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1. US20060275830 - Isolation of Proteins Involved in Posttranscriptional Gene Silencing and Methods of Use

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

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Claims

1. A method of isolating nucleic acid encoding a plant polypeptide active in PTGS comprising the steps of:
i) selecting a bait nucleic acid which encodes a bait protein active in PTGS in plants or suppressive of PTGS in plants;
ii) preparing a cDNA prey library from a plant or plant tissue wherein the plant or plant tissue actively exhibits PTGS;
iii) conducting a yeast two-hybrid assay with the bait and prey nucleic acids, wherein prey cDNA that yields a true positive yeast two-hybrid assay result encodes a polypeptide active in PTGS in the plant or plant tissue,
wherein the bait nucleic acid comprises a sequence of SEQ.ID.NO. 5 or a sequence of a degenerate code homolog of SEQ.ID.NO. 5.
2. The method of claim 1 further comprising preparing a cDNA prey library from a monocot plant.
3. The method of claim 2 wherein the monocot is selected from the group consisting of sugarcane, corn, sorghum and rice.
4. The method of claim 1 further comprising the step of confirming interaction between the bait protein and the polypeptide active in PTGS using a farwestern blot assay.
5. The method of claim 1 further comprising the step of confirming interaction between the bait protein and the polypeptide active in PTGS using a pull down assay.
6. The method of claim 1 further comprising the step of confirming interaction between the bait protein and the polypeptide active in PTGS using an in planta assay.
7. The method of claim 6 wherein the in planta assay is conducted in embryonic calli of the plant.
8. An isolated nucleic acid molecule comprising a nucleic acid molecule having at least 93% identity with the nucleic acid sequence of SEQ.ID.NO. 5 and operable to encode a RING zinc finger protein.
9. An isolated nucleic acid molecule comprising a nucleic acid molecule having the nucleic acid sequence of SEQ.ID.NO. 5 or the nucleic acid sequence of a degenerate code homolog of SEQ.ID.NO. 5.