Sains Malaysiana 47(12)(2018): 2961–2968

http://dx.doi.org/10.17576/jsm-2018-4712-04

 

PEG-4000 Increased the Mating Efficiency of Yeast-Two Hybrid Screening Process using PmF-box1 as Bait

(PEG-4000 Meningkatkan Kecekapan Pengawanan bagi Proses Penyaringan Yis-Dua Hibrid menggunakan PmF-box1 sebagai Umpan)

 

NUR ATHIRAH ABD HAMID¹ & ISMANIZAN ISMAIL²*

 

¹Institute of Systems Biology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

 

²Centre of Biotechnology and Functional Food, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

 

Diserahkan: 30 Mei 2018/Diterima: 19 September 2018

 

ABSTRACT

Protein degradation can occur through Ubiquitin 26S-Proteosome System (UPS). The degradation can be mediated by the SCF E3 ubiquitin ligase complex consisting of Skp1, Cullin, and F-box protein as the main components. The F-box protein at the C-terminal domain functions to recognize the targeted protein to be ubiquitinated and degraded via UPS. A stress-responsive F-box gene, PmF-box1 from Persicaria minor was categorized in the F-box containing kelch repeat (FBK) family; a family that specific to plant kingdom. To identify the targeted protein of PmF-box1, yeast-two hybrid system (Y2H) was used. In the Y2H screening process, mating efficiency is very important to fish out the interacting proteins. Therefore, one modification was conducted to increase the mating efficiency. In this screening, PmF-box1 was used as a bait to screen for the Y2H library which was constructed using RNA from plant samples treated with abscisic acid (ABA) and polyethylene glycol (PEG)-8000 and control sample. Autoactivation and toxicity tests of bait were performed before the Y2H screening. Tests on PmF-box1 showed that it is not toxic to the yeast and cannot autoactivate the yeast reporter genes. Mating efficiency was improved from 2.07% to 9.15% after addition of PEG-4000 in the mating culture compared to the original protocol, which it also increased the colony number in the screening step afterward. Additionally, bands of gene with different sizes were observed on electrophoresis gel after colony PCR analysis from the improved technique. Those genes may code for potential interacting proteins that needs further identification and confirmation.

 

Keywords: PmF-box1; polyethylene glycol-4000; yeast-two hybrid

 

ABSTRAK

Degradasi protein boleh berlaku melalui Sistem Ubikuitin 26S-Proteasome (UPS). Degradasi tersebut boleh berlaku berperantarakan kompleks SCF E3 ubikuitin ligase yang mengandungi Skp1, Cullin dan protein F-box sebagai komponen utama. Protein F-box pada domain C-terminal berfungsi untuk mengenal pasti protein yang disasarkan untuk diubikuitinasi dan didegradasi melalui UPS. Salah satu gen F-box yang bergerak balas terhadap tekanan, PmF-box1 daripada Persicaria minor telah dikategorikan dalam famili F-box yang mengandungi ulangan kelch (FBK); merupakan famili yang khusus kepada alam tumbuhan. Untuk mengenal pasti protein yang disasarkan oleh PmF-box1, sistem yis-dua hibrid (Y2H) telah digunakan. Dalam proses penyaringan Y2H, kecekapan pengawanan adalah sangat penting untuk mencari protein-protein yang berinteraksi. Oleh itu, satu pengubahsuaian telah dijalankan untuk meningkatkan kecekapan pengawanan. Dalam penyaringan ini, PmF-box1 telah digunakan sebagai umpan untuk menyaring perpustakaan Y2H yang telah dibina menggunakan RNA daripada sampel tumbuhan yang dirawat dengan asid absisik (ABA) dan polietilena glikol (PEG)-8000 dan sampel kawalan. Ujian autopengaktifan dan ketoksikan umpan telah dijalankan sebelum penyaringan Y2H. Ujian tersebut menunjukkan bahawa PmF-box1 tidak toksik terhadap yis dan tidak dapat mengautoaktifkan gen-gen pelapor di dalam yis. Kecekapan pengawanan dapat ditingkatkan daripada 2.07% kepada 9.15% selepas PEG-4000 ditambahkan ke dalam kultur pengawanan berbanding dengan protokol asal dan ia juga telah meningkatkan bilangan koloni dalam langkah penyaringan selepas itu. Tambahan lagi, jalur-jalur gen dengan saiz yang berbeza telah diperhatikan di atas gel elektroforesis setelah analisis PCR koloni daripada teknik yang telah ditambah baik. Gen-gen tersebut mungkin mengekodkan protein-protein berinteraksi yang berpotensi yang memerlukan pengenalpastian dan pengesahan selanjutnya.

 

Kata kunci: PmF-box1; polietilena glikol-4000; yis-dua hybrid

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