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All Journal Vegetalika BIOTROPIA - The Southeast Asian Journal of Tropical Biology
Supena, Ence Darmo Jaya
Unknown Affiliation
Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Immunology & microbiology Veterinary

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Optimasi Ekstraksi RNA dan Teknik Kloning: Studi Kasus Kloning Gen Heading Date 3a pada Kelapa Sawit Polosoro, Aqwin; Enggarini, Wening; Kusumanegara, Kusumawaty; Hadiarto, Toto; Miftahudin, Miftahudin; Supena, Ence Darmo Jaya
Vegetalika Vol 13, No 2 (2024)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22146/veg.92085

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
Agrobacterium-Mediated Genetic Transformation of Seaweed Kappaphycus alvarezii Using Gα Gene and Callus Cultures Sulistiani, Erina; Suharsono, Suharsono; Supena, Ence Darmo Jaya; Miftahudin, Miftahudin
BIOTROPIA Vol. 26 No. 1 (2019): BIOTROPIA Vol. 26 No. 1 April 2019
Publisher : SEAMEO BIOTROP

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (133.176 KB) | DOI: 10.11598/btb.2019.26.1.989

Abstract

Cottonii seaweed (Kappaphycus alvarezii Doty) is one of the most important commercial sources of carrageenan, which is widely used in the pharmaceutical and food industries. A major problem in the cultivation of this seaweed is ice-ice disease, which is caused by extreme changes in environmental conditions such as temperature and seawater salinity. Gene transformation to produce transgenic Kappaphycus lines that are tolerant to environmental stress is a potential solution to this problem. The Gα gene, which encodes the heterotrimeric G-protein alpha subunit, plays an important role in tolerance to biotic and abiotic environmental stress. This study aimed to: (a) introduce the Gα gene into the callus cells of K. alvarezii and regenerate transformed callus cells into transgenic plantlets; and (b) determine the appropriate concentration of acetosyringone and Agrobacterium tumefaciens strain for successful gene transfer into the callus of K. alvarezii. The callus cells of K. alvarezii were transformed using Agrobacterium tumefaciens strains LBA4404 and EHA105 carrying the expression vector pGWB502-Gα under the control of the CaMV-35S promoter. The calli and A. tumefaciens were co-cultivated in different concentrations of acetosyringone (20, 40, and 60 mg/L). The regeneration of transformed callus cells into transgenic plantlets was successfully achieved using the somatic embryogenesis technique. The results showed that the highest percentage of putative transgenic micropropagule formation occurred at acetosyringone concentrations of 20–40 mg/L. Polymerase chain reaction (PCR) analysis of twenty regenerated plantlets indicated that the Gα gene was successfully introduced into the genomic DNA of all samples. The highest transformation efficiency was obtained from the 20–40 mg/L acetosyringone co-cultivation treatment (22–28%). The transformation efficiency produced by Agrobacterium tumefaciens EHA105 (23%) was not significantly different from that produced using strain LBA4404 (15%).
Co-Authors Kusumanegara, Kusumawaty Miftahudin . Polosoro, Aqwin Suharsono Suharsono Sulistiani, Erina Toto Hadiarto Wening Enggarini