Sains Malaysiana 47(12)(2018): 2975–2983

http://dx.doi.org/10.17576/jsm-2018-4712-06

 

Optimization Method for Proteomic Analysis of the Larva and Adult Tissues of Plutella xylostella (L.) (Lepidoptera: Plutellidae)

(Pengoptimuman Kaedah Proteomik bagi Tisu Larva dan Dewasa Plutella xylostella (L.) (Lepidoptera: Plutellidae))

 

MAIZOM HASSAN¹*, NORAZILA YUSOFF¹, WAN MOHD AIZAT¹, NURUL WAHIDA OTHMAN² & IDRIS ABD GHANI²

 

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

 

²School of Environmental and Natural Resources Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

 

Diserahkan: 30 Mei 2018/Diterima: 19 September 2018

 

ABSTRACT

Plutella xylostella (L.) (Lepidoptera: Plutellidae), the major insect pest of cruciferous crops worldwide shows significant resistance to almost all classes of insecticides. In order to effectively prevent and manage the insecticidal resistance, it is crucial to understand the physiological adaptation of insects against insecticides. Identification of insect protein that interacting with insecticides and characterization of their modification in resistant strains can be done by using differential proteomics approach. This study focuses on optimizing a sensitive and rapid method for the extraction of high quality protein of both larva and adult tissues of P. xylostella to be used in two-dimensional gel electrophoresis. Five extraction methods were evaluated for protein concentration, yields and resolving patterns of one-dimensional and two-dimensional electrophoresis. The results showed that trichloroacetic acid/acetone extraction methods with two different concentrations of 2-mercaptoethanol produced the highest protein concentration and yield for both adult and larva tissues, respectively. Meanwhile, trichloroacetic acid/acetone with dithiothreitol extraction method gave better separation of spots and intensity for both larva and adult tissues compared to other methods tested. As such, we concluded that trichloroacetic acid/acetone with dithiothreitol successfully yielded high total protein concentration and good separation of two-dimensional electrophoresis gel spots in both adult and larva P. xylostella.

 

Keywords: Optimization; Plutella xylostella (L.); protein extraction; proteomics; two-dimensional gel electrophoresis

ABSTRAK

Plutella xylostella (L.) (Lepidoptera: Plutellidae), serangga perosak utama tanaman krusifer di seluruh dunia telah menunjukkan kerintangan yang ketara terhadap hampir semua kelas racun serangga. Pemahaman mengenai penyesuaian fisiologi serangga terhadap racun serangga adalah penting bagi pengurusan yang efektif dan pencegahan kerintangan terhadap racun serangga. Pengenalpastian protein serangga yang berinteraksi dengan racun serangga dan pencirian pengubahsuaian protein serangga yang rintang dapat dilakukan dengan menggunakan pendekatan proteomik pembezaan. Kajian ini memberi tumpuan kepada pengoptimuman kaedah yang lebih sensitif dan cepat untuk pengekstrakan protein berkualiti tinggi daripada kedua-dua tisu larva dan dewasa P. xylostella untuk digunakan dalam elektroforesis gel dua dimensi. Lima kaedah pengekstrakan telah dinilai berdasarkan kepekatan protein, jumlah protein dan corak gel elektroforesis satu dimensi dan dua dimensi. Keputusan menunjukkan bahawa kaedah pengekstrakan asid trikloroasetik/aseton dengan dua kepekatan 2-merkaptoetanol yang berbeza menghasilkan kepekatan dan jumlah protein paling tinggi bagi kedua-dua tisu larva dan dewasa. Sementara itu, kaedah pengekstrakan asid trikloroasetik/aseton dengan dithiothreitol memberikan pemisahan titik protein dan keamatan yang lebih baik bagi kedua-dua tisu larva dan dewasa berbanding kaedah lain. Oleh itu, dapat disimpulkan bahawa pengekstrakan menggunakan kaedah asid trikloroasetik/aseton dengan dithiothreitol berjaya menghasilkan kepekatan jumlah protein yang tinggi dan pemisahan protein yang baik untuk gel elektroforesis dua dimensi bagi kedua-dua tisu dewasa dan larva P. xylostella.

 

Kata kunci: Elektroforesis gel dua dimensi; pengekstrakan protein; pengoptimuman; Plutella xylostella (L.); proteomic

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