By Gary D. Foster, Sally Taylor
A finished number of state of the art thoughts for producing transgenic crops which are immune to plant viruses through the cloning and expression of the coat protein gene. Its unfailingly reproducible tools, perfected by way of hands-on masters, hide the complete procedure from virus isolation, RNA extraction, and cloning coat protein genes, to the advent of the coat protein gene into the plant genome and the trying out of transgenic vegetation for resistance. tools for trying out for transformation through PCR and Southern blotting, the detection of RNA transcripts via Northern blotting, and the creation of protein by way of Western research are supplied, as are equipment for tough the transgenic vegetation produced and for detecting and measuring the degrees of virus.
Read Online or Download Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology) PDF
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Additional info for Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology)
We have often found that DNA Isolated from maize plants infected with severe isolatesof MSV contains such high amountsof RF-DNA that further treatment to enrich for RF-DNA is unnecessary However, plants showmg milder symptoms will usually yield lessRF-DNA 50 Palmer, Schnippenkoetter, and Rybicki 8 It may be difficult to resuspend large amounts of plant total nucletc acids m such a small volume of buffer P 1 If larger volumes are required, scale up the amounts of P2 and P3 used, proportionally 9 A high concentration of residual plant DNA after the alkaline lysis procedure may congest the column To remedy this problem, precipitate the supernatant from step 5 with 0 8 vol of isopropanol, pellet the DNA m a microcentrifuge and repeat the alkaline denaturation procedure (steps l-5) 10 Trace amounts of contammation with genomic DNA are unavoidable, however, the RF-DNA preparation is usually clean enough for standard molecular mampulations hke restriction mappmg, clonmg, and even direct DNA sequencing References 1 Lazarowitz, S G (1992) Gemmtvuuses genome structure and gene function Crlt Rev Plant Scl 11,327-349 2 Timmermans, M C P , Das, 0 P, and Messing, J.
A , and Summers,M D , eds), Springer-Verlag, Vienna, pp 158-l 65 4 Rybicki, E P (1994)A phylogenetic and evolutionary Justification for three genera of Gemmivmdae Arch Vcrol 139,49-77 5 Bisaro, D M (1996) Gemunvu-usreplication, m Eukaryotzc DNA Replzcatlon (De Pamphtlis, M , ed ), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 6 Laufs, J , Traut, W, Heyraud, F, Matzeit, V, Rogers, S G , Schell, J , and Gronenborn, B (1995) In wtro cleavage and Joining at the viral origin of rephcanon by the replication initiator protem of tomato yellow leaf curl virus Proc Nat1 Acad Scz USA 92,3879-3883 7 Laufs, J , Jupm, I , David, C , Schumacher, S , Heyraud-Nitschke, F , and Gronenborn, B (1995) Gemimvirus replication genetic and biochemical characterisation of Rep protein function, a review Blochunze 77, 765-773 8 Stanley, J.
Covey et al. 56 3. Sterilized glass rod with tip flattened obliquely 4. Sodium phosphate buffer, pH 7 2 Stock solutions, 0 5M Na2HP04 (17 91 g/100 mL), 0 SMNaH,PO, (7 8 g/100 mL) For 200 mL of buffer, mix 144 mL of 0 5M Na,HP04 with 56 mL 0 5M NaH,PO,, cool to 4’C 5 Solid sodium sulfite (0 75%). Weigh out 1 5 g per 200 mL extraction buffer 6 Solid urea (1M). Weigh out 12 g for 200 mL buffer 7. 10% Trlton X-100 (stock solution) 8. DNase buffer. 50 mMTris-HCl, pH 7 5, 5 mA4MgC1, 9. Phenol:chloroform mixture.