J. Ache Gana

Document Type

Thesis - University Access Only

Award Date

1996

Degree Name

Doctor of Philosophy (PhD)

Department / School

Plant Science

Abstract

Proteins associated with the cell membranes may play a crucial role in maintaining membrane integrity during freeze stress. Therefore, in order to further understand the mechanism of freeze resistance, isolation and characterization of cDNAs for membrane proteins is important. This study describes the isolation of a wheat cDNA clone (pTACR7) by PCR. Its nucleotide sequence was very high in homology (67-87 %) with the coding regions of various barley and rye cDNAs for cold-regulated genes. Deduced amino acid sequence of pT ACR 7 is highly hydrophobic with a single transmembrane domain and an amino acid bias of leucine (19%). Northern blot hybridization analysis with the cDNA clone (pTACR7) on ABA or cold-treated tissue revealed that TACR7 is cold-regulated but not ABA-regulated. Differential expression of the TACR7 gene at the transcript level in freeze resistant (FR) versus freeze susceptible (FS) lines was obtained by evaluating seedlings, crown tissue, and callus at 2 °C temperature at different time intervals. Transcript for T ACR 7 gene was found to accumulate as early as six hours in the cold, suggesting it might be involved in early signaling events of cold acclimation. Other environmental stresses such as salt, dehydration, and heat resulted in no accumulation of T ACR 7 transcript. T ACR 7 is thus considered a cold-specific regulated gene. Southern blot analysis of T ACR 7 gene in FR and FS plants showed polymorphism at the Bel I restriction sites. Also, more than one band was obtained with EcoR I, Hind III, and Bel I, suggesting more than a single copy in the genome or possibly a small gene family. To aid in purification, a recombinant protein was developed with six histidine His(6) molecules as an N-terminal tag to TACR7 protein. The His(6)-TACR7 protein was expressed in E. coli and represented 0.5% of total bacteria protein. The His(6)- T ACR 7 protein was used as antigen for polyclonal antibody production. Antibodies present in the rabbit in the pre-immune serum cross-hybridized with the recombinant protein in addition to anti-His(6)-TACR7 sera from subsequent boosts. Serum from other rabbits (controls) and animals not immunized with His(6)-TACR7 protein showed that antibodies to the recombinant protein are not universal in all rabbits or other organisms. This suggests that the rabbit may have been under stress and therefore it probably produced antibodies to stress proteins that have similar antigenic determinants as T ACR 7 protein, being itself a stress protein. Such antibodies will be used in the future to determine accumulation kinetics of T ACR 7 protein in cold-treated plants.

Library of Congress Subject Headings

Winter wheat -- Frost resistance

Winter wheat -- Genetics

DNA

Membrane proteins

Format

application/pdf

Number of Pages

89

Publisher

South Dakota State University

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Rights Statement

In Copyright