Karakterisasi Dan Analisis Ekspresi Gen GpPAL, GpDFR Dan GpCHS Tanaman Daun Dewa (G. pseudochina L. (DC)) Pasca Perlakuan Silver Nanoparticle Secara In Vitro

Pratami, Aiziah Ayu (2022) Karakterisasi Dan Analisis Ekspresi Gen GpPAL, GpDFR Dan GpCHS Tanaman Daun Dewa (G. pseudochina L. (DC)) Pasca Perlakuan Silver Nanoparticle Secara In Vitro. Masters thesis, Institut Teknologi Sepuluh Nopember.

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Abstract

Tanaman daun dewa (Gynura pseudochina) merupakan genus Gynura dan termasuk ke dalam famili Asteraceae, yang diketahui mengandung senyawa bioaktif yaitu flavonoid. Salah satu jenis senyawa flavonoid yang dominan terdapat pada tanaman tersebut adalah antosianin. Antosianin merupakan pigmen berwarna yang juga berfungsi sebagai antioksidan, sehingga memiliki potensi untuk dikembangkan sebagai sumber biofarmaka Indonesia. Upaya peningkatan kandungan metabolit sekunder tanaman dapat dilakukan salah satunya dengan aplikasi elisitor. Pada saat ini, elisitor yang sedang berkembang adalah elisitor berbasis nanoteknologi, diantaranya adalah silver nanopartikel (AgNPs). Hasil penelitian sebelumnya mengungkapkan bahwa AgNPs terbukti dapat meningkatkan kandungan flavonoid dan antosianin total tanaman daun dewa. Namun demikian, pembuktian pengaruh AgNPs tersebut di level molekuler masih belum dilakukan. Hal ini dikarenakan minimnya informasi genetik tanaman daun dewa. Beberapa gen yang terlibat dalam biosintesis flavonoid diantaranya adalah GpPAL., GpDFR, dan GpCHS. Penelitian ini bertujuan untuk mengisolasi fragmen gen DFR dan CHS, mengetahui hubungan kekerabatan antara gen-gen tersebut menggunakan pohon filogenetik serta mengetahui ekspresi genetik dari gen PAL, DFR dan CHS pada tanaman daun dewa pasca perlakuan silver nanopartikel secara in vitro dengan menggunakan real time PCR. Tahapan penelitian yang dilakukan meliputi isolasi RNA, konstruksi pohon filogenetik dan analisa ekspresi gen menggunakan qRT-PCR. Berkaitan dengan analisa ekspresi gen, sampel yang digunakan adalah hasil kultur in vitro tanaman daun dewa pasca pemberian AgNP dengan konsentrasi sebesar 0 µg/L, 30 µg/L, 60 µg/L, dan 90 µg/L. Fragmen cDNA gen GpPAL, GpDFR, dan GpCHS berhasil diisolasi dengan ukuran 667 bp, 637 bp, dan 947 bp. Hasil konstruksi filogenetik menunjukkan bahwa GpDFR berkerabat dekat dengan GbDFR dengan indeks similaritas sebesar 98,36%. GpCHS menunjukkan kekerabatan dekat dengan GbCHS dengan indeks similaritas sebesar 98,31%. Hasil analisis ekspresi genetik menunjukkan bahwa GpPAL pada tanaman daun dewa yang diberi perlakuan AgNP 60 µg/L mengalami peningkatan nilai ekspresi relatif mRNA sebesar 3,51 kali lipat (fold) dibanding kontrol. Sementara itu, gen DFR dan CHS mengalami penurunan nilai ekspresi relatif mRNA pada tanaman daun dewa pasca pemberian AgNP dibanding dengan kontrol.
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Dewa leaf plant (Gynura pseudochina) is a genus of Gynura and belongs to the Asteraceae family, which is known to contain bioactive compounds, namely flavonoids. One of the dominant types of flavonoid compounds found in these plants is anthocyanins. Anthocyanin is a colored pigment that also functions as an antioxidant, so it has the potential to be developed as a source of Indonesian biopharmaceuticals. Efforts to increase the content of plant secondary metabolites can be done, one of which is the elicitor application. At this time, elicitors that are being developed are nanotechnology-based elicitors, including silver nanoparticles (AgNPs). The results of previous studies revealed that AgNPs were proven to increase the total flavonoid and anthocyanin content of Dewa leaf plants. However, the evidence for the effect of these AgNPs at the molecular level has not been carried out. This is due to the lack of genetic information on Dewa leaf plants. Several genes involved in flavonoid biosynthesis include GpPAL., GpDFR, and GpCHS. This study aims to isolate the DFR and CHS gene fragments, determine the relationship between these genes using a phylogenetic tree and determine the genetic expression of the PAL, DFR and CHS genes in leaf dewa plants after silver nanoparticle treatment in vitro using real time PCR. The stages of the research include RNA isolation, phylogenetic tree construction and gene expression analysis using qRT-PCR. In relation to gene expression analysis, the sample used was the result of in vitro culture of Dewa leaves after administration of AgNP with concentrations of 0 g/L, 30 g/L, 60 g/L, and 90 g/L. The cDNA fragments of GpPAL, GpDFR, and GpCHS genes were isolated with sizes of 667 bp, 637 bp, and 947 bp. The results of the phylogenetic construction show that GpDFR is closely related to GbDFR with a similarity index of 98.36%. GpCHS shows a close relationship with GbCHS with a similarity index of 98.31%. The results of genetic expression analysis showed that GpPAL in Dewa leaf plants treated with AgNP 60 g/L experienced an increase in the relative expression value of mRNA by 3.51 times (fold) compared to controls. Meanwhile, DFR and CHS genes decreased the relative expression value of mRNA in Dewa leaf plants after administration of AgNP compared to controls.

Item Type:	Thesis (Masters)
Uncontrolled Keywords:	AgNPs, Elisitor, Flavonoid, Gynura pseudochina, Metabolit sekunder, Elicitors, Flavonoids, Gynura pseudochina, Secondary metabolites
Subjects:	Q Science > QH Biology Q Science > QH Biology > QH541 Ecology
Divisions:	Faculty of Mathematics and Science > Biology > 46101-(S2) Master Thesis
Depositing User:	Eko Sulistiono
Date Deposited:	24 Sep 2025 02:36
Last Modified:	24 Sep 2025 02:36
URI:	http://repository.its.ac.id/id/eprint/128399

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