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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 Received: 30 May 2018/Accepted: 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;
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