US 7,001,739 B2 · Isolation of proteins involved in posttranscriptional gene silencing and methods of use

Abstract

The present invention includes a method for detecting and isolating sugarcane proteins that interact with the HC-Pro and P1 proteins of SrMV and other proteins involved in gene silencing, particularly in sugarcane. The method uses a two hybrid assay with an HC-Pro, P1, or other silencing-related protein-containing bait protein and a prey protein containing a polypeptide encoded by a DNA molecule in a cDNA library. The method also includes identification of false positives through reverse two-hybrid assays and using in vitro techniques such as farwestern blots or pull down assays where plant physiological conditions may be replicated. Finally, interactions may be confirmed in planta. Some novel proteins used in and discovered using the these methods are also identified. Methods of using viral and plant proteins to regulate silencing in plants such as sugarcane are also discussed.

Claims9

Dependent claims

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 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. 1 or a sequence of a degenerate code homolog of SEQ. ID. NO. 1.

1 more element

Dependencies are inferred from the claim text.

Isolation of proteins involved in posttranscriptional gene silencing and methods of use

Life of this patent

Isolation of proteins involved in posttranscriptional gene silencing and methods of use

Life of this patent

Abstract

Claims9