Cloning and Characterisation of the cDNA Clones of three Genes that are differentially Expressed during Dormancy-Breakage in the Seeds of Douglas Fir ( Pseudotsuga menziesii)

Abstract

Subtractive hybridisation methodology and differential screening have been used to isolate cDNA clones of genes that show enhanced expression during cold, moist dormancy-breakage treatment (stratification) of Douglas fir (Pseudotsuga menziesii) seeds. This paper describes the isolation and characterisation of three of these cDNA clones. Sequence analysis reveals a high degree of similarity between the three cDNA clones and Late Embryogenesis Abundant (LEA) protein genes. Each of the three Douglas fir clones encodes a different class of LEA protein (Dure et al., 1989). One of the genes (DF65), showing strong sequence similarity to a dehydrin (class 2 LEA) gene, is expressed in seeds treated at 4 °C but not in those seeds treated at 20 °C. The other two genes (DF6 and DF77), which are highly similar to class 1 and class 3 LEA genes respectively, were expressed in a similar pattern during dormancy-breakage, with expression at 4 °C being maintained at a high level for four weeks of treatment, whereas in seeds incubated at 20 °C, expression level decreased after one week of treatment. It is shown that there is a family of genes related to DF65 and DF77 expressed in Douglas fir seeds. Possible roles for LEA proteins in dormancy-breakage of seeds is discussed.