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

Received: 16 June 2009 / Accepted: 5 May 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

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*Corresponding author; email: nms@ukm.my