| 陈博雯1，2，潘俊霞3，蔡文侠3，付珊3，姜伟3.尾叶桉GLU4 中4-香豆酰辅酶A连接酶
基因克隆及原核表达[J].广东农业科学,2014,41(12):150-155 |
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尾叶桉GLU4 中4-香豆酰辅酶A连接酶
基因克隆及原核表达 |
| Gene cloning and prokaryotic expression of 4-coumarate:coenzyme A ligase in Eucalyptus urophylla clone GLU4 |
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| DOI： |
| 中文关键词: 尾叶桉 4-香豆酰辅酶A 连接酶 生物信息学分析 原核表达 |
| 英文关键词: Eucalyptus urophylla 4-coumarate: coenzyme A ligase bioinformatics analysis prokaryotic expression |
| 基金项目:广西优良用材林资源培育重点实验室开放课题
基金(12A0302）；国家自然科学基金(31100473） |
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| 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 cDNA 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 Cinnamomum 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 transformed 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. |
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