Konstruksi Vektor CRISPR/Cas9 untuk Pengeditan Gen StTOPP6 pada Tanaman Kentang (Solanum tuberosum L.) IPB CP1

Abstract

Perkembangan teknologi genome editing memungkinkan penyuntingan susceptible gene (S-gene) sebagai metode menjanjikan untuk memodifikasi tanaman menjadi tahan penyakit. Protein TOPP6 pada tanaman kentang dapat berinteraksi dengan efektor RipAS dari Ralstonia solanacearum yang mendorong virulensi sehingga menyebabkan tanaman mengalami gejala penyakit layu bakteri. Penelitian ini bertujuan mengkonstruksi vektor rekombinan CRISPR/Cas9 pembawa sgRNA dari gen StTOPP6. Keberadaan gen target dari kentang kultivar IPB CP1 telah dikonfirmasi dan dua sgRNA telah didesain. Oligo sgRNA telah disisipkan ke dalam plasmid pHSE401 diikuti dengan transformasi ke sel kompeten Escherichia coli TOP10. Verifikasi penyisipan sgRNA dilakukan dengan PCR koloni dan PCR plasmid yang menghasilkan amplikon berukuran sesuai target 478 pb. Transformasi konstruksi vektor rekombinan pHSE401_Cr1 ke Agrobacterium tumefaciens LBA4044 berhasil dilakukan dengan triparental mating yang ditandai dengan tumbuhnya koloni bakteri pada media seleksi dan munculnya pita berukuran 478 pb pada hasil PCR koloni.

The development of genome editing technology has enabled susceptible-gene (S-gene) editing techniques to emerge as a promising method for developing plants disease resistant. In potatoes, the type one phosphatase protein 6 (TOPP6) interacts with the RipAS effector of Ralstonia solanacearum, enhancing effector virulence, leading to bacterial wilt symptoms in the plant. This study aimed to construct a CRISPR/Cas9 recombinant vector carrying sgRNA to disrupt the StTOPP6 gene. The presence of the StTOPP6 gene in the potato cultivar IPB CP1 was confirmed and two high-precision sgRNAs were designed. These sgRNAs were inserted into the pHSE401 plasmid and transformed into Escherichia coli TOP10 cells. Verification of sgRNA insertion was conducted using colony PCR and plasmid PCR. The resulting amplicon showed an expected band size 478 bp. The recombinant vector construct, pHSE401_Cr1, was subsequently transformed into Agrobacterium tumefaciens LBA4044 via triparental mating. The sgRNA construct in pHSE401 plasmid was successfully transformed into A. tumefaciens LBA4044, it was confirmed by the growth of bacterial colonies on selection medium and the appearance of a 478 bp band in colony PCR results.