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ISOLASI DAN KARAKTERISASI GEN PENYANDI β-AMYRIN SYNTHASE YANG TERLIBAT DALAM BIOSINTESIS SAPONIN PADA Talinum paniculatum (Jacq.) Gaertn.

ISOLATION AND CHARACTERIZATION OF GENE ENCODING β-AMYRIN SYNTHASE INVOLVED IN SAPONIN BIOSYNTHESIS FROM Talinum paniculatum (Jacq.) Gaertn.

Master Theses from JBPTITBPP / 2018-03-14 09:38:29
Oleh : IKA QURROTUL AFIFAH - NIM : 21115006, S2 - Biotechnology-SITH
Dibuat : 2018-03-14, dengan 1 file

Keyword : Talinum paniculatum, saponin, β-Amyrin synthase, TpbAS, RACE PCR, metabolic engineering.

Talinum paniculatum atau gingseng Jawa merupakan tanaman yang banyak dimanfaatkan untuk pengobatan tradisional. Tanaman bergenus Talinum diketahui menghasilkan saponin tipe oleanan seperti talinumosida I. Golongan saponin ini memiliki berbagai aktivitas farmakologi seperti antikanker, antidiabetes, dan antiinflamasi. Penelitian ini bertujuan untuk mengisolasi dan mengkarakterisasi gen penyandi β-Amyrin synthase (TpbAS) yang terlibat dalam reaksi siklisasi oksidoskualen membentuk kerangka saponin tipe oleanan β-Amyrin dan menganalisis hubungan antara level ekspresi gen TpbAS dan produksi saponin pada tiap organ tanaman. Gen TpbAS diisolasi menggunakan primer degenerate dan PCR 5’/3’ Rapid Amplification cDNA Ends (RACE). Analisis ekspresi gen TpbAS pada berbagai organ dilakukan secara semi kuantitatif menggunakan Reverse Transcriptase PCR (RT-PCR), sedangkan kandungan saponin dianalisis secara kuantitatif dengan spektrofotometer. Urutan DNA pengkode (Coding DNA Sequence/ CDS) TpbAS yang berhasil diisolasi terdiri dari 2298 pasang basa yang mengkode protein dengan 765 residu asam amino. Selain itu, ditemukan elemen regulator berupa urutan (GA)n berulang dan urutan AATAAA di daerah 5’/3’ Untranslated Region (UTR) yang diduga berperan dalam proses transkripsi dan ekspresi protein. Deduksi urutan asam amino menunjukkan adanya motif DCTAE, QW dan WCYCR yang sangat lestari pada semua golongan enzim oksidoskualen siklase. Hasil konstruksi pohon filogenetik juga menunjukkan bahwa TpbAS memiliki kekerabatan yang sangat dekat dengan enzim golongan oksidoskualen siklase tanaman lain. Analisis menujukkan bahwa tingkat ekspresi gen TpbAS berkorelasi positif dengan konsentrasi saponin. Hal ini menghasilkan prediksi bahwa saponin disintesis dan diakumulasikan pada organ yang sama dan menujukkan level paling tinggi di daun. Hasil penelitian ini merupakan langkah awal dalam meningkatkan produksi saponin dan memproduksi tanaman dengan sifat tertentu untuk memenuhi kebutuhan industri farmasi melalui pendekatan metabolic engineering.


Deskripsi Alternatif :

Talinum paniculatum or Javanese ginseng is a plant that is widely used in traditional medicine. The genus Talinum is known to produce oleanane-type triterpene saponins such as talinumoside I. This type of saponins has various pharmacological activities such as anticancer, antidiabetic, and anti inflammatory. The aims of this study were to isolate the gene encoding β-Amyrin synthase (bAS), a key enzyme involved in the cyclization reaction of 2,3-oxidosqulane producing backbone of oleanane-type saponin β-Amyrin and to analyze the correlation between the TpbAS gene expression level and saponin production in various plant organs. The TpbAS gene was isolated using degenerate primers and PCR 5 '/ 3' Rapid Amplification cDNA Ends (RACE). TpbAS gene expression level was analyzed in various organs using Reverse Transcriptase PCR (RT-PCR), whereas the saponin content was measured by spectrophotometer. The Coding DNA Sequence (CDS) of TpbAS consists of 2298 base pairs encoding a protein with 765 amino acid residues. In addition, regulatory elements were also identified in the form of (GA)n sequences and AATAAA sequences in the 5 '/ 3' Untranslated Region (UTR) which are predicted to play an important role in the process of transcription and protein expression. The amino acid sequence deduction showed DCTAE, QW, and WCYCR motifs that are conserved in all classes of oxidosqualene cyclase enzymes. The phylogenetic tree analysis also showed that the TpbAS gene has a very close relation to other plant oxidosqualene cyclase groups. Analysis showed that the level of TpbAS gene expression has a positive correlation with saponin concentration. These predicted that saponin is synthesized and accumulated in the same organs with the highest value in leaves. These findings are essential as an initial step to boost the saponin content and produce plant with valuable trait for pharmaceutical industry supply through metabolic engineering approach.


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