尾叶桉GLU4肉桂酰-辅酶A还原酶基因克隆及原核表达  

Cloning and prokaryotic expression of cinnamoyl Co-A reductase of Eucalyptus urophylla clone GLU4

作　　者：陈博雯[1,2] 盖颖[1] 蒋湘宁[1] 

机构地区：[1]北京林业大学生物科学与技术学院,北京100083 [2]广西林科院广西优良用材林资源培育重点实验室,广西南宁530002

出　　处：《中南林业科技大学学报》2014年第11期71-76,97,共7页Journal of Central South University of Forestry & Technology

基　　金：国家自然科学基金项目(31400522);广西林业科技项目(桂林科字[2014]第33号);广西优良用材林资源培育重点实验室开放课题基金项目(12A0302)

摘　　要：肉桂酰-辅酶A还原酶(Cinnamoyl Co-A Reductase,CCR)是木质素合成中的关键酶,根据植物中CCR保守序列设计引物,以尾叶桉GLU4嫩茎为材料克隆到其CCR基因,命名为Eu CCR。该基因g DNA长2 918bp,c DNA长1 045 bp,CDS区编码336个氨基酸。EuCCR核酸序列与Gen Bank已登录的桉属植物CCR基因同源性达到96%以上,与伞房属、杯果木属植物CCR的同源性达85%以上,其编码的氨基酸序列经比对发现具有完整FR_SDR_e结构域及NADP结合位点和底物结合位点,与可可树等植物中CCR基因编码序列同源性也在84%以上,确定为CCR基因。对EuCCR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EuCCR进行原核表达,重组质粒成功表达分子量约36 kD的目的蛋白。本研究从尾叶桉GLU4中克隆得到EuCCR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。Cinnamoyl Co-A reductase is a key enzyme in lignin synthesis pathway. A CCR gene was cloned from the immature stem of Eucalyptus urophylla clone GLU4 and named EuCCR by using specific primers based on the highly conserved sequences of plant CCR. The gDNA and eDNA sequence of EuCCR were 2918 bp and 1045 bp respectively, which CDS encodes 336 amino acid residues. EuCCR had more than 96% sequence homology with Eucalyptus that had been logged in GenBank, and its homology with Angophora and Corymbia were over 85%. EuCCR encoding sequence was analyzed, the result shows it has an entire FR_SDR_e domain, a NADP binding site and a substrate binding site. The homology with Theobroma cacao and others were over 84%. The physicochemical property, structure of EuCCR and its phylogenetic analysis were analyzed by using bioinformatics tools and MEGA. The SDS-PAGE analysis showed that EuCCR was transformed into pQE30/M15 system and fusion protein with molecular weighting about 39 kD was successfully expressed in transformant. The cloning and expression of EuCCR gene provided some effective resources for enzymology research and further transgenic research.

关 键 词：尾叶桉 肉桂酰-辅酶A还原酶 生物信息学分析 原核表达 

分 类 号：S792.39[农业科学—林木遗传育种]