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Cloning and Heterologous Expression of Cyanobacterial Genes Encoding Phosphinothricin N- acetyltransferase in E. coli.

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Název: Cloning and Heterologous Expression of Cyanobacterial Genes Encoding Phosphinothricin N- acetyltransferase in E. coli.
Alternate Title: Fosfinotrisin N-asetiltransferazı Kodlayan Siyanobakteriyel Genlerin E. coli de Klonlanması ve Heterolog İfadesi. (Turkish)
Autoři: ÖZKUL, Kübra, YILMAZ, Ebru, KURT KIZILDOĞAN, Aslıhan
Zdroj: Yuzuncu Yil University Journal of the Institute of Natural & Applied Sciences / Yüzüncü Yl Üniversitesi Fen Bilimleri Enstitüsü Dergisi; 2025, Vol. 30 Issue 2, p815-829, 15p
Témata: HERBICIDE resistance, ESCHERICHIA coli, MOLECULAR cloning, GLUFOSINATE, BACTERIAL enzymes, ACETOLACTATE synthase
Abstract (English): Phosphinothricin N-acetyltransferase (Pat) is a bacterial enzyme that is introduced into plants to confer resistance to herbicides containing phosphinothricin (PPT). The expression of the Pat protein in transgenic plants allows them to tolerate the herbicide glufosinate. Although the enzyme has been identified in cyanobacteria through sequence analysis, its activities have not yet been thoroughly investigated. In this study, we cloned the putative Pat enzyme-encoding genes alr4468 and sll1647 from Anabaena sp. PCC7120 and Synechocystis sp. PCC6803, respectively, into the His-Tagged pET28a+ expression vector. The recombinant vectors containing these genes were introduced into protease-free E. coli BL21 cells. We successfully expressed the Alr4468 and Sll1647 polypeptides in the recombinant E. coli BL21 cultures that were induced with 1 mM IPTG. The highest in vitro Pat activities were measured using enzyme assays from IPTG-induced cell crude extracts, showing increases of 3.47 times for rAlr4468 and 2.53 times for rSll1647 compared to controls. As a result, the alr4468 and sll1647 genes from cyanobacteria, which exhibit similar physiological properties to those of high-structured plants, may serve as potential sources for developing herbicide-resistant transgenic plants in future research. [ABSTRACT FROM AUTHOR]
Abstract (Turkish): Fosfinotrisin N-asetiltransferaz (Pat), fosfinotrisin (PPT) içeren herbisitlere direnç sağlamak için bitkilerde aktarılan bakteriyel bir enzimdir. Pat proteininin transgenik bitkilerde ifade edilmesi, glufosinat herbisitine karşı tolerans sağlar. Siyanobakterilerdeki bu enzim dizi analizi ile tanımlanmış olmasına rağmen, aktiviteleri henüz belirlenmemiştir. Bu çalışmada, Anabaena sp. PCC7120 ve Synechocystis sp. PCC6803'de Pat enzimini kodladığı varsayılan sırasıyla alr ve sll genleri His-Taq pET28a+ ekspesyon vektörüne klonlandı. İlgili genleri içeren rekombinant vektörler proteaz içermeyen E. coli BL21 hücrelerine transfer edildi. Alr4468 ve Slll647 polipeptitleri 1 mM IPTG ile indüklenmiş rekombinant E. coli BL21 kültürlerinde başarıyla ifade edildi. En yüksek in vitro Pat aktiviteleri IPTG ile indüklenmiş kaba hücre ekstraktları kullanılarak yapılan enzim deneylerinde elde edilmiş ve kontrollerden sırasıyla 3.47 (rAlr4468 için) ve 2.53 (rSll1647 için) kat daha yüksek bulunmuştur. Sonuç olarak, yüksek yapılı bitkilerle benzer fizyolojik özelliklere sahip olan siyanobakterilerin alr4468 ve sll1647 genleri, gelecekteki çalışmalarda herbisite dirençli yerli transgenik bitkilerin üretimi için potansiyel kaynaklar olarak kullanılabilirler. [ABSTRACT FROM AUTHOR]
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Databáze: Complementary Index
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Abstrakt:Phosphinothricin N-acetyltransferase (Pat) is a bacterial enzyme that is introduced into plants to confer resistance to herbicides containing phosphinothricin (PPT). The expression of the Pat protein in transgenic plants allows them to tolerate the herbicide glufosinate. Although the enzyme has been identified in cyanobacteria through sequence analysis, its activities have not yet been thoroughly investigated. In this study, we cloned the putative Pat enzyme-encoding genes alr4468 and sll1647 from Anabaena sp. PCC7120 and Synechocystis sp. PCC6803, respectively, into the His-Tagged pET28a+ expression vector. The recombinant vectors containing these genes were introduced into protease-free E. coli BL21 cells. We successfully expressed the Alr4468 and Sll1647 polypeptides in the recombinant E. coli BL21 cultures that were induced with 1 mM IPTG. The highest in vitro Pat activities were measured using enzyme assays from IPTG-induced cell crude extracts, showing increases of 3.47 times for rAlr4468 and 2.53 times for rSll1647 compared to controls. As a result, the alr4468 and sll1647 genes from cyanobacteria, which exhibit similar physiological properties to those of high-structured plants, may serve as potential sources for developing herbicide-resistant transgenic plants in future research. [ABSTRACT FROM AUTHOR]
ISSN:13005413
DOI:10.53433/yyufbed.1633412