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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 (CCR) is a key enzyme in lignin synthesis. According to the CCR conserved sequence in plant, CCR genes were cloned from the tender shoots of GLU4 Eu CCR. The gene g DNA length of 2 918bp, c DNA length 1 045 bp, CDS coding for 336 amino acids. The homology between the EuCCR nucleic acid sequence and the GenBank registered Eucalyptus CCR gene was more than 96%, and the homology with the CCR genera of Corymbutan and Cuprum genus was over 85%. The amino acid sequence of EuCCR was found by comparison It possesses the complete FR_SDR_e domain and NADP binding site and substrate binding site. Its homology with the CCR gene in plants such as cacao is more than 84%, which is identified as CCR gene. The physical and chemical properties and structure of EuCCR protein sequence were analyzed by bioinformatics, and phylogenetic tree analysis of gene sequence was carried out by MEGA software. EuCCR was expressed in pQE30 / M15 system. The recombinant plasmid successfully expressed the target protein with molecular weight of about 36 kD. In this study, the EuCCR gene was cloned from Eucalyptus urophylla GLU4 and prokaryotic expressed, which laid the foundation for the enzymatic analysis of the gene and the regulation of the lignin synthesis of Eucalyptus urophylla.