 |
 |
 |
 |
 |
 |
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
Received: 30 May 2018/Accepted: 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
REFERENCES
Albers, M., Kogl, M.,
Kober, I. & Loser, E. 2002. Method of Stimulating the Mating of
Microorganisms in Liquid Medium. Google Patents.
Arabidopsis Interactome
Mapping Consortium. 2011. Evidence for network evolution in an Arabidopsis interactome
map. Science 333(6042): 601-607.
Auerbach, D. &
Stagljar, I. 2005. Yeast two-hybrid protein-protein interaction networks. In Proteomics
and Protein- Protein Interactions: Biology, Chemistry, Bioinformatics, and Drug
Design, edited by Waksman, G. New York, USA: Springer. pp. 19-31.
Baharum, S.N., Bunawan,
H., Ghani, M.A., Mustapha, W.A. & Noor, N.M. 2010. Analysis of the chemical
composition of the essential oil of Polygonum minus Huds. using
two-dimensional gas chromatography-time-of-flight mass spectrometry (GC-TOF
MS). Molecules 15(10): 7006-7015.
Cao, S. & Yan, L.
2013. Construction of a high-quality yeast two-hybrid (Y2H) library and its
application in identification of interacting proteins with key vernalization
regulator TaVRN-A1 in wheat. BMC Research Notes 6: 81.
Chen, R., Guo, W., Yin,
Y. & Gong, Z.H. 2014. A novel F-box protein CaF-box is involved in
responses to plant hormones and abiotic stress in pepper (Capsicum annuum L.). International Journal of Molecular Sciences 15(2): 2413-2430.
Chen, Y., Xu, Y., Luo,
W., Li, W., Chen, N., Zhang, D. & Chong, K. 2013. The F-box protein OsFBK12
targets OsSAMS1 for degradation and affects pleiotropic phenotypes, including
leaf senescence, in rice. Plant Physiology 163(4): 1673-1685.
Dharmasiri, N.,
Dharmasiri, S., Weijers, D., Lechner, E., Yamada, M., Hobbie, L., Ehrismann, J.
S., Jurgens, G. & Estelle, M. 2005. Plant development is regulated by a
family of auxin receptor F box proteins. Development Cell 9(1): 109-119.
Endo, M., Takesako, K.,
Kato, I. & Yamaguchi, H. 1997. Fungicidal action of aureobasidin A, a
cyclic depsipeptide antifungal antibiotic, against Saccharomyces cerevisiae. Antimicrobial Agents and Chemotherapy 41(3): 672-676.
Fields, S. & Song, O.K. 1989. A novel
genetic system to detect protein-protein interactions. Nature 340:
245-246.
Gagne, J.M., Downes, B.P., Shiu, S.H.,
Durski, A.M. & Vierstra, R.D. 2002. The F-box subunit of the SCF E3 complex
is encoded by a diverse superfamily of genes in Arabidopsis. Proceedings
of the National Academy of Science U S A 99(17): 11519-11524.
Gor, M.C., Ismail, I., Mustapha, W.A.W.,
Zainal, Z., Noor, N.M., Othman, R. & Mohamed-Hussein, Z.A. 2010.
Identification of cDNAs for jasmonic acid-responsive genes in Polygonum
minus roots by suppression subtractive hybridization. Acta Physiologiae
Plantarum 33(2): 283-294.
Hackbusch, J., Richter, K., Muller, J.,
Salamini, F. & Uhrig, J.F. 2005. A central role of Arabidopsis thaliana ovate
family proteins in networking and subcellular localization of 3-aa loop
extension homeodomain proteins. Proceedings of the National Academy of
Science USA 102(13): 4908-4912.
Iantcheva, A., Boycheva, I., Vassileva,
V., Revalska, M. & Zechirov, G. 2015. Cyclin-like F-box protein plays a
role in growth and development of the three model species Medicago
truncatula, Lotus japonicus, and Arabidopsis thaliana. Research
and Reports in Biology 2015: 117-130.
Kipreos, E.T. & Pagano, M. 2000. The
F-box protein family. Genome Biology 1(5): reviews/3002.3001.
Lacount, D.J., Vignali, M., Chettier, R.,
Phansalkar, A., Bell, R., Hesselberth, J.R., Schoenfeld, L.W., Ota, I.,
Sahasrabudhe, S., Kurschner, C., Fields, S. & Hughes, R.E. 2005. A protein
interaction network of the malaria parasite Plasmodium falciparum. Nature 438(7064): 103-107.
Li, Y., Liu, Z., Wang, J., Li, X. &
Yang, Y. 2016. The Arabidopsis kelch repeat F-box E3 ligase ARKP1 plays
a positive role for the regulation of abscisic acid signaling. Plant
Molecular Biology Reporter 34(3): 582-591.
Lin, J.S. & Lai, E.M. 2017.
Protein-protein interactions: Yeast two-hybrid system. In Bacterial Protein
Secretion Systems: Methods and Protocols, edited by Journet, L. &
Cascales, E. New York: Springer Nature. pp. 177-187.
López-Gómez, R. & Gómez-Lim, M.A.
1992. A method for extracting intact RNA from fruits rich in polysaccharides
using ripe mango mesocarp. HortScience 27(5): 440-442.
Mazzucotelli, E., Belloni, S., Marone,
D., Leonardis, A.M.D., Guerra, D., Fonzo, N.D., Cattivelli, L. &
Mastrangelo, A.M. 2006. The E3 ubiquitin ligase gene family in plants:
Regulation by degradation. Current Genomics 7: 509-522.
Othman, M.H.C., Hadi, N.A., Zainal, Z.,
Kiat, C.J., Naeem-Ul- Hassan, M., Zain, C.R.C.M. & Ismail, I. 2017.
Expression profile of gene encoding Kelch repeat containing F-box protein
(PmF-box1) in relation to the production of green leaf volatiles. Australian
Journal of Crop Science 11(04): 406-418.
Rain, J.C., Selig, L., Reuse, H.D.,
Battaglia, V., Reverdy, C., Simon, S., Lenzen, G., Petel, F., Wojcik, J.,
Schächter, V., Chemama, Y., Labigne, A. & Legrain, P. 2001. The
protein-protein interaction map of Helicobacter pylori. Nature 409:
211-215.
Schumann, N., Navarro-Quezada, A.,
Ullrich, K., Kuhl, C. & Quint, M. 2011. Molecular evolution and selection
patterns of plant F-box proteins with C-terminal kelch repeats. Plant
Physiology 155(2): 835-850.
Shen, H., Luong, P. & Huq, E. 2007.
The F-box protein MAX2 functions as a positive regulator of photomorphogenesis
in Arabidopsis. Plant Physiology 145(4): 1471-1483.
Stefanowicz, K., Lannoo, N. & Van
Damme, E.J.M. 2015. Plant F-box proteins-judges between life and death. Critical
Reviews in Plant Sciences 34(6): 523-552.
Takesako, K., Ikai, K., Haruna, F., Endo,
M., Shimanaka, K., Sono, E., Nakamura, T., Kato, I. & Yamaguchi, H. 1991.
Aureobasidins, new antifungal antibiotics. Taxonomy, fermentation, isolation
and properties. The Journal of Antibiotics (Tokyo) 44(9): 919-924.
Takesako, K., Kuroda, H., Inoue, T.,
Haruna, F., Yoshikawa, Y., Kato, I., Uchida, K., Hiratani, T. & Yamaguchi,
H. 1993. Biological properties of aureobasidin A, a cyclic depsipeptide
antifungal antibiotic. The Journal of Antibiotics (Tokyo) 46(9):
1414-1420.
Uetz, P., Giot, L., Cagney, G.,
Mansfield, T.A., Judson, R.S., Knight, J.R., Lockshon, D., Narayan, V.,
Srinivasan, M., Pochart, P., Qureshi-Emili, A., Li, Y., Godwin, B., Conover,
D., Kalbfleisch, T., Vijayadamodar, G., Yang, M., Johnston, M. & Fields, S.
2000. A comprehensive analysis of protein-protein interactions in Saccharomyces
cerevisiae. Nature 403: 623-627.
Walhout, A.J.M., Sordella, R., Lu, X.,
Hartley, J.L., Temple, G.F., Brasch, M.A., Thierry-Mieg, N. & Vidal, M.
2000. Protein interaction mapping in C. elegans using proteins involved
in vulval development. Science 287(5450): 116-122.
Wang, W., Liu, G., Niu, H., Timko, M.P.
& Zhang, H. 2014. The F-box protein COI1 functions upstream of MYB305 to
regulate primary carbohydrate metabolism in tobacco (Nicotiana tabacum L.
cv. TN90). Journal of Experimental Botany 65(8): 2147-2160.
Wang, Z., Dai, L., Jiang, Z., Peng, W.,
Zhang, L., Wang, G. & Xie, D. 2005. GmCOI1, a soybean F-Box protein gene,
shows ability to mediate jasmonate-regulated plant defense and fertility in Arabidopsis. Molecular Plant-Microbe Interactions 18(12): 1285-1295.
Yan, Y.S., Chen, X.Y., Yang, K., Sun,
Z.X., Fu, Y.P., Zhang, Y.M. & Fang, R.X. 2011. Overexpression of an F-box
protein gene reduces abiotic stress tolerance and promotes root growth in rice. Molecular Plant 4(1): 190-197.
Zhang, X., Gou, M., Guo, C., Yang, H.
& Liu, C.J. 2015. Down-regulation of Kelch domain-containing F-box protein
in Arabidopsis enhances the production of (poly)phenols and tolerance to
ultraviolet radiation. Plant Physiology 167(2): 337-350.
Zhang, X., Gou, M. & Liu, C.J. 2013.
Arabidopsis Kelch repeat F-box proteins regulate phenylpropanoid biosynthesis
via controlling the turnover of phenylalanine ammonia-lyase. Plant Cell 25(12):
4994-5010.
*Corresponding author; email: maniz@ukm.edu.my
|
|||
|
