Optimasi Ekstraksi RNA dan Teknik Kloning: Studi Kasus Kloning Gen Heading Date 3a pada Kelapa Sawit

https://doi.org/10.22146/veg.92085

Aqwin Polosoro(1), Wening Enggarini(2), Kusumawaty Kusumanegara(3), Toto Hadiarto(4), Miftahudin Miftahudin(5), Ence Darmo Jaya Supena(6*)

(1) 1Program Studi Biologi Tumbuhan, Fakultas Matematika dan Ilmu Pengetahuan Alam, Institut Pertanian Bogor, Jl. Agathis, Kampus IPB Dramaga, Bogor 16680 2Pusat Riset Rekayasa Genetika, Badan Riset dan Inovasi Nasional, Kawasan Sains dan Teknologi Soekarno, Jl. Raya Jakarta-Bogor Km. 46, Cibinong, Bogor 16911
(2) Pusat Riset Rekayasa Genetika, Badan Riset dan Inovasi Nasional, Kawasan Sains dan Teknologi Soekarno, Jl. Raya Jakarta-Bogor Km. 46, Cibinong, Bogor 16911
(3) Pusat Riset Hortikultura dan Perkebunan, Badan Riset dan Inovasi Nasional, Kawasan Sains dan Teknologi Soekarno, Jl. Raya Jakarta-Bogor Km. 46, Cibinong, Bogor 16911
(4) Pusat Riset Rekayasa Genetika, Badan Riset dan Inovasi Nasional, Kawasan Sains dan Teknologi Soekarno, Jl. Raya Jakarta-Bogor Km. 46, Cibinong, Bogor 16911
(5) Program Studi Biologi Tumbuhan, Fakultas Matematika dan Ilmu Pengetahuan Alam, Institut Pertanian Bogor, Jl. Agathis, Kampus IPB Dramaga, Bogor 16680
(6) Program Studi Biologi Tumbuhan, Fakultas Matematika dan Ilmu Pengetahuan Alam, Institut Pertanian Bogor, Jl. Agathis, Kampus IPB Dramaga, Bogor 16680
(*) Corresponding Author

Abstract


Pembungaan memegang peranan penting bagi tumbuhan karena memfasilitasi rekombinasi genetik, sehingga mendukung perkembangan keragaman genetik yang penting. Keluarga protein phosphatidylethanolamine binding proteins (PEBP) memainkan peran penting dalam mengatur waktu pembungaan dan dormansi benih di beragam spesies tanaman. Penelitian ini bertujuan untuk merancang vektor biner dengan membangun pCAMBIA1300 yang menggabungkan rangkaian gen EgHd3a dari kelapa sawit. Proses konstruksi gen meliputi ekstraksi RNA, sintesis cDNA, amplifikasi gen EgHd3a, kloning gen menjadi vektor kloning, subkloning ke dalam vektor biner pCAMBIA1300, dan diakhiri dengan validasi gen melalui analisis sekuens. Pada ekstraksi RNA, metode PCL-Chisam telah terbukti efektif melalui ekstraksi berulang, meningkatkan kualitas dan kuantitas total RNA. Dalam proses kloaning, metode konvensional menghadapi tantangan dalam memilih lokasi pembelahan yang tepat. Untuk mengatasi kendala ini, penggunaan enzim dengan overhang yang kompatibel diusulkan sebagai solusi potensial. Secara khusus, penggantian BamHI dari BglII telah secara efektif mengatasi tantangan ini. Konfirmasi integrasi fragmen gen ke dalam plasmid pCAMBIA1300 dicapai melalui pengurutan. Meskipun perbedaan diidentifikasi dalam rangkaian EgHd3a-2, perubahan ini tidak berdampak pada asam amino yang dikodekan, sehingga menjaga integritas rangkaian protein


Keywords


kelapa sawit; kloning; Hd3a; pembungaan.

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DOI: https://doi.org/10.22146/veg.92085

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