Isolation of a putative endogenous endo-ß-1,4-glucanase (cellulase) from the midgut diverticulae of the wood-boring crustacean, Limnoria quadripunctata
J Dymond, M J Guille, S M Cragg
Over the last 75 years, many workers have conducted research into the digestive biochemistry of Limnoria. Cellulolytic enzymes have been detected in crude extracts, although whether these are of endogenous or microbial origin has been in question. To elucidate the source of these enzymes, RNA was isolated from the midgut diverticulae of Limnoria quadripunctata. Reverse transcription polymerase chain reaction (RT-PCR) was performed using degenerate primers derived from conserved regions of known endoglucanases. This produced a fragment of cDNA that was extended by rapid amplification of complementary DNA ends (RACE). The overlapping cDNA fragments were cloned and sequenced, and the amino acid sequence of a putative protein was deduced. BLAST analysis was performed to determine the similarity between this sequence, and those of known proteins. The results indicated a conserved structural domain of a glycosyl hydrolase, and the greatest sequence homology was to endo-ß-1,4-glucanases from termite species. In situ hybridisation was performed using labelled sense and antisense probes. This indicated the presence of a cellulase-encoding mRNA in the tissues of the midgut diverticulae. These results provide the strongest evidence to date of endogenous endoglucanase production by Limnoria.
Keywords: Cellulase, endoglucanase, Limnoria, crustacean, in situ hybridisation