 and MTRS2. The
primer pairs used successfully produced expected DNA fragments
of 270 bp, 1.1 kb and 170 bp, respectively. Sequencing and sequence analysis of
the 170 bp fragment was 100 % identical to the eiMT1. Gene expression study via
reverse transcription-polymerase chain reaction approach showed that the
transgene was successfully expressed in 11 of the 19 transgenic plants.
Keywords:
eiMT1; Eleusine
indica; metallothionein; transformation; tobacco
RUJUKAN
Alonso, J. M.,
Stepanova, A.N., Leisse, T.Kim, C.Chen, H., Shinn, P., Stevenson, D.K.,
Zimmerman, Barajas, P. & Cheuk, R. 2003. Genome-wide insertional
mutagenesis of Arabidopsis thaliana. Science 301: 653-657.
Choi,
D., Kim, H.M., Yun, H.K., Park, A., Kim, W.T. & Bok, S.H. 1996. Molecular
cloning of a metallothionein-like gene from Nicotiana glutinosa L. and
its induction by wounding and tobacco mosaic virus infection. Plant Physiol. 112: 353-359.
Cobbett,
C. & Goldsbroug, P. 2002. Phytochelatins and metallothioneins: roles in
heavy metal detoxification and homeostasis. Annu. Rev. Plant Bio. 53:
159-182.
Day,
C.D., Lee, E., Kobayashi, J., Holappa, L.D., Albert, H. &
Ow, D.W. 2000. Transgene integration into the same chromosome location can
produce alleles that express at a predictable level, or alleles that are
differentially silenced. Genes & Development 14: 2869-2880.
Doyle, J. &
Doyle, L. 1987. A rapid DNA isolation procedure for small quantities of fresh
leaf tissue. Phytochem. Bull. 19: 11-15.
Eapen,
S. & D’Souza, S.F. 2005. Prospects of genetic engineering of plants for
phytoremediation of toxic metals. Biotechnol. Adv. 23: 97-114.
Eapen,
S., Singh, S. & S.F. D’Souza, S.F. 2007. Advances in development of
transgenic plants for remediation of xenobiotic pollutants. Biotechnol. Adv. 25: 442-451.
Fabrik,
I., Ruferova, Z., Hilscherova, K., Adam, V., Trnkova, L. & Kizek, R. 2008.
A determination of metallothionein in larvae of freshwater midges (Chironomus
riparius) using Brdicka reaction. Sensors. 8: 4081-4094.
Gehring,
M. & Henikoff, S. 2007. DNA methylation dynamics in plant genomes. Biochim.
Biophys. Acta 1769: 276-286.
Gelvin,
S.B. & Kim, S.I. 2007. Effect of chromation upon Agrobacterium T-DNA
integration and transgene expression. Biochim. Biophys. Acta 1769: 410-421.
Hassinen,
V.H., Tervahauta, A.I., Halimaa, P., Plessl, M., Peraniemi, S., Schat, H.,
Aarts, M.G., Servomaa, K. & Karenlampi, S. 2007. Isolation of Zn responsive
genes from two accessions of the hyperaccumulator plant Thaspi
caerulescens. Planta 225: 977-989.
Hsieh, J.L.,
Chen, C.Y., Chiu, M.H., Chein, M.F., Chang, S., Endo, G. & Huang, C.C.
2009. Expressing a bacterial mercuric ion binding protein in plant for
phytoremediation of heavy metals. J. Hazard. Mat. 161:
920-925.
Hyun,
P., In-Young, A., Heeseon, J.C., Sei, H.P. & Hye, E.L.
2006. Cloning, expression and characterizationof metallothionein from the
Antarctic clam Laternula elliptica. Prot. Expres. Purif. 52:
82-88.
Lam,
A.L., Pazin, D.E. & Sullivan, B.A. 2005. Control of gene expression and
assembly of chromosomal subdomains by chromatin regulators with antagonistic
functions. Chromosoma 114: 242-251.
Ma, M.,
Lau, P.S., Jia,
Y.T., Tsang, W.K., Samuel, L.K.S. Nora, T.F.Y. & Wong, Y.S. 2003. The
isolation and characterization of Type 1 metallothionein (MT) cDNA from a
heavy-metal-tolerant plant, Festuca rubra cv. Merlin. Plant Sci. 164:
51-60.
Macek,
T., Kotrba, P., Svatos, A., Novakova, M., Demnerova, K. & Mackova, M. 2007.
Novel roles for genetically modified plants in environmental protection. Trends
Biotech. 26:
146-152.
Macek,
T., Macková, M., Pavlíková, D., Száková, J., Truksa, M., Singh Cundy, A.,
Kotrba, P., Yancey, N. & d Scouten, W.H. 2002. Accumulation of Cadmium by
Transgenic Tobacco. Acta Biotech. 22: 101-106.
McCafferty,
H.R.K., Moore, P.H. & Zhu, Y.2008. Papaya transformed with the Galanthus
nivalis GNA gene produces a biologically active lectin with spider mite
control activity. Plant Sci. 3: 385-393.
Meyer,
P. 1995. Understanding and controlling transgene expression. Trends
Biotechnol.
13: 332-337.
Nik
Marzuki, S., Shaiful, A.S., Chin, I.S., Belinda, M.Y.S., Mushrifah, I., Shahrul
Hisham, Z.A. & Shahidan, S. 2006. Molecular cloning and sequencing of
metallothionein gene from Eleusine Indica. Malays.
Appl. Biol. 35:
71-74.
Oller, A.L.W.,
Agostini, E., Talano, M.A., Capozucca, C., Milrad, S.R., Tigier, H.A. &
Medina, M.I. 2005. Overexpression of a basic peroxidase in transgenic tomato (Lycopersicon
esculentum Mill. cv. Pera) hairy roots increases phytoremediation of
phenol. Plant Sci. 169: 1102-1111.
Pilon-Smits,
E. 2005. Phytoremediation. Annu. Rev. Plant Biol. 56: 15-39.
Sridevi,
G., Parameswari, C., Sabapathi, N., Raghupathy, V. & Veluthambi. K. 2008.
Combined expression of chitinase and β-1,3-glucanase genes in indica rice
(Oryza sativa L.) enhances resistance against Rhizoctonia solani.
Plant Sci. 175: 283-290.
Suzuki,
M.M. & Bird, A. 2008. DNA methylation landscapes: provocative insights from
epigenomics. Nat. Rev. Gen. 9(6): 465-76.
Vasâk,
M. 2005. Advances in metallothionein structure and functions. J. Trace
Elem. Med.
Biol. 19:
13-17.
Watt,
D.A. 2003. Aluminium-responsive genes in sugarcane: identification and analysis
of expression under oxidative stress. J. Exp. Bot. 54: 1163-1174.
Wong,
M.H. & Lau, W.M. 1985. Root growth of Cynodon dactylon and Eleusine
indica collected from motorways at different concentrations of lead. Environ.
Res. 36(2): 257-267.
Zhang,
X. 2008. The epigenetic landscape of plants. Science 320: 489-492.
*Pengarang
untuk surat-menyurat; email: nms@ukm.my
|