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Sains Malaysiana 52(4)(2023):
http://doi.org/10.17576/jsm-2023-5204-08
Functional
Characterization of a Novel Synthetic Herbicide Resistance Gene in a Model
Plant
(Pencirian
Fungsian Gen Rintangan Herbisid Sintetik Novel dalam Model Tumbuhan)
SARWAT1, SHEHLA
SHOUKAT1,2, MUHAMMAD AMIR ZIA2 & SHAUKAT ALI2,*
1PARC Institute of Advanced Studies
in Agriculture, Islamabad, Pakistan
2National Institute for Genomics and
Advanced Biotechnology, P.O. Box 45000, National Agricultural Research Centre, Islamabad, Pakistan
Received:
4 April 2022/Accepted: 17 March 2023
Abstract
As crop losses can occur owing to
the abrupt growth of uncontrollable weeds in the field, research is currently
being conducted in Pakistan to eradicate herbs. To carry out our proposed
research domain, we have designed a novel synthetically modified EPSPS gene
that has a potent role in herbicide-resistance development. The novel
codon-optimized synthesized mEPSPS sequence was inserted into the pXCSG-mYFP
plant expression vector. Results of colony PCR (1400 bp) confirmed the
integration of genes into bacteria. For functional validation of
pXCSG-mYFP-EPSPS, transient expression in tobacco (Nicotiana benthamiana)
in comparison with PBSF-16 was done. Benth infiltration results showed that
transient expression was successfully confirmed through ELISA and western blot
analysis via anti-YFP antibody in tobacco leaves. For further validation of
transient expression, the stable transformation results of the pXCSG-mYFP-EPSPS
vector showed that successful transformation was done via two days of
co-cultivation followed by selection and regeneration of transformed tobacco
plants. The regenerated tobacco plants were then confirmed through
gene-specific-based PCR. After PCR-based confirmation, western blot further
validates the 26 kDa anti-YFP antibodies expression in transformed tobacco
plants. Another important finding of the study was the bar and PAT gene
real-time expression elucidating that the bar gene was 4.9-fold more expressive
under the 35S promoter than the PAT gene under the nos promoter.
Keywords: EPSPS gene; functional
characterization; molecular analysis; tobacco; transformation
Abstrak
Memandangkan kepentingan
penyelidikan semasa di Pakistan adalah untuk menghapuskan herba kerana
kehilangan hasil boleh berlaku disebabkan oleh pertumbuhan mendadak rumpai yang
tidak terkawal dalam amalan lapangan. Untuk melaksanakan domain penyelidikan
kami yang dicadangkan, kami telah mereka bentuk gen EPSPS yang diubah suai
secara sintetik novel yang mempunyai peranan yang kuat dalam pembangunan tahan
herbisid. Urutan mEPSPS tersintesis yang dioptimumkan kodon novel telah
dimasukkan ke dalam vektor ekspresi tumbuhan pXCSG-mYFP. Keputusan koloni PCR
(1400 bp) mengesahkan integrasi gen ke dalam bakteria. Untuk pengesahan fungsi
pXCSG-mYFP-EPSPS ekspresi sementara dalam tembakau (Nicotiana benthamiana)
berbanding dengan PBSF-16 telah dilakukan. Keputusan penyusupan Benth
mendedahkan bahawa ekspresi sementara telah berjaya dilakukan melalui
pengesahan ELISA dan analisis pemblotan western melalui antibodi anti-YFP dalam
daun tembakau. Untuk pengesahan lanjut bagi ekspresi sementara, keputusan
transformasi stabil bagi vektor pXCSG-mYFP-EPSPS menunjukkan bahawa
transformasi yang berjaya dilakukan melalui penanaman bersama selama dua hari
diikuti dengan pemilihan dan penjanaan semula tumbuhan tembakau yang telah
diubah. Tumbuhan tembakau yang dijana semula kemudiannya disahkan melalui PCR
berasaskan gen khusus. Selepas pengesahan berasaskan PCR, pemblotan western
mengesahkan lagi ekspresi antibodi anti-YFP 26 kDa dalam loji tembakau yang diubah.
Satu lagi penemuan penting dalam kajian ini ialah ekspresi masa nyata gen bar
dan PAT yang menjelaskan bahawa gen bar adalah 4.9 kali ganda lebih ekspresif
di bawah promoter 35S berbanding gen PAT di bawah promoter nos.
Kata kunci: Analisis molekul;
EPSPS; pencirian fungsi; tembakau; transformasi
REFERENCES
Chitty, J.A., Allen,
R.S. & Larkin, P.J. 2006. Opium poppy (Papaver somniferum). In Agrobacterium Protocols Volume 2. Methods in Molecular Biology. Vol 344, edited by
Wang, K. Humana Press. https://doi.org/10.1385/1-59745-131-2:383
Chou, T.C. & Moyle,
R.L. 2014. Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. BMC Plant Biol. 14: 92. https://doi.org/10.1186/1471-2229-14-92
Conze, T., Shetye, A.,
Tanaka, Y., Gu, J., Larsson, C., Göransson, J., Tavoosidana, G., Söderberg, O.,
Nilsson, M. & Landegren, U. 2009. Analysis of genes, transcripts, and
proteins via DNA ligation. Annu. Rev. Anal. Chem. Palo Alto Calif. 2:
215-239.
Deb, C.R. & Imchen,
T. 2010. An efficient in vitro hardening of tissue culture raised
plants. Biotechnology 9: 79-83.
Délye, C., Jasieniuk,
M. & Le Corre, V. 2013. Deciphering the evolution of herbicide resistance
in weeds. Trends in Genetics 29(11): 649-658. https://doi.org/10.1016/j.tig.2013.06.001
Fartyal, D., Agarwal,
A., James, D., Borphukan, B., Ram, B., Sheri, V., Agrawal, P.K., Achary, V.
& Reddy, M.K. 2018. Developing dual herbicide tolerant transgenic rice
plants for sustainable weed management. Scientific Reports 8(1): 11598. https://doi.org/10.1038/s41598-018-29554-9
Feys,
B.J., Wiermer, M., Bhat, R.A., Moisan, L.J., Medina-Escobar, N., Neu, C. &
Parker, J.E. 2005. Arabidopsis SENESCENCE-ASSOCIATED GENE101 stabilizes and signals within an ENHANCED DISEASE SUSCEPTIBILITY1 complex in plant innate immunity. The Plant Cell 17(9):
2601-2613.
Greenfield, A. 2021.
Cloning, mitochondrial replacement and genome editing: 25 years of ethical
debate since Dolly. Reproduction. doi: 10.1530/rep-20-0635
Horvath, D.P., Kudrna,
D., Talag, J., Anderson, J.V., Chao, W.S., Wing, R., Foley, M.E. &
Doğramaci, M. 2013. BAC library development and clone characterization for
dormancy-Responsive DREB4A, DAM, and FT from Leafy Spurge (Euphorbia
esula) identifies differential splicing and conserved promoter motifs. Weed
Science 61(2): 303-309. http://www.jstor.org/stable/43699803
Hughes, R.A. &
Ellington, A.D. 2017. Synthetic DNA synthesis and assembly: Putting the
synthetic in synthetic biology. Cold Spring Harbor Perspectives in Biology 9(1): a023812. https://doi.org/10.1101/cshperspect.a023812
Landy, A. 1989.
Dynamic, structural, and regulatory aspects of lambda site-specific
recombination. Annual Review of Biochemistry 58: 913-949. https://doi.org/10.1146/annurev.bi.58.070189.004405
Norkunas, K., Harding,
R., Dale, J. & Dugdale, B. 2018. Improving agroinfiltration-based transient
gene expression in Nicotiana benthamiana. Plant Methods 14(1).
doi: 10.1186/s13007-018-0343-2
Osman, G.H., Soltane,
R., Saleh, I., Abulreesh, H.H., Gazi, K.S., Arif, I.A., Ramadan, A.M.,
Alameldin, H.F., Osman, Y.A. & Idriss, M. 2019. Isolation, characterization,
cloning and bioinformatics analysis of a novel receptor from black cut worm (Agrotis
ipsilon) of Bacillus thuringiensis vip 3Aa toxins. Saudi Journal
of Biological Sciences 26(5): 1078-1083. doi: 10.1016/j.sjbs.2018.06.002
Owen, M.J., Goggin,
D.E. & Powles, S.B. 2012. Non-target-site-based resistance to
ALS-inhibiting herbicides in six Bromus rigidus populations from Western
Australian cropping fields. Pest Manag. Sci. 68: 1077-1082.
Pouyet, F., Mouchiroud,
D., Duret, L. & Sémon, M. 2017. Recombination, meiotic expression and human
codon usage. Elife 6. doi: 10.7554/elife.27344
Quinn, J.P. 1990.
Evolving strategies for the genetic engineering of herbicide resistance in
plants. Biotechnology Advances 8(2): 321-333. https://doi.org/10.1016/0734-9750(90)91068-r
Reece-Hoyes, J.S. &
Walhout, A. 2018. Gateway recombinational cloning. Cold Spring Harbor
Protocols 2018(1): pdb.top 094912. https://doi.org/10.1101/pdb.top094912
Steinrücken, H.C. &
Amrhein, N. 1980. The herbicide glyphosate is a potent inhibitor of
5-enolpyruvylshikimic acid-3-phosphate synthase. Biochemical and Biophysical
Research Communications 94: 1207-1212. https://doi.org/10.1016/0006-291X(80)90547-1
Tyurin, A.A.,
Suhorukova, A.V., Kabardaeva, K.V. & Goldenkova-Pavlova, I.V. 2020.
Transient gene expression is an effective experimental tool for the research
into the fine mechanisms of plant gene function: Advantages, limitations, and
solutions. Plants 9(9): 1187. https://doi.org/10.3390/plants9091187
Vargas, R.N. &
Wright, S.D. 2005. UCIPM Pest Management Guidelines: Cotton. Division of
Agriculture and Natural Resources, University of California, Statewide IPM
Program Publication 3444.
Webster,
T.M. & Sosnoskie, L.M. 2010. Loss of glyphosate efficacy: A changing weed
spectrum in Georgia cotton. Weed Science 58(1): 73-79.
Welch, M.,
Govindarajan, S., Ness, J.E., Villalobos, A., Gurney, A., Minshull, J. &
Gustafsson, C. 2009. Design parameters to control synthetic gene expression in Escherichia
coli. PLoS ONE 4(9): e7002. https://doi.org/10.1371/journal.pone.0007002
Wilson, R.G. &
Orloff, S.B. 2008. Winter annual weed control with herbicides in
alfalfa-orchard grass mixtures. Weed Technology 22(1): 30-33. http://www.jstor.org/stable/25194987
Wohlleben, W., Arnold,
W., Broer, I., Hillemann, D., Strauch, E. & Pühler, A. 1988. Nucleotide
sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces
viridochromo genes Tü494 and its expression in Nicotiana tabacum. Gene 70(1): 25-37. https://doi.org/10.1016/0378-1119(88)90101-1
Zheng, L., Yang, J.,
Chen, Y., Ding, L., Wei, J. & Wang, H. 2021. An improved and efficient
method of Agrobacterium syringe infiltration for transient
transformation and its application in the elucidation of gene function in
poplar. BMC Plant Biol. 21: 54. https://doi.org/10.1186/s12870-021-02833-w
Zobiole, L.H.S.,
Junior, R.S.D.O., Kremer, R.J., Muniz, A.S. & Junior, A.D.O. 2010. Nutrient
accumulation and photosynthesis in glyphosate-resistant soybeans is reduced
under glyphosate use. Journal of Plant Nutrition 33(12): 1860-1873.
*Corresponding author; email: shaukat_parc@yahoo.co.in
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