Plant Genome IV Conference Plant Genome IV Conference
The 78 kDa glucose-regulated/immunoglobulin heavy chain binding protein (GRP 78/BiP) is an endoplasmic reticulum (ER)-localized chaperone protein that plays an important role in the folding and assembly of proteins in the ER. Using the polymerase chain reaction (PCR) technique we have cloned an homolog of GRP 78 gene from Arabidopsis thaliana. By testing various PCR parameters, we were able to repeatedly amplify a 1.6 kilobase fragment from the genomic DNA of Arabidopsis. An annealing temperature at 55C and a magnesium concentration of 2.5 mM appeared to be critical. The 5' degenerate primer sequence was based on the sequence homologies among mammalian GRP 78, maize b-70, yeast Kar-2, tobacco BiP, and hsp 70. The 3' primer sequence was derived from the ER-anchoring HDEL peptide sequence plus a 9-base region from a 226 bp fragment of an expressed sequence tag library of Arabidopsis. The size of the amplified sequence is comparable to that of the maize b-70, also a mammalian homolog of GRP 78. Its small size suggests that the amplified gene sequence contains few if any introns. Using an antibody against HDEL sequence, we have also screened the cDNA expression library (Genebank Z17760) which was derived from various types of Arabidopsis tissues. Many positive clones were identified, and were probed with several different GRP 78 homologs.