Sains Malaysiana 38(3): 287-294(2009)

Identification and Characterisation of a Copper-inducible Metallothionein

Gene from Cockle, Anadara granosa

(Pengenalpastian dan Pencirian Gen Metalotionin yang Diaruh oleh Logam

Kuprum dalam Kerang, Anadara granosa)  

Kok-Kee Wong¹, Noor-Arniwati Mat-Daud², Roohaida Othman², Zubir Din³, Kiew-Lian Wan² & Salmijah Surif²*

¹School of Environmental & Natural Resource Sciences

Faculty of Science & Technology, Universiti Kebangsaan Malaysia

43600 UKM Bangi, Selangor DE, Malaysia

²School of Biosciences & Biotechnology, Faculty of Science & Technology

Universiti Kebangsaan Malaysia, 43600 UKM Bangi

Selangor DE, Malaysia

³Centre for Estuarine & Coastal Studies, Universiti Sains Malaysia

11800 Minden, Penang, Malaysia

Diserahkan: 1 Julai 2008 / Diterima: 20 Oktober 2008

ABSTRACT

The cockle, Anadara granosa, was experimentally exposed to low (0.1 mg/L) and sublethal (1.0 mg/L) doses of copper (Cu) for a period of 24 hrs. Significant increase in Cu concentrations in whole tissues and hepatopancreas compared to control animals were observed. In order to study the effect of copper exposure at molecular levels, a subtractive cDNA library was constructed from the hepatopancreas of cockles exposed to 1.0 mg/L Cu. Screening of the subtractive cDNA library using reverse northern analysis resulted in several differentially expressed genes, including one that codes for metallothionein (MT). The complete coding sequence of the MT gene (designated as AnaMT2) reveals an open reading frame of 234 bp in length that encodes a 77 amino acid polypeptide as revealed by the deduced amino acid composition. Although showing similarities with other molluscan MTs, AnaMT2 can be distinguished by its lower glycine and higher asparagine and proline content. Expression analysis of the AnaMT2 by northern analysis indicated higher mRNA level in cockle exposed to 1.0 mg/L Cu and was undetectable in those treated with 0.1 mg/L. This suggests that AnaMT2 represents a primarily inducible MT not highly expressed under basal conditions.  

Keywords:   Cockle copper; metallothionein; subtractive hybridisation

ABSTRAK

Kerang, Anadara granosa didedahkan kepada logam kuprum (Cu) pada dos rendah (0.1 mg/L) dan dos sub-maut (1.0 mg/L) untuk tempoh 24 jam. Peningkatan aras Cu yang signifikan diperhatikan pada kedua-dua tisu keseluruhan kerang serta tisu hepa-opankreas yang terdedah kepada Cu berbanding sampel kawalan. Untuk mengkaji kesan Cu pada aras molekul, satu perpustakaan cDNA subtraktif telah dibina menggunakan tisu hepatopankreas kerang yang didedahkan kepada 1.0 mg/L Cu. Penyaringan perpustakaan cDNA subtraktif ini, menggunakan pemblotan northern berbalik, menghasilkan beberapa gen yang diekspres secara berbeza berbanding kawalan, termasuklah gen yang mengkodkan metalotionin (MT). Gen MT jujukan penuh (AnaMT2) ini mempunyai jujukan rangka terbuka ORF bersaiz 234 pb dan mengekodkan 77 asid amino polipeptida melalui komposisi asid amino yang dideduksi. Walaupun terdapat persamaan dengan MT daripada kumpulan moluska, AnaMT2 ini dapat dibezakan daripada yang lain daripada segi kandungan asparagina dan prolina yang secara relatifnya lebih tinggi dan glisina yang lebih rendah. Kajian pengekspresan gen AnaMT2 menggunakan teknik pemblotan northern menunjukkan aras pengekspresan mRNA agak tinggi dalam tisu terdedah kepada 1.0 mg/L Cu dan tidak dapat dikesan dalam sampel yang didedahkan kepada 0.1 mg/L Cu. Ini mencadangkan bahawa AnaMT2 adalah MT yang diinduksi logam dan kurang diekspres  dalam keadaan biasa.

Kata kunci: Kerang; kuprum; metalotionin; penghibridan subtraktif

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*Pengarang untuk surat-menyurat; email: salmij@ukm.my