Cloning of srp gene from the gametophytes of Laminaria japonica and its expression in Escherichia coli

A male differentially expressed cDNA library subtracted from the female gametophytes of Laminaria japonica was constructed by use of suppression subtractive hybridization(SSH).A clone b9 was found to be a new differentially expressed gene using Southern dot-blotting.With the designed gene-specific primers on basis of Clone b9 sequence,a full length of cDNA was cloned by use of rapid amplification of cDNA ends(RACE).This gene was composed of 831 bp in sequence including a 182 bp 5′-untranslated region(UTR),a 199 bp 3′-UTR with a poly(A)tail,and a 450 bp open reading frame(ORF).There was no difference in sequence of this gene no matter where it was cloned from the females or the males.Protein homology searching result showed that it was homologous to Nostoc punctiforme SpoIID/LytB domain-containing protein(sporulation resulting in the formation of spores)with 30% similarity.This gene,therefore,was named sporulation-related protein gene(srp)(GenBank accession No.EF490313).The srp gene was ligated into pET-28a expression vector and was consequentially transformed into Escherichia coli BL21 competent cells.A recombinant protein was successfully expressed in prokaryotic cells with molecular weight of about 19.3 ku,and its expression was positively related to the content of inducer ITPG.Mass spectrometry analysis verified that the sequence of expressed recombinant protein was the same as the putative one on the basis of srp gene ORF sequence.The recombinant protein was purified from the transformed E.coli and its polyclonal antibody was obtained by inoculating immunologically rabbits.The SRP protein was proved to be present in the crude extracts from both the female and male gametophytes of L.japonica by using Western blot with the preparative antibody.The results lay a foundation for the further study on the isolation and characterization of SRP protein from gametophytes and on the investigation of function of SRP protein during the growth and development of L.japonica gametophytes.