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TRANSFORMASI GEN

TRANSFORMASI GEN. Ir. Makhziah, MP UPN “Veteran” Jawa Timur. Transformasi Gen. Masuknya gen target (DNA rekombinan) ke dalam sel inang. Proses Transfer Gen: Biologis Mekanis Kimia. A. TRANSFER GEN SECARA BIOLOGIS Hasil cloning gen dikonjugasi, antara:

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TRANSFORMASI GEN

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  1. TRANSFORMASI GEN Ir. Makhziah, MP UPN “Veteran” Jawa Timur

  2. Transformasi Gen Masuknya gen target (DNA rekombinan) ke dalam sel inang. Proses Transfer Gen: • Biologis • Mekanis • Kimia

  3. A. TRANSFER GEN SECARA BIOLOGIS • Hasil cloning gen • dikonjugasi, antara: • pBR322 + pGV 3850 • (E. coli) (Agrobacterium) • Jenis Agrobacterium: • Agrobacterium Tumefacient • Agrobacterium Rhizogen

  4. Agrobacterium tumefaciens (At) • Penyebab tumor • “CROWN GALL” • sintesis asam amino “OPINE” • sumber carbon & nitrogen

  5. Nama Plasmid : Ti (Tumor inducing) • sifat : stabil dalam At. pada suhu < 30 C • Segmen Ti : • Transfer DNA (T-DNA) • Virulence (Vir) • Konjugasi • Replikasi plasmid

  6. 2. Agrobacterium Rhizogene (Ar) • Penyebab ” Hairy Root “ • Nama Plasmid : Ri (Root Inducing) • Segmen : sama dg At • Transfer DNA (T-DNA) • Virulence (Vir) • Konjugasi • Replikasi plasmid

  7. MEKANISME TRANSFER DNA menginfeksi sel tanaman Agrobacterium Eksudate Asetosyringone (AS) Induksi transkripsi gen-gen vir. Aktifasi: Gen vir. A Gen vir. G Gen vir. D “Single stranded nick” “ Gen vir. E Lepasnya single stranded “T-DNA” Integrasi dalam sel tanaman

  8. MEKANISME TRANSFER GEN MELALUI Ti PLASMID

  9. IDENTIFIKASI TRANSFORMASI Plasmid dimasuki gen ketahanan antibiotik (selectable marker) misal: KANAMYCIN RESISTANCE b. Analisis DNA PCR (Polymerase Chain Reaction)

  10. B. SECARA MEKANIS • a. Microinjection • Cara : • Immobilisasi : • 1. Immobilisasi protoplas Polylisine • 2. Holding Pipette mobilization • Efisiensi Transformasi tergantung : • Lokasi injeksi • Bentuk fisik DNA • Konsentrasi DNA

  11. TEKNIK MIKRO INJEKSI

  12. MIKRO INJEKSI pada KENTANG & BAWANG MERAH Microinjection into the cell of a potato callus. Using fine cannulae, DNA that codes for a fluorescent protein was inserted into onion cells. Two days after the injection, the green dye can be seen. The integration of the foreign DNA into the onion genome was successful

  13. b. Electroporation Listrik tegangan tinggi (± 2500 V) “Reversible Break-Down” membran protoplas DNA masuk ke protoplas Faktor-faktor yang mempengaruhi : Voltage Waktu Umur sel/jaringan Bagian tanaman

  14. PROSES TRANSFER GEN CARA ELEKTROPORASI

  15. PERALATAN ELEKTROPORASI

  16. ELEKTROPORASI PADA JAGUNG

  17. Particle Bombardment • Digunakan untuk memasukkan DNA/ gen ke dalam tanaman yang sulit diregenerasikan dari protoplas • Particle : • Gold • Coated DNA/Gene • Tungsten • Yang digunakan gas : Helium

  18. PERALATAN PARTICLE BOMBARDMENT

  19. MEKANISME PENEMBAKAN GEN by PARTICLE BOMBARDMENT

  20. PENEMBAKAN GEN PADA TANAMAN PISANG

  21. The End

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