Molecular Cloning and in silico Characterization of the Arabidopsis thaliana Phytochelatin Synthase 1 (AtPCS1) Gene for Potential Phytoremediation Applications

Öz

The PCS1 gene encodes phytochelatin synthase 1, a member of the phytochelatin synthase family, which catalyzes the biosynthesis of glutathione-derived peptides known as phytochelatins. These peptides bind heavy metal ions and play a crucial role in heavy metal detoxification and tolerance mechanisms in plants. Due to its central role in metal homeostasis, PCS1 represents an important molecular target for studies related to plant stress responses and phytoremediation. In this study, the PCS1 gene was isolated and cloned from Arabidopsis thaliana. Successful amplification was achieved from A. thaliana using high-fidelity Pfu polymerase. The amplified PCS1 gene was ligated into the plant expression vector pBIN61 using T4 DNA ligase and subsequently transformed into Escherichia coli JM109 by heat shock. Recombinant clones were selected on kanamycin-containing medium and verified by colony PCR and restriction enzyme analysis. Confirmed plasmids harboring the PCS1 gene were isolated and preserved as glycerol stocks at −80 °C. Positive constructs were then transferred into Agrobacterium tumefaciens strain LBA4404 via electroporation. In addition to experimental cloning, in silico analyses were conducted to characterize the physicochemical properties, conserved domain architecture, and sequence conservation of the PCS1 protein, providing complementary insights into its structural and functional features. The resulting Agrobacterium strains carrying the PCS1 gene constitute a valuable resource for future plant transformation studies, and PCS1-expressing transgenic plants may serve as promising tools for the phytoremediation of heavy metal–contaminated environments.

Anahtar Kelimeler

• Arabidopsis thaliana
• gene cloning
• PCS1
• Phytochelatin synthase
• phytoremediation

Etik Beyan

The authors declare that this study does not require ethical approval as it does not involve human or animal participants, and only involves molecular studies on plant materials.

Potansiyel Fitoremediyasyon Uygulamaları için Arabidopsis thaliana Fitokelatin Sentaz 1 (AtPCS1) Geninin Moleküler Klonlanması ve in silico Karakterizasyonu

Öz

PCS1 geni, fitokelatinler olarak bilinen ve glutatyon türevi peptitlerin biyosentezini katalize eden, fitokelatin sentaz ailesinin bir üyesi olan fitokelatin sentaz 1'i kodlar. Bu peptitler, ağır metal iyonlarını bağlayarak bitkilerdeki ağır metal detoksifikasyonu ve tolerans mekanizmalarında kritik bir rol oynarlar. Metal homeostazındaki merkezi rolü nedeniyle PCS1, bitki stres tepkileri ve fitoremediasyon ile ilgili çalışmalar için önemli bir moleküler hedef teşkil etmektedir. Bu çalışmada, PCS1 geni Arabidopsis thaliana'dan izole edilmiş ve klonlanmıştır. A. thaliana'dan yüksek doğruluklu (high-fidelity) Pfu polimeraz kullanılarak başarılı bir amplifikasyon gerçekleştirilmiştir. Çoğaltılan PCS1 geni, T4 DNA ligaz kullanılarak pBIN61 bitki ekspresyon vektörüne ligasyonu yapılmış ve ardından ısı şoku yöntemiyle Escherichia coli JM109 suşuna transforme edilmiştir. Rekombinant klonlar, kanamisin içeren besiyerinde seçilmiş; koloni PCR ve restriksiyon enzim analizi ile doğrulanmıştır. PCS1 genini taşıyan doğrulanmış plazmitler izole edilmiş ve -80 °C'de gliserol stokları olarak muhafaza edilmiştir. Pozitif yapılar daha sonra elektroporasyon yoluyla Agrobacterium tumefaciens LBA4404 suşuna aktarılmıştır. Deneysel klonlamaya ek olarak, PCS1 proteininin fizikokimyasal özelliklerini, korunmuş domain mimarisini ve dizi korunmuşluğunu karakterize etmek amacıyla in silico analizler yürütülmüş; böylece proteinin yapısal ve işlevsel özelliklerine dair tamamlayıcı bilgiler sağlanmıştır. PCS1 genini taşıyan nihai Agrobacterium suşları, gelecekteki bitki transformasyon çalışmaları için değerli bir kaynak oluşturmaktadır ve PCS1 ekspresyonu yapan transgenik bitkiler, ağır metal ile kirlenmiş ortamların fitoremediyasyonu için gelecek vadeden araçlar olarak hizmet edebilir.

Anahtar Kelimeler

• Arabidopsis thaliana
• gen klonlama
• PCS1
• Fitokelatin sentaz
• fitoremediasyon

Etik Beyan

Bu çalışma insan veya hayvan denekleri içermediği; yalnızca bitki materyalleri üzerinde yürütülen moleküler çalışmaları kapsadığı için etik kurul onayı gerektirmemektedir.

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