菊芋果聚糖1-外切水解酶（1-FEH）基因克隆、表达和调控模式研究

项目名称： 菊芋果聚糖1-外切水解酶（1-FEH）基因克隆、表达和调控模式研究

项目编号： No.31201842

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 畜牧学与草地科学、兽医学、水产学

项目作者： 梁明祥

作者单位： 南京农业大学

项目金额： 23万元

中文摘要： 果聚糖1-外切水解酶（1-FEH）参与植物果聚糖的水解，提高植物抗逆境性能。菊芋作为一个富含果聚糖和耐受性高的草本植物，针对其体内1-FEH的研究目前还比较缺乏，菊芋1-FEH基因信息及逆境下的功能和调控研究几乎空白。因此，本项目拟克隆出菊芋多个1-FEH基因，并利用毕赤酵母异源表达系统鉴定并明确菊芋各1-FEH酶的基本特性。利用实时PCR和Western Blot技术测定菊芋各1-FEH在植物全生育期和不同逆境处理下的时空表达特征，同期检测植物中1-FEH酶活和植物果聚糖组成成分变化，明确各1-FEH在菊芋正常生长和逆境中的功能和作用方式。通过克隆各菊芋1-FEH的启动子，并在洋葱和菊芋外植体瞬时系统分别表达，探索菊芋各1-FEH在不同处理下表达差异的内在原因。通过本项目可以明确菊芋各1-FEH基因的功能与调控模式，为提高植物抗逆性能及工业化生产果糖类产品奠定理论基础。

中文关键词： 果聚糖水解酶；菊芋；基因表达；酶活；非生物胁迫

英文摘要： Fructan 1-exohydrolases (1-FEHs) participate in the degradation of plant fructan and contribute to abiotic tolerance. Among the angiosperms that store high amount of fructans, Jerusalem artichoke (Helianthus tuberosus) is one of the most noted due to abiotic resistance. Until now only limited information about Jerusalem artichoke's 1-FEHs was available. In the present work, cloning and function of Ht1-FEHs will be provided. The recombinant Ht1-FEHs will be expressed in Pichia pastoris for determination of some of its physical and kinetic properties. Through Real-Time PCR technique and Western Blot, the expression level of identified Ht1-FEHs will be quantified in a time-course experiment with or without abiotic treatment. Both 1-FEHs enzyme activity and fructan content will be monitored also. The regulatory mechanism behind different Ht1-FEHs expression was demonstrated through promoter analysis by transient expression assay(onion epidermal cell and Jerusalem artichoke tissue explant). In a conclusion, characterization of the Ht1-FEHs may provide tools to study abiotic tolerance in plants and to increase its industrial application in the future.

英文关键词： Fructan exohydrolase；Jerusalem artichoke；Gene expression；Enzyme Activity；abiotic stress