ISOLATION OF A FULL-LENGTH CDNA-ENCODING CALRETICULIN FROM A PCR LIBRARY OF IN-VITRO ZYGOTES OF MAIZE

A full-size cDNA clone (1614 bp) encoding calreticulin was isolated fr om a PCR-based cDNA library of maize in vitro zygotes. Calreticulin is a major Ca2+ storage protein located mainly in the lumen of the endop lasmic reticulum but also in the nucleus and/or cytoplasm of some cell s. A differential screening between cDNA libraries originating from 10 4 in vitro zygotes (18 h after in vitro fertilization) and 128 unferti lized egg cells was performed to isolate newly expressed genes or gene s expressed more abundantly after fertilization. The expression of the isolated cDNA clone is enhanced after fertilization and strongly corr elated to cell division. Sequence comparison to a shorter maize calret iculin cDNA isolated from a conventional cDNA library proves the abili ty and reproducibility of the recently described method for PCR based cDNA library construction from a few plant cells [12]. It is further s hown that calreticulins in maize are probably transcribed from a small gene family differentially expressed in abundance in diverse tissues. The deduced amino acid sequence encodes an acidic protein (pi 4.17) o f 48 kDa sharing 77-92% and 50-54% homology to other plant and animal calreticulins, respectively. The described calreticulin gene represent s to our knowledge the first cDNA clone isolated from a RT/PCR cDNA li brary originating from only a few plant cells and is the first gene is olated from zygotes of higher plants.