CLONING AND EXPRESSION OF 2 GENES ENCODING AUXIN-BINDING PROTEINS FROM TOBACCO

Two genes encoding the auxin-binding protein (ABP1) of tobacco (Nicoti ana tabacum L.), both of which possess the characteristics of a lumina l protein of the endoplasmic reticulum (ER), were isolated and sequenc ed. These genes were composed of at least five exons and four introns. The two coding exons showed 95% sequence homology and coded for two p recursor proteins of 187 amino acid residues with molecular masses of 21 256 and 21 453 Da. The deduced amino acid sequences were 93% identi cal and both possessed an amino-terminal signal peptide, a hydrophilic mature protein region with two potential N-glycosylation sites and a carboxyl-terminal sorting signal, KDEL, for the ER. Restriction mappin g of the cDNAs encoding tobacco ABP1, previously purified by amplifica tion of tobacco cDNA libraries by polymerase chain reaction (PCR) usin g specific primers common to both genes, indicated that both genes wer e expressed, although one was expressed at a higher level than the oth er. Genomic Southern blot hybridization showed no other homologous gen es except for these two in the tobacco genome. The apparent molecular mass of the mature form of tobacco ABP1 was revealed to be 25 kDa by S DS polyacrylamide gel electrophoresis using affinity-purified anti (to bacco ABP1) antibodies raised against a fusion protein with maltose-bi nding protein. Expression of the recombinant ABP1 gene in transgenic t obacco resulted in accumulation of the 25 kDa protein. A single point mutation of an amino acid residue at either of the two potential N-gly cosylation sites resulted in a decrease in the apparent molecular mass and produced a 22 kDa protein. Mutations at both sites resulted in th e formation of a 19.3 kDa protein, suggesting that tobacco ABP1 is gly cosylated at two asparagine residues.