Amplified Restriction Fragment Length Polymorphism-Based mRNA Fingerprinting Using a Single Restriction Enzyme That Recognizes a 4-bp Sequence

Abstract

Using amplified restriction fragment length polymorphism (AFLP) technology, we have developed a new protocol for the fingerprinting of mRNA that allows systematic comparison of the differential expression of genes between mRNA samples. The major advantage of our protocol is the use of only a single restriction enzyme that recognizes a 4-bp sequence but allows screening of large numbers of different cDNAs. Using this new protocol, we compared mRNA samples obtained from the flower buds of two lines of the common morning glory (Ipomoea purpurea) with red and white flowers, respectively. Approximately 50 bands were observed in each lane of a denaturing polyacrylamide gel and the results were highly reproducible, as indicated by the results of analysis of two sets of independent mRNA samples. Two cDNA fragments, which were differentially amplified in the samples from the two lines, were shown to have been derived from a single gene that was actively expressed in the buds of red flowers but not in those of white flowers. A full-length cDNA of this gene was cloned from a bud cDNA library. Sequence analysis showed that this cDNA carries a sequence highly homologous to the chalcone synthase (CHS) genes, the key enzyme in the flavonoid biosynthetic pathway.