Sains Malaysiana 52(4)(2023): 1133-1143

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 SHOUKAT^1,2, MUHAMMAD AMIR ZIA² & SHAUKAT ALI^2,*

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

Diserahkan: 4 April 2022/Diterima: 17 Mac 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

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^*Pengarang untuk surat-menyurat; email: shaukat_parc@yahoo.co.in