当归转录因子AsMYB44的克隆与功能研究

Cloning and functional research of MYB transcription factor AsMYB44 from Angelica sinensis

为明确AsMYB 44调控次生代谢产物生物合成的功能,从当归(Angelica sinensis)中分离克隆得到MYB转录因子AsMYB 44,对AsMYB 44进行生物信息学分析,利用根癌农杆菌介导的遗传转化体系在Samsun烟草中过表达,并对转基因烟草进行多组学联合分析。结果表明,AsMYB 44基因CDS(coding sequence)全长903 bp,编码300个氨基酸,具有完整的SANT、MYB domains结构域。系统发育树结果表明,AsMYB 44与拟南芥AtMYB 11(AF062863.1)、AtMYB 12(AF062864.1)和丹参SmMYB 97(KF059561.1)同源性较高。AsMYB 44转基因烟草中差异表达基因主要富集在苯丙烷合成代谢途径,其中苯丙氨酸解氨酶基因(PAL)、肉桂酸-4-羟化酶基因(C4H)、4-香豆酸:辅酶A连接酶基因(4 CL)、咖啡酸3-O-甲基转移酶基因(COMT)、肉桂醇脱氢酶基因(CAD)等表达量上调。广泛靶向代谢组学结果显示,转基因烟草中酚酸、脂质、黄酮类化合物、生物碱等代谢产物含量变化显著。AsMYB 44转录因子可正向调控酚酸的代谢合成,这可以为当归酚酸次生代谢物的生物合成机制提供理论基础。

In order to study the mechanism of biosynthetic regulation of Angelica sinensis phenolic acids,a MYB transcription factor AsMYB 44 gene was cloned from Angelica sinensis and the relevant bioinformatics analysis of the sequence was carried out using biological software.The Agrobacterium tumefaciens-mediated genetic transformation system was overexpressed in Samsun tobacco,and a multi-omics combined analysis of the transgenic tobacco was performed.The CDS(coding sequence)of the AsMYB 44 was 903 bp,encoding 300 amino acids,and with the complete SANT and MYB domains.Phylogenetic analysis showed that AsMYB 44 had higher homology with Arabidopsis thaliana AtMYB11(AF062863.1),AtMYB12(AF062864.1)and Salvia miltiorrhiza SmMYB 97(KF059561.1).The results of transcriptome sequencing showed that the differentially expressed genes were mostly enriched in the phenylpropane synthesis and metabolism pathway in AsMYB 44 transgenic tobacco.Expression levels of phenylalanine ammonia lyase gene(PAL),cinnamic acid-4-hydroxyl(C4H),4-coumaric acid:coenzyme A ligase gene(4 CL),caffeic acid 3-O-methyltransferase gene(COMT),cinnamyl alcohol dehydrogenase gene(CAD)and other structural genes were upregulated.Metabolomic analysis showed that the contents of metabolites such as phenolic acids,lipids,alkaloids,flavonoids and other compounds in transgenic tobacco changed significantly.These results indicated that the AsMYB 44 transcription factor could positively regulate the metabolic synthesis of phenolic acids,which provided a theoretical basis for the biosynthesis mechanism of phenolic acid secondary metabolites in Angelica sinensis.