Gene cloning, nucleotide analysis, and overexpression in Escherichia coli of a substrate-specific indole-3-acetyl-L-alanine hydrolase from Arthrobacter ilicis

摘要

Indole-3-acetic acid (IAA) is an essential plant hormone and plays many important roles in plant growth and development. In plants IAA is found mostly as conjugates, thus studies of IAA-conjugated hydrolases may provide clues on the function and metabolism of IAA during plant growth and development. We studied an alanine-conjugated IAA hydrolase in bacteria Arthrobacter ilicis in order to clone IAA conjugate hydrolase genes., and a gene coding for an IAA-Ala hydrolase was successfully cloned and sequenced without the need of protein preparation and analysis. The procedure involved the design of universal degenerate PCR primers for IAA amidohydrolases with similar sequence alignment to IAA amidohydrolases found in the GenBank database. Then, more pairs of specific PCR primers were generated based on degenerate PCR products. Real time PCR was performed to determine which PCR products were inducible under specific conditions. A colony screening procedure was performed later to screen a partial genomicDNA library. Based on this study, a gene with an ORF of 1218 nucleotides was found and then overexpressed in E. coli. The enzyme activity assay confirmed this gene as IAA-Ala hydrolase gene.