大豆橙花叔醇合酶基因克隆及功能鉴定

Cloning and functional characterization of soybean gene Gm NES encoding nerolidol synthase

通过同源检索得到大豆橙花叔醇合酶(nerolidol synthase,Gm NES)基因Glyma13g32380,采用RT-PCR方法从经水杨酸(SA)处理的大豆材料中,克隆到c DNA片段,其包括完整的ORF(1 605bp)。共编码534个氨基酸,蛋白分子量约62.338 k D。该蛋白序列与番茄等植物的NES序列同源性较高,包含DDXXD保守结构域。将该ORF构建入p ET28a表达载体,在大肠杆菌BL21(DE3)中表达为可溶性蛋白。通过与法尼基焦磷酸合酶基因在大肠杆菌中进行微生物代谢工程共表达,气质联用检测到橙花叔醇的生成。同时还检测到SA模拟生物胁迫处理诱导大豆叶片产生橙花叔醇,并且在1.0 mmol/L SA处理下,Gm NES基因表达量明显上调。本研究克隆了大豆橙花叔醇合酶基因,并鉴定了其生化功能及其在植物体内的生理相关性,为研究大豆萜类代谢及对病虫害的防御反应和抗性品种选育提供了理论依据。

Nerolidol and its metabolic derivative DMNT are terpenoids,involved in plant defence against pathogen infection and herbivory. Nerolidol synthase（ NES） gene was obtained through homology blast from Phytozome database with accession number Glyma13g32380,named Gm NES. The c DNA of Gm NES was amplified from soybean leaf with 1. 0 mmol / L salicylic acid（ SA） treatment,which included the complete open reading frame（ ORF） of 1 605 bp,encoding 534 amino acids with predicted molecular weight of 62. 338 k D. Alignment analysis showed that the deduced protein had extensive homology with NES from other plants,containing a conserved DDXXD domain. The ORF was ligated into p ET28 a vector,and expressed in E. coli BL21（ DE3） as soluble protein. Coexpression of Gm NES with trans farnesyl diphosphate（ FPP） synthase from E. coli through metabolic engineering resulted in nerolidol formation with GC- MS analysis in comparison to the authentic standard. In the mean time,SA treatment induced Gm NES gene expression in soybean leaves and nerolidol accumulation,and constitutive gene expression and trace amount of nerolidol were observed in control. In this study,the nerolidol synthase gene of soybean was successfully cloned,and their functional role was identified as to catalyze nerolidol formation from trans- FPP,as well as the physiological relevance in planta,which sheds light on the soybean terpenoid meatablism in defense response and breeding for resistance.