启动子探针载体的构建及橡胶树白粉菌启动子筛选鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2018-0584

生物技术通报 ›› 2019, Vol. 35 ›› Issue (1): 51-57.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0584

启动子探针载体的构建及橡胶树白粉菌启动子筛选鉴定

徐良向, 刘耀, 廖小淼, 王义, M.J.N.Rajaofera, 何其光, 刘文波, 林春花, 缪卫国

海南大学热带农林学院热带农林生物灾害绿色防控教育部重点实验室,海口 570228

收稿日期:2018-06-28 出版日期:2019-01-26 发布日期:2019-01-23
作者简介:徐良向,男,硕士研究生,研究方向：病原微生物;E-mail：1054283820@qq.com
基金资助:
国家自然科学基金项目（31660033,31560495）,国家重点研发计划（2018YFD0201105）,海南自然科学基金创新研究团队项目（2016CXTD002）,海南省重点研发计划项目（ZDYF2016208）,现代农业产业技术体系建设专项资金项目（CARS-33-GW-BC1）

Construction of Promoter Probe Vectors,and Screening and Identification of Oidium heveae Promoters

XU Liang-xiang, LIU Yao, LIAO Xiao-miao, WANG Yi, M.J.N. RAJAOFERA, HE Qi-guang, LIU Wen-bo, LIN Chun-hua, MIAO Wei-guo

Institute of Tropical Agriculture and Forestry,Hainan University/Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests（Hainan University）,Ministry of Education,Haikou 570228

Received:2018-06-28 Published:2019-01-26 Online:2019-01-23

摘要/Abstract

摘要： 旨在构建适于快速检测橡胶树白粉病菌（Oidium heveae）HO-73启动子的探针载体。以卡那霉素抗性基因Kan作为报告基因,在pUC19骨架载体,构建获得启动子探针载体pUC19-K,连入CaMV35S启动子做启动子探针载体功能验证;利用启动子探针载体pUC19-K对预测的启动子片段LY1、LY2、LY3、LY4进行筛选鉴定。将CaMV35S启动子连入到启动子探针载体中,得到可检测启动子活性的启动子探针载体pUC19-K;应用生物信息学软件对部分橡胶树白粉菌HO-73全基因组数据预测,得到4个理论上具有活性的启动子序列LY1、LY2、LY3、LY4,利用构建的启动子活性探针载体进行活性比较,卡那耐受性实验检测发现含LY2和LY3的菌株随卡那霉素浓度的升高耐受性更强,最终得到2个活性较强的启动子LY2和LY3。以上结果表明,构建的启动子活性探针载体可以有效、灵敏地用于HO-73强启动子的筛选和启动子活性检测。

关键词: 橡胶树白粉菌, 启动子, 探针载体

Abstract: This study aims to construct a probe vector suitable for rapidly detecting the promoter of Oidium heveae HO-73. The kanamycin-resistance gene was used as a reporter gene to construct the promoter probe vector pUC19-K in the pUC19 backbone vector,and it was ligated with the CaMV35S promoter to verify the function of the promoter probe vector. The promoter probe vector pUC19-K was employed to screen and identify the predicted promoter fragments LY1,LY2,LY3,and LY4. The CaMV35S promoter was ligated into the promoter probe vector to obtain the promoter probe vector pUC19-K that can detect the promoter activity. Using bioinformatics software to predict the whole genome data of Oidium heveae HO-73,4 theoretically active promoter sequences,LY1,LY2,LY3,and LY4,were obtained,and their activities were compared using the constructed promoter activity probe vector. The Kana tolerance test showed that strains containing LY2 and LY3 were more tolerant with increasing concentrations of kanamycin,resulting in two more active promoters,LY2 and LY3.The above results indicate that the constructed promoter activity probe vector can be effectively and sensitively used for screening the strong promoter of HO-73 and detecting promoter activity.

Key words: Oidium heveae, promoter, probe vector

徐良向, 刘耀, 廖小淼, 王义, M.J.N.Rajaofera, 何其光, 刘文波, 林春花, 缪卫国. 启动子探针载体的构建及橡胶树白粉菌启动子筛选鉴定[J]. 生物技术通报, 2019, 35(1): 51-57.

XU Liang-xiang, LIU Yao, LIAO Xiao-miao, WANG Yi, M.J.N. RAJAOFERA, HE Qi-guang, LIU Wen-bo, LIN Chun-hua, MIAO Wei-guo. Construction of Promoter Probe Vectors,and Screening and Identification of Oidium heveae Promoters[J]. Biotechnology Bulletin, 2019, 35(1): 51-57.

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启动子探针载体的构建及橡胶树白粉菌启动子筛选鉴定

Construction of Promoter Probe Vectors,and Screening and Identification of Oidium heveae Promoters

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