中文摘要：

从已构建好的海带雄配子体抑制消减cDNA文库中,通过Southern点杂交及序列分析,发现一未知功能的差异表达基因片段（克隆b9）。首先根据该克隆序列设计基因特异性引物,利用cDNA末端快速扩增技术,自雄配子体中克隆了一条长831bp的cDNA序列,其中开放阅读框450bp,5＇-非翻译区182bp,3＇-非翻译区199bp且具有明显的poly（A）尾巴。同样利用PCR方法自雌配子体中克隆了该基因的cDNA序列,它与雄配子体的完全一致。蛋白同源搜索结果显示,该基因编码蛋白与点形念珠藻含有SpoIID/LytB结构域蛋白（SpoIID/Lyt Bdomain-containing protein：一种孢子形成时起作用的蛋白质）具有30%相似性,故暂命名为孢子形成相关蛋白基因（sporulation-related protein gene,srp）（GenBank登录号：EF490313）。然后构建了srp基因原核表达载体pET-28a-srp,并将其转化至大肠杆菌BL21中进行表达,获得了分子量大小约19.3ku的目的蛋白,且表达量与诱导物IPTG的量成正比。通过高效液相色谱-质谱分析证实,重组蛋白的氨基酸序列与推测的目的蛋白一致。最后纯化重组了SRP蛋白并制备其多克隆抗体,利用该抗体并运用Western印迹技术,在海带配子体中证实SRP蛋白的存在。

英文摘要：

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.japoni