Use of a fast protein electrophoretic purification procedure for N-terminal sequence analysis to identify S-locus related proteins in stigmas of Brassica oleracea.
In the cruciferous plant Brassica oleracea L. (cabbage), the S-locus specific glycoproteins (SLSGs) isolated only in stigmas are considered to play an important role in the normal prevention of self-fertilization. Recent molecular data have shown that the gene encoding these glycoproteins (the SLG gene) belonged to a multigenic family consisting of about 10 homologous copies among which another member is expressed, the S-locus related gene (SLR1gene). Our aim was to determine whether the SLR1-gene proteins were expressed in the stigmatic tissues. We first identified the putative SLSGs or SLR1-proteins by Con A-peroxidase detection of glycoproteins separated after isoelectric focusing in polyacrylamide gels. We describe a fast purification procedure for the glycoproteins of interest, based on analytical isoelectric focusing, electrophoresis, and electroblotting of proteins onto polyvinylidene difluoride membranes. Blotted proteins were sequenced for N-terminal amino acid determination. By comparison of the N-terminal sequences of the purified proteins with the peptide sequence predicted from the SLR1-cDNA, we demonstrate the expression of SLR1-like proteins in stigmas of B. oleracea.
Gaude
T
T
RCAP-INRA, UCB Lyon I, Villeurbanne, France.
Denoroy
L
L
Dumas
C
C
D000595:Amino Acid Sequence; D001937:Brassica / Q000235:genetics*; D002874:Chromosome Mapping; D004586:Electrophoresis / Q000379:methods*; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics* / Q000302:isolation & purification
D010940:Plant Proteins