桑树4CL基因家族的筛选和Mm4CL2的功能研究  

Screening of 4CL Family Genes in Mulberry and Functional Study of Mm4CL2

作　　者：侯冲 晁楠 戴明洁 刘利 Hou Chong;Chao Nan;Dai Mingjie;Liu Li(School of Biotechnology,Jiangsu University of Science and Technology,Zhenjiang Jiangsu 212100,China;Sericultural Research Institute,Chinese Academy of Agricultural Sciences,Zhenjiang Jiangsu 212100,China)

机构地区：[1]江苏科技大学生物技术学院,江苏镇江212100 [2]中国农业科学院蚕业研究所,江苏镇江212100

出　　处：《蚕业科学》2022年第1期18-24,共7页ACTA SERICOLOGICA SINICA

基　　金：农业农村部农作物种质资源保护与利用项目(19200382);科技部、财政部国家科技资源共享服务平台课题(NCGRC2020-041);财政部和农业农村部国家现代农业产业技术体系项目。

摘　　要：4-香豆酸辅酶A连接酶(4-coumarate:coenzyme A ligase)是苯丙烷类代谢途径中的重要限速酶。在川桑基因组数据库的基础上利用全基因组筛选方法鉴别出6个Mn4CL同源基因,命名为Mn4CL1~Mn4CL6。蛋白质功能域分析发现6个Mn4CL蛋白均包含保守结构域BoxⅠ和BoxⅡ,都属于ANL酶超家族。系统发生分析表明,Mn4CL1、Mn4CL2、Mn4CL4与已报道的参与木质素生物合成的4CL聚类在一起,属于第Ⅰ类;Mn4CL3属于第Ⅱ类,参与黄酮类化合物的生物合成;Mn4CL5和Mn4CL6属于4CL类似蛋白。以此为基础,从湖桑32号叶片中克隆出一条长为1641 bp的cDNA序列,命名为Mm4CL2,构建重组质粒pET-28a-Mm4CL2并成功诱导表达出Mm4CL2蛋白。初步的酶学活性分析发现Mm4CL2蛋白能够分别催化香豆酸、咖啡酸和阿魏酸形成其相应的肉桂酰CoA,而不能催化芥子酸。本研究初步鉴定出一个桑树4CL基因Mm4CL2,为基因工程定向选育低木质素的桑树品种提供了新的候选基因。4-Coumarate:coenzyme A ligase is an important rate-limiting enzyme in the phenylpropane metabolic pathway.In this study,six Mn4CL homologous genes,named Mn4CL1-Mn4CL6,were identified by genome-wide screening from the genomic database of Morus notabilis.Analysis on protein function domains showed that six Mn4 CL proteins contained conserved BoxⅠand BoxⅡstructure domains,belonging to ANL enzyme family.Phylogenetic analysis showed that Mn4 CL1,Mn4 CL2 and Mn4 CL4 clustered with the reported 4 CL which was involved in lignin biosynthesis,belonging to the CladeⅠ;Mn4 CL3 belonged to the CladeⅡ,participating in the biosynthesis of flavonoids;Mn4 CL5 and Mn4 CL6 belonged to 4 CL-like proteins.On this basis,a 1641 bp cDNA sequence named Mm4CL2 was cloned from the leaf of Husang 32,and the recombinant plasmid pET-28 a-Mm4 CL2 was constructed and successfully induced to express Mm4 CL2 protein.Preliminary enzymatic activity analysis showed that Mm4 CL2 could catalyze coumaric acid,caffeic acid and ferulic acid to form their cinnamyl CoA respectively,but could not catalyze erucic acid.Results showed that Mm4CL2 might be a new candidate gene for genetic engineering selection of low lignin mulberry varieties in the future.

关 键 词：桑树 木质素 4-香豆酸辅酶A连接酶 基因家族 酶学活性 

分 类 号：S888.2[农业科学—特种经济动物饲养]