枇杷悬浮细胞AS基因的瞬时表达载体构建与亚细胞定位  

Transient Expression Vector Construction of AS Gene from Suspension Cells of Eriobotrya japonica and Its Subcellular Localization

作　　者：李惠华[1] 曾碧玉[1] 王伟[1] 常强[1] 苏明华[1] LI Hui-Hua;ZENG Bi-Yu;WANG Wei;CHANG Qiang;SU Ming-Hua(Fujian Key Laboratory of Physiology and Biochemistry for Subtropical Plant,Fujian Institute of Subtropical Botany,Xiamen 361006,China)

机构地区：[1]福建省亚热带植物研究所福建省亚热带植物生理生化重点公共实验室

出　　处：《农业生物技术学报》2019年第11期1923-1932,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31501735)

摘　　要：枇杷(Eriobotrya japonica)细胞悬浮培养可以产生丰富的五环三萜类化合物。香树素合成酶(amyrine synthase,AS)是三萜合成途径中的关键酶,决定主要代谢和次生代谢的分支点。枇杷AS基因的功能未见报道。本研究以AS基因过表达质粒(pDONR207-AS)为模板,经PCR扩增获得AS基因的正向开放阅读框,经酶切及与载体的连接,成功构建表达载体pBWA(V)HS-AS-GFP-Tnos。以枇杷悬浮细胞(AS分离的同源系统)为受体材料,在农杆菌(Agrobacterium tumefaciens)GV3101的介导下进行瞬时表达,通过共转化的GFP在激光共聚焦显微镜下成像,分析AS蛋白的亚细胞定位;采用气相色谱-质谱联合分析AS催化产物。研究结果显示,AS在枇杷悬浮细胞中定位于内质网;枇杷悬浮细胞中AS的催化产物含α-香树素和β-香树素,AS属于多功能酶。本研究可为明确五环三萜类化合物合成途径以及枇杷悬浮细胞的转基因应用提供参考依据。Suspension culture of Eriobotrya japonica(loquat)can produce rich pentacyclic triterpenoids.Amyrine synthase(AS),a key enzyme in the triterpenoid synthesis pathway,determines the branching point of major metabolism and secondary metabolism.But the function of the AS in Eriobotrya japonica has not been reported before.In this study,using the AS overexpression plasmid pDONR207-AS as the template,the positive open reading frame of the AS gene was amplified by PCR,then ligated into the fusion expression vector pBWA(V)HS-GFP after digestion to construct the expression vector pBWA(V)HS-AS-GFP-Tnos.Next,using loquat suspension cells(homologous system with AS isolated)as receptor materials,transient expression was carried out under the mediation of Agrobacterium tumefaciens GV3101.Finally,the subcellular localization of AS protein were analyzed by imaging of co-transformed GFP under laser confocal microscopy,and the catalytic products of AS protein were analyzed by conjoint analysis of gas chromatography-mass spectrometry.Results were as follows:The subcellular localization of AS protein was in the endoplasmic reticulum in loquat.And the catalytic products of AS protein containedα-amyrin andβ-amyrin,which indicated that AS of loquat belonged to the multifunctional enzyme.This study provides a reference for improvement of the synthesis of pentacyclic triterpenoid and for the further transgenic application in suspension cells of loquat.

关 键 词：香树素合成酶(AS) 枇杷 悬浮细胞 瞬时表达 亚细胞定位 

分 类 号：S667.3[农业科学—果树学]