Sains Malaysiana 47(7)(2018): 1465–1471

http://dx.doi.org/10.17576/jsm-2018-4707-14

Transformasi Gen Proteolisis 6 (PRT6) Berperantarakan Agrobacterium tumefaciens ke dalam Kotiledon Tomato kultivar Micro Tom

(Agrobacterium tumefaciens Mediated Transformation of the Proteolysis 6 (PRT6) Gene
into Cotyledons of Tomato cv. Micro Tom)

INTAN ELYA SUKA¹, NUR FARHANA ROSLAN¹, BEE LYNN CHEW², HOE HAN GOH³,

ZAMRI ZAINAL^1,3 & NURULHIKMA MD ISA^1*

¹Pusat Pengajian Biosains dan Bioteknologi, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

²Pusat Pengajian Sains Kajihayat, Universiti Sains Malaysia, Minden, 11800 Georgetown, Penang, Malaysia

³Institut Biologi Sistem, Universiti Kebangsaan Malaysia, 43600 UKM Bangi Selangor Darul Ehsan, Malaysia

Received: 15 September 2017/Accepted: 7 March 2018

ABSTRAK

Gen Proteolisis 6 (PRT6) merupakan gen yang memainkan peranan penting dalam tapak jalan N-end rule dan berfungsi sebagai enzim E3 ligase. PRT6 berperanan dalam pengenalan protein sasaran bagi proses degradasi. Objektif utama kajian ini adalah untuk mentransformasi konstruk RNAi PRT6 ke dalam tomato berperantarakan Agrobacterium tumefaciens. Ini bertujuan untuk memahami peranan tapak jalan N-end rule semasa proses pemasakan buah. Beberapa faktor yang memberi kesan kepada transformasi seperti masa ko-penanaman dan juga kepekatan antibiotik yang digunakan telah dioptimumkan. Keputusan kajian menunjukkan pengeraman kotiledon selama 48 jam pada medium ko-penanaman dapat meningkatkan penghasilan kalus sebanyak 61% manakala penggunaan 500 mg/L antibiotik karbenisilin dalam medium regenerasi pucuk dapat mengurangkan kontaminasi A. tumefaciens sehingga 5.2%. Selain itu, strain A. tumefaciens C58 merupakan strain A. tumefaciens yang paling sesuai digunakan sebagai perantara dalam kajian ini. Tindak balas berantai polimerase (PCR) telah dijalankan pada pucuk yang terhasil untuk mengesahkan integrasi fragmen PRT6 ke dalam genom tomato. Berdasarkan analisis PCR, kesemua tujuh pucuk putatif transgenik adalah merupakan transforman positif.

Kata kunci: Antibiotik; A. tumefaciens; kalus; ko-penanaman; Proteolisis 6

ABSTRACT

Proteolysis 6 (PRT6) gene plays an important role in the N-end rule pathway which functions as an E3 ligase enzyme. PRT6 functions to recognise target proteins for degradation. The main objective of this study is to transform the PRT6 RNAi construct through Agrobacterium tumefaciens into tomato. The purpose of this study was to understand the role of the N-end rule pathway during fruit ripening. Several factors affecting transformation efficiency such as co-cultivation time and concentration of antibiotics were optimised. The results from this study showed that pre-cultured cotyledons incubated for 48 h in co-cultivation medium increased the callus formation to 61% while using 500 mg/L carbenicillin antibiotic in the shoot regeneration medium reduced the contamination of A. tumefaciens to 5.2%. Besides, A. tumefaciens strain C58 was shown to be the most suitable A. tumefaciens strain to be used in this study. Polymerase Chain Reaction (PCR) was performed on the regenerated shoots to confirm integration of the PRT6 fragment into the tomato genome. Based on the PCR analysis, all putative transgenic shoots were positive transformants.

Keywords: Antibiotic; A. tumefaciens; callus; co-cultivation; Proteolysis 6

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