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Molecular cloning and characterization of an endo-β-mannanase gene expressed in the lettuce endosperm following radicle emergence

Endo-β-mannanase (EC 3.2.1.78), an enzyme that mobilizes the endosperm cell walls of the lettuce (Lactuca sativa L.) seed, increases in activity in the micropylar and lateral regions of this tissue following the completion of germination. Its complementary DNA (cDNA) sequence (LsMan1) was determined using the polymerase chain reaction (PCR) with degenerate primers. The 3‘-end of the cDNA sequence was obtained by 3‘-end rapid amplification of cDNA ends (RACE), and the 5‘-end sequence by genome walking and 5‘-end RACE. The predicted amino acid sequence from the cDNA has a high identity with endo-β-mannanases present in other species (e.g. 67% identity with coffee β-1,4-mannan endohydrolase, 62% identity with tomato fruit endo-β-mannanase). Southern blot analysis suggests the presence of several members of an endo-β-mannanase gene family in the lettuce genome. Several isoforms of the enzyme, including three major ones, were detected by isoelectric focusing. Based on Northern blot analysis, accumulation of the endo-β-mannanase mRNA occurred only after lettuce seeds had germinated, and increased thereafter, although enzyme activity persisted after transcription declined.