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PMID: 9290642 Published · ppublish English Comparative Study Journal Article

Cloning and characterization of the calreticulin gene from Ricinus communis L.

Plant molecular biology ·Vol. 34 ·No. 6 ·1997-08-00 ·Pages 897-911

Coughlan SJ, Hastings C, Winfrey R

Abstract

A full-length cDNA encoding a calreticulin-like protein was isolated by immune-screening a germinating castor bean endosperm cDNA library with antisera raised to the total lumenal fraction of purified plant endoplasmic reticulum. The calcium-binding properties of the recombinant protein were characterized and shown to be essentially identical to those reported for the mammalian calreticulin. Calcium overlays and immune blot analysis confirmed the endoplasmic lumenal identity of this reticuloplasmin. Probing protein blots of endoplasmic reticulum subfractions with radio-iodinated calreticulin showed specific associations with various polypeptides including one identified as the abundant reticuloplasmin protein disulfide isomerase. Characterization of the corresponding genomic clones revealed that calreticulin is encoded by a single gene of 3 kb in castor. The full genomic sequence reveals the presence of 12 introns, 12 translated exons, and one exon containing the last three amino acids of the translated sequence and the 3'-untranslated region of the gene. Northern blot analysis of RNA isolated from various organ tissues showed a basal constitutive level of expression throughout the plant, but more abundant mRNA being detected in tissues active in secretion. This was confirmed by analysis of transgenic tobacco plants containing 1.8 kb of 5'-untranslated genomic sequence fused to the beta-glucuronidase reporter gene (GUS) showed a more localized pattern of expression. Activity being localized to the vasculature (phloem, root hairs and root tip) in vegetative tissue, and being strongly expressed in the floral organs including the developing and germinating seed.

MeSH Terms
Amino Acid Sequence Base Sequence Calcium/metabolism Calcium-Binding Proteins/genetics Calreticulin Castor Bean/genetics Cell Compartmentation Chromatography, Affinity DNA, Complementary/genetics Endoplasmic Reticulum/genetics Escherichia coli/genetics Gene Expression Gene Expression Regulation, Plant Gene Library Genes, Plant Genes, Reporter Molecular Sequence Data Plants, Toxic Promoter Regions, Genetic/genetics Protein Binding RNA, Messenger/isolation & purification RNA, Plant/isolation & purification Recombinant Proteins/metabolism Ribonucleoproteins/genetics Seeds/genetics Sequence Analysis, DNA Sequence Homology Tissue Distribution Transformation, Genetic
Chemicals
Calcium-Binding Proteins Calreticulin DNA, Complementary RNA, Messenger RNA, Plant Recombinant Proteins Ribonucleoproteins Calcium
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Coughlan S J
Trait and Technology Development Department, Pioneer-Hi-Bred International, Johnston, IA 50131-1004, USA.
Hastings C
Winfrey R
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Article Info
Journal
Plant molecular biology
Abbr.
Plant Mol Biol
ISSN
0167-4412
DOI
10.1023/a:1005822327479
Published
1997-08-00
Pages
897-911
Language
English
Region
Netherlands
NLM ID
9106343
Subset
IM
Databases
GENBANK
U74630, U74631
External Links
PubMed source DOI: 10.1023/a:1005822327479 View journal details
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