Rekayasa Gen Tanaman 2008
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Transcript of Rekayasa Gen Tanaman 2008
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PRINSIP REKAYASA GENETIKA PADA TANAMAN
Suharsono- Pusat Penelitian Sumberdaya Hayati dan Bioteknologi, IPB
- Departemen Biologi, IPBE-mail: [email protected]; [email protected]
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Transformasi Tanaman dengan Agrobacterium tumefaciens
Agrobacterium tumefaciens:- gram negatif- fitopatogen- mentransformasi sel tanaman tumor
Tumor:transfer, integrasi dan ekspresi gen yang terdapat di T-DNA ke dalam genom sel tanaman
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Proses pembentukan tumor
Tanaman luka acetosyringone,hydroxyacetosyringone induksi gen vir
Vir: •25 gen di dalam 7 operon•di luar T-DNA•fungsi: transfer & integrasi T-DNA di genom tanaman
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Plasmid TiT-DNA1. iaaM (tms1)2. iaaH (tms2) 3. tmr (ipt)4. gen-gen penyandi opine •diekspresikan setelah terintegrasi ke genom tanaman•ditransfer dalam bentuk utas tunggal linier, ujung 5’membawa RB dan ujung 3’ LB
sekuen spesifik RB: 25 pb
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Sintesis auxin dan sitokinine
trp 2-monooxygenase
iaaM (tms1)
i-3-a hydrolase
iaaH (tms2)
isopentenyl transferase
tmr (ipt)
isopentenyl adenosine monophosphate
isopentenyl diphosphate
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Opine
• Sumber N dan C bagi A.tumefaciens
• Beberapa jenis• Tidak bermanfaat bagi
tanaman
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Kendala pemanfaatan pTi alami
1. Sel tidak dapat diregenerasigen fitohormon harus dibuang
2. Regenerasi sel terhambat karena N, dan Cdigunakan untuk membentuk opine
gen opine harus dibuang3. Ukuran pTi terlalu besar
segmen DNA yang tidak penting harus dibuang4. Tidak dapat melakukan replikasi di E. coli
Ori dari E. coli harus disisipkan ke pTi
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Vektor derivatif dari pTi
Mengandung1. gen penanda seleksi (hpt, npt)2. Ori di E. coli (kadang: + Ori di A. t)3. RB dari T-DNA (sering: + LB)4. Situs pengklonan (MCS)
Vektor ekspresi pTi-tidak mempunyai vir1. Vektor biner2. vektor kointegrasi
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1. Vektor biner•Ori E. coli, A.t•Tidak ada vir•A.t resipien:-disarmed pTi: tidak ada T-DNA,ada vir
2. Vektor kointegrasiRekombinasi vektor pengklonan + disarmed pTi vektor (vir, T-DNA)
Vektor ekspresi pTi
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Transfer gen dengan A. tumefaciens
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Metode fisik untuk mentransfer gen ke tanaman
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Tanaman yang telah ditransformasi secara genetik
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Biolistik
•partikel diselimuti DNA+ CaCl, spermidine/PEG•efisiensi: DNA linier>sirkuler; besar<kecil (plasmid besar terfragmentasi YAC)
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Tanaman transgenik menggunakan biolistik
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Biolistik dengan YAC
GOI= 150 kb (beberapa gen) dapat diintegrasikan ke genom tanaman
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GEN REPORTER• menyeleksi sel yang tertransformasi• kuantifikasi ekspresi gen2 macam:• gen penanda seleksi dominan• gen yang produknya dapat dideteksi:
#β-D-glucuronidase:*5-bromo-4-chloro-3-indolyl β-D-glucuronideacid biru*4-methylumbelliferyl β-D-glucuronidefluorescent (fluorometer)
#GFP (green fluorescent protein): penanda in vivo; sinar UV atau biru (tanpa substrat) fluorescent hijau (tanaman tipe liar fluorescent merah keunguan)
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Gen reporter dan penanda seleksi di tanaman
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Manipulasi ekspresi gen di tanaman
•Pilih PromoterPromoter 35S CaMV: konstitutif kuat, di seluruh jaringan, sepanjang hidupPromoter SSU rubisco: aktif di daun (jaringan fotosintetik)Pathogenesis-related promoter
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Faktor penentu tingkat ekspresi lainnya•enhancer: terletak di hulu promoter (1-ratusan bp)•intron: stabilitas mRNA•terminator
Perbandingan promoter 35S dengan promoter komposit
Promoter komposit: p35S+7 enhancer+ Ω TMV+tnosGen reporter: β-glucuronase
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Produksi protein di kloroplast/mitokondria
Strategi:•Gen penyandi fusi protein sasaran+peptida signal inti sel•Gen penyandi protein sasaran kloroplast/mitokondria
Sel daun: 50-100 kloroplast @ 10-100 kopi DNAinsersi gen di kloroplast produksi protein > insersi di kromosomCara: GOI+marker diapit oleh sekuen DNA kloroplast microprojectile bombardment. Rekombinasi GOI terintegrasi ke genom kloroplat
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Sekresi protein
Produksi protein di tanaman mahaluntuk purifikasi (90%)Tanaman sebagai bioreaktor eksudasi protein
lewat (apoplast) akar
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APLIKASI REKAYASA GENETIKA TANAMAN
Tujuan utama bioteknologi tanaman: merakit kultivar baru
Perakitan tanaman resisten terhadap seranggaStrategi:•insektisidal protoksin oleh Bacillus thuringiensis•inhibitor α-amilase•inhibitor protease•lektin
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Contoh sifat yang diperbaiki
Kapas-BtKapas biasa
Ulat menyerang tongkol jagung Penggerek batang
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BtNon Bt
Kapas
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Pepaya resisten Vs peka Ring Spot Virus
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Meningkatkan ekspresi
•memotong gen (tersisa N-terminal yang mengandung toksin)
•menggunakan promoter kuat
Perbandingan aa protoksin dari berbagai strain B.t:98% ujung N terkonservasi; ujung C bervariasiaktivitas: 646 aa pertama dari ujung N (protoxin: 1156 aa)
Gen protoxin yang dipotong tomat
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Ekspresi toxin Bt
Manduca sextaHeliothis zeaKeiferia lycopersicella
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Usaha menaikkan ekspresi protoksin
1.Mutagenesis situs terarah: mengubah sekuen yang menghambat transkripsi dan translasi 95.6% smdg WT 10 x produk WT
2.Modifikasi sekuen sintesis secara kimia kodon yang biasa di tanaman 78.9% smdg WT
100 x produk WT3.Menggunakan promoter SSU rubisco + peptida
transit + sekuen termodifikasi over produksi di kloroplast produk sangat tinggi (1% protein daun)
4. Introduksi gen protoksin ke kloroplast 2-3% protein daun
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Keuntungan ekspresi protoksin di kloroplast
1. Jumlah kloroplast tiap sel banyak multi kopi gen protoksin produk tinggi
2. Tidak ada resiko penyebaran gen ke lingkungannya melalui polen
Kerugian:Serangga dapat menyerang akar, buah, bunga, batang yang tidak mempunyai kloroplast
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Ekspresi gen penyandi protoksin B. thuringiensis di tanaman
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Strategi lain mencegah serangan serangga
- Cowpea trypsin inhibitor•menghambat H. virescens
- Potato proteinase inhibitor II•penggerek batang merah muda (Sesamia inferens)
- Inhibitor α-amylase•kumbang Callosobruchus maculatus, C. chinensis
-Cholesterol oxidase•Anthonomus grandis grandis (coleoptera)
-Lektin-Tryptophan decarboxylase•kupu putih (Bemisia tabaci)
-Avidin
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Strategi lainnya untuk mencegah serangan serangga
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Pencegahan resistensi serangga terhadap B.t
B.t. sebagai agen seleksi serangga resisten Cara pencegahan:•Ekspresi protoksin Bt dibatasi pada waktu singkat
promoter PR-1a Bt diinduksi oleh pathogen atau asam salisilat atau asam poliakrilat•Fusi 2 gen toksin protein toksin hibrid•Kombinasi: Bt + insektisida lain (inhibitor α-amilase)•Kombinasi: Bt + insektisida kimiawi dosis rendah•Strategi pengungsian (refuge). 20% area non-Bt. Serangga yang tahan di area Bt (80%) jumlahnyasedikit (99.9% mati) kawin dengan serangga sensitif di area 20% non-Bt dilusi ketahanan
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Tanaman resisten terhadap virus
Resistensi alami:•mencegah transmisi virus•mencegah terbentuknya virus•mengatasi gejala virus
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Proteksi dengan protein mantel virus
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Ekspresi protein mantel
Tanaman transgenik:•mengekspresikan protein mantel (RNA sense) resisten•dengan antisense resisten pada dosis virus rendah
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Resistensi terhadap banyak virusProtein mantel CuMV, Zucchini yellow mosaic virus, WMV2 Cucurbita pepo resisten terhadap ketiga virus
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Tanaman resisten terhadap herbisida
Tanaman resisten herbisida:1. Menghambat penyerapan herbisida2. Produksi protein sasaran herbisida secara
berlebihan protein aktif masih ada 3. Mengurangi kemampuan protein sasaran
mengikat herbisida4. Menginaktivasi herbisida melalui metabolisme2-4: melalui perakitan tanaman transgenik
EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) aa aromatik; sasaran glyphosateEPSPS mutan E. coli tanaman tanaman resisten terhadap glyphosate
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Perakitan tanaman resisten terhadap herbisida
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•Nitrilase (dari Klebsiella ozaenae) menginaktivasi herbisida bromoxynile tanaman resisten•Herbisida bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) menghambat fotosintesis
Resisten herbisida bromoxynil
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Tanaman resisten terhadap fungi dan bakteriekspresi pathogenesis-related (PR) protein:
β-1,3-glukanase, chitinase, thaumatin-like protein, protease inhibitor, asam salisilat
•chitinase menghidrolisis β-1,4 dari chitin N-acetyl-D-glucosamine (dinding sel fungi)
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Ekspresi asam salisilat di tanaman•Resisten terhadap fungi dan bakteri•Cara: Ekspresi gen penyandi enzim isochorismate synthase dan isochorismate lyase
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Tanaman toleran terhadap cekaman dan senesen
Ekspresi SOD:•tahan panas, herbisida methyl viologen (paraquat), layu (bunga potong)
Ekspresi glutation peroksidase (-s-transferase):•tahan garam, beku
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Toleran terhadap cekaman garam/kering
•perlu osmoprotectant: gula, alkohol, prolin, trehalose, D-ononitol, mannitol, sorbitol, glisin betain, 3-methylsulfoniopropionate
•ekspresi choline dehydrogenase (betA) betain
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Penundaan buah masak dan bunga layuBuah masak:•ekspresi polygalacturonase dan cellulase•diinduksi
dihambat: masak buah tertunda (layu bunga, gugur daun tertunda)
Bagaimana menghambat ekspresi?•RNA antisense thd gen penyandi•mengalihkan jalur sintesis
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Mengalihkan jalur sintesis etilenuntuk menghambat sintesis etilen
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Degradasi ACC sintesis etilen dihambat
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Manipulasi genetik pigmentasi bunga
Antosianin (flavonoid) pigmen bungaChrysanthemum + cDNA CHS sense/antisense bunga putih
Astaxanthin:•pink (salmon, udang)•dari bakteri & mikroalga•mencegah telur rusak karena UV•meningkatkan daya hidup & laju tumbuh antioksidan
Produksi astaxanthin:-ekspresi cDNA β-caroten ketolase (β-C-4 oxygenase) alga hijau (Haematococcus pluvialis)
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Modifikasi kandungan nutrisi tanaman
memperbaiki:-kualitas nutrisi: jagung, kacang polong (kandungan aa di biji)-komposisi asam lemak dari tanaman penghasil minyak-rasa buah dan sayuran
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Meningkatkan kandungan lisin
Over produksi lisin: menghentikan feedback inhibition oleh aspartokinase & dihydrodipicolinic acid synthase
ekspresi gen DHDPS lysin-feedback-insensitive dari Corynebacterium & AK lysin-feedback-insensitive dari E. colidikendalikan promoter biji biji mengandung 2-5 x lisin
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Kandungan lipid
Kanola transgenik: antisense gen stearate desaturase dari Brasicca akumulasi asam stearat(tidak menjadi asam oleat)
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Mengganggu RNA tanamanModifikasi fenotipe: downregulation dari ekspresi gen menghambat mRNACara:•ekspresi kopi tambahan (cosuppression)•ekspresi antisens•ribozim molekul kecil RNA endoribonuklease spesifik
stearoyl-ACP 9 desaturase: asam stearat asam oleat;stearat meningkat margarin dan minyak goreng
+ ribozim: asam oleat dihambat akumulasi asam stearat
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Vitamin AMamalia: β-carotene (provitamin A) vitamin Aβ-carotene: pigmen karotenoid pada membran tanaman fotosintetik
Ekspresi β-carotene di beras via A. t:1: Phytoene synthase (psy) (pglutellin) & phytoene desaturase (crt) (p35S) tanpa smg + peptidatransit plastid2: Lycopene b-cyclase (lcy) (pglutellin) + peptida transit plastid1+2 padi beras mengandung β-carotene shg berwarna kuning/emas
golden rice
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Modifikasi rasa dan tampilan tanaman
Polifenol oksidase: oksidasi monofenol/o-difenol o-quinon perubahan warna buah/sayuran
Perubahan warna dihambat dengan menghambat sintesis polifenol oksidase
Kentang:antisense polifenol oksidase +p35S/pGBSS
resisten thd black spotSense polifenol oksidase tidak tahan
(Granule-bound starch synthase)
Mencegah perubahan warna
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PemanisMonellin: -Dioscoreophyllum cumminsii-3000 x lebih manis darpd sukrosa-Dimer: rantai A 45 aa, B 50 aa dengan ikatan lemah nonkovalen-Rusak oleh: panas, asam
Pemanis rendah kalori
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Kandungan pati
Kentang: 20-30 amilosa (rantai lurus); 70-80% amilopektin (rantai bercabang) rasio keduanya: perubahan sifat fisik & kimia pati
Amilosa: ikatan α-1,4
Amilopektin: ikatan α-1,4 dan α-1,6
Biosintesis pati
Glukan+glukan α-1,6 amilopektin
p35S + antisense startch branching enzyme amilosa naik: 28% 60-80%
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Tanaman sebagai bioreaktor
Produksi agen terapetik di tanaman transgenik (skala lab)
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Antibodi
Keunggulan tanaman drpd bakteri: integrasi DNA di genom tanaman lebih stabil, protein mengalami pemrosesan spt di sel hewan, lebih murah (produksi antobodi per gram di sel hibridoma: $ 5000, bakteri: $ 1000, tanaman: $ 10-100), dapat disimpan di biji shg tahan lama pada suhu ruang
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Produksi biopolimer
Biosintesis poly (3-hydroxybutyric acid) dari acetyl-CoA Alcaligenes eutrophus
Acetyl Co-A Acetoacetyl Co-A D-3-Hydroxybutyryl co-A PHA
Acetoacetyl Co-A reductase Poly(3-hydroxybutyric acid) synthase3-ketothiolase
PHA: bioplastik, di A. eutrophus 1 operon tidak dikenal tanaman ekspresi 3 gen terpisah: A. thaliana target ke kloroplast@ gen + peptida transit di kloroplast ssu rubisco + p35S pTi biner1 x 2 tanaman (1+2) x 3 tanaman (1+2+3) 1 mg /g daun segar PHA
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Mengubah kandungan ligninLignin:tinggi mutu pulp dan hijauan pakan rendahrendah: antisense 4-Coumarate:coenzyme A ligase
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PERKEMBANGAN REKAYASA GENETIKA TANAMAN
• Adopsi tanaman transgenik pesat– 1996: 2.8 jt ha (AS, Kanada, Argentina,
Brasil, Afsel, China) 2003: 67.7 jt ha (18 negara)
– 4 komoditas penting: kedelai, jagung, kapas, kanola
– 2 sifat: resisten herbisida, resisten hama(penggerek)
• 2006: + negara, + komoditas (pepaya, padi, alfalfa, labu), + sifat (toleran virus
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Tahun Jumlah negara Luas (juta ha)1996 6 1.71997 8 11.01998 8 27.81999 14 39.92000 13 44.22001 13 52.62002 16 58.72003 18 67.72004 20 81.02005 21 90.02006 22 102.0
Jumlah negara dan luas tanaman transgenik(James 1996-2006)
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Melibatkan > 10 jt petani (90% petani kecil)
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No Crop 2002 (%)
2005 (million ha) % 2006
(million ha) %
1 Soybean 62
21
12
5
-
-
-
100
54.4 50 58.6 57
2 Maize 21.2 24 25.2 25
3 Cotton 9.8 11 13.4 13
4 Canola 4.6 5 4.8 5
5 Alfalfa - - <0.1 <1
6 Rice <0.1 <1 <0.1 <1
7 Others <0.1 <1 <0.1 <1
TOTAL 90.0 100 102.0 100
Luas tanaman transgenik di dunia, berdasarkan jenis tanaman
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No Trait 2005 (million ha) % 2006
(million ha) %
1 Herbicide Tolerance (HT) 63.7 71 69.9 68
2 Insect Resistance (Bt) 16.2 18 19.0 19
3 Bt/HT 10.1 11 13.1 13
4 Virus Resistance <0.1 <1 <0.1 <1
TOTAL 90.0 100 102.0 100
James 2006
Luas tanaman transgenik dunia, berdasarkan sifat