Differential Screening in a cDNA-Library from Spruce for Clones Associated with Forest Decline Reveals Accumulation of Ribulose-l,5-Bisphosphate Carboxylase Small Subunit mRNA

In this work we identified mRNA species in spruce (Picea abies L., Karst), which are expressed in altered concentration if the symptoms of forest decline are present. A cDNA library was constructed and ‘damage-associated’ clones were identified by differential screening. A procedure for polyA+-RNA preparation from needles of spruce was established which yielded a RNA fraction of sufficient purity for gel electrophoresis, hybridization and most importantly, for reverse transcription and the consecutive steps of molecular cloning. RNA isolated from a symptomless and a damaged tree was combined and a cDNA library in phage λ gt 10 was constructed. Three series of differential screening were carried out. In the first series radioactively labeled cDNA samples were prepared from poly A+-RNA from a symptomless tree and a damaged tree. Differential hybridization of 10,000 cDNA clones with the two different samples yielded 37 cDNAs with different levels of hybridization signals. In a second series samples for hybridization tests were prepared from a symptomless and from a damaged tree at three different seasons, July, September, and December. These six different samples were used to search for significant hybridization differences in 2,000 clones. Five clones could be identified which showed hybridization differences independent of the annual season. In a third series, we tried to differentiate between genetic variations of individual trees and differences solely induced by the damage. Two mixed samples were prepared, one from an ensemble of 10 symptomless and one of 10 damaged trees. Six cDNA clones could be identified, which exerted clear differences with the mixed probes from the symptomless ensemble and the damage ensemble, respectively. In Northern hybridizations the cDNA clones could be attributed to mRNAs of well defined length. After sequence analysis of the cDNA clones and homology search in a gene data bank two sequences could be identified. One clone codes for the small subunit of ribulose-l,5-bisphosphate carboxylase (Rubisco SS) and another for the subunit II of photosystem I. By quantitative Northern