中文摘要：

4-香豆酰辅酶A连接酶（4-coumarate：coenzyme A ligase,4CL）是木质素合成中的关键酶,根据植物中4CL保守序列设计引物,以尾叶桉GLU4嫩茎为材料克隆到其4CL基因,命名为Eu4CL.该基因gDNA长5 534 bp,cDNA长1 640 bp,CDS区编码544个氨基酸.Eu4CL核酸序列与GenBank已登录的桉属植物4CL基因同源性达到98％,与毛竹等其他科属植物4CL的同源性达77％以上,其编码的氨基酸序列经比对发现具有完整4CL结构域及AMP结合位点、CoA结合位点,与土肉桂等植物中4CL基因编码序列同源性也在79％以上,确定为4CL基因.对Eu4CL编码蛋白序列理化性质及结构进行生物信息学分析,利用MEGA软件对基因序列进行系统进化树分析.采用pQE30/M15系统对Eu4CL进行原核表达,重组质粒成功表达目的蛋白,分子量约60 ku.本研究从尾叶桉GLU4中克隆得到Eu4CL基因并原核表达,为该基因的酶学分析以及利用该基因转化调控尾叶桉木质素合成奠定基础.

英文摘要：

4-coumarate： coenzyme A ligase is a key enzyme in lignin synthesis pathway. A 4CL gene was cloned from the young stem of Eucalyptus urophylla clone GLU4 and named Eu4CL using specific primers based on the highly conserved sequences of plant 4CL. The gDNA and eDNA sequence were 5 534 bp and 1 640 bp respectively, while CDS encoded 544 amino acid residues. Eu4CL had 98% sequence homology with Eucalyptus that had been logged in GenBank, and its homology with Phyllostachys and others were over 77%. Eu4CL encoding sequence was analyzed, the result showed it has an entire 4CL domain, an AMP binding site and a CoA binding site, the homology with Cinnarnomum osmophloeum and others were over 79%. The physicochemical property, structure of Eu4CL and its phylogenetic analysis were analyzed using bioinformatics tools and MEGA. The SDS-PAGE analysis showed that Eu4CL was transfomled into pQE30/M15 system and fusion protein with molecular weight about 60 ku was successfully expressed in transformant. The cloning and prokaryotic expression of Eu4CL gene provided effective resources for enzymology research and futher research of transgenic.