厚壳贻贝M7lysin分子的克隆与表达

The cloning and expression of M7 lysin in Mytilus coruscus

M7 lysin位于贻贝精子顶体中,是溶解卵黄膜、促进受精作用的重要蛋白质,决定了贻贝种间精卵识别的特异性。采用同源克隆法得到厚壳贻贝M7 lysin分子,并在原核生物中对该分子进行重组表达。结果表明,扩增产物为540 bp左右的片段,进一步测序发现其开放阅读框cDNA为543 bp,与贻贝、地中海贻贝、盖勒贻贝具有较高的相似性;在线翻译所得蛋白质片段中含有180个氨基酸,分子量为20 ku,等电点为8.48;通过M7 lysin氨基酸序列构建系统进化树发现,贻贝和地中海贻贝亲缘关系最近,其次为盖勒贻贝,最后是厚壳贻贝;将M7lysin分子在E.coli Rosseta(DE3)中融合表达,得到包括载体氨基酸序列在内的25 ku蛋白质,符合预期分子质量。

M7 lysin is located in the mussel sperm acrosome.As an important protein for fertilization,it can dissolve vitelline membrane and determine the specificity of sperm-egg recognition of mussel.At present,the M7 lysin sequences of Mytilus edulis Linnaeus,M.galloprovincialis and M.trossulus have been recognized,but it hasn＇t been reported in M.coruscus.In this study,we cloned M7 lysin of M.coruscus with homology cloning method,and it was expressed in E.coli Rosseta（DE3）.The results showed that the amplified product was about 540 bp fragment.The further sequencing revealed that the cDNA of open reading frame was 543 bp,and it had high similarity with those of M.edulis,M.galloprovincialis and M.trossulus.The protein included 180 amino acids through online translation,its molecular weight was 20 ku,and its isoelectric point was 8.48.The phylogenetic tree from the amino acid sequence of M7 lysin showed that M.edulis and M.galloprovincialis had closest relationship,followed by M.trossulus,and finally M.coruscus.The results suggested that M7 lysin could be used as molecular markers to study mussel＇s evolution.The protein of 25 ku was showed in SDS-PAGE when M7 lysin was expressed in the prokaryotes,which included the amino acid sequences in vector.This band was consistent with the expected molecular weight.Disulfide bonds＇ positions in M7 lysin were highly conservative,which was similar to C-type lectin carbohydrates identification area（CRD）.We speculated that M7 lysin dissolved the yolk membrane through combining its sugar and sugar-based protein.The conclusions helped us to reveal the mussel＇s reproductive mechanism,and further provide a reference for the cross breeding of mussels.