橡胶树白粉菌（HO-73）启动子WY172不同长度片段的克隆及表达活性分析

doi:10.13560/j.cnki.biotech.bull.1985.2019-0728

生物技术通报 ›› 2020, Vol. 36 ›› Issue (1): 29-36.doi: 10.13560/j.cnki.biotech.bull.1985.2019-0728

橡胶树白粉菌（HO-73）启动子WY172不同长度片段的克隆及表达活性分析

殷金瑶, 王义, 徐良向, 朱利, 王晨, 刘文波, 缪卫国

海南大学植物保护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:2019-08-16 出版日期:2020-01-26 发布日期:2020-01-08
作者简介:殷金瑶,女,硕士研究生,研究方向：植物病原真菌学;E-mail：1050376472@qq.com
基金资助:
国家自然科学基金项目（31660033,315604958,31760499）,海南省自然科学基金创新研究团队项目（2016CXTD002）,973前期专项（2011CB111612）,现代农业产业技术体系建设专项资金项目（CARS-34-BC1）,海南省重点研发计划项目（ZDYF2016208）,海南大学科研启动基金项目（kyqd1535）

Cloning and Expression Analysis of Different-length Fragments of Oidium heveae（HO-73）Promoter WY172

YIN Jin-yao, WANG Yi, XU Liang-xiang, ZHU Li, WANG Chen, LIU Wen-bo, MIAO Wei-guo

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

Received:2019-08-16 Published:2020-01-26 Online:2020-01-08

摘要/Abstract

摘要： 旨在克隆橡胶树白粉菌启动子WY172及其上游2K序列上4个不同长度缺失片段,以分析启动子各片段的表达活性。基于实验室前期研究基础,以WY172上游2K序列作为研究对象进行渐变缺失突变,得到4个不同长度的可能具有启动子活性的片段,结合WY172,选用pBI121载体作为骨架,分别替换GUS基因前的CaMV35S启动子,并分别构建重组表达载体,通过ATMT法转化农杆菌;利用GUS染色法和酶活性检测,分析WY172启动子及不同长度片段的酶活性。分别构建了pBI121-WY172、pBI121-WY172Q、pBI121-WY172Q1、pBI121-WY172Q2、pBI121-WY172Q3共5个重组的植物表达载体,所有植物表达载体烟草瞬时表达GUS染色均有蓝色出现,且蓝色程度均强于阳性对照CaMV35S启动子,其中pBI121-WY172Q3的GUS染色相对最深;GUS酶活性测定结果显示所有缺失突变片段都具有调控基因表达的启动子活性,且启动活性均强于CaMV35S启动子,WY172Q3调控GUS基因表达的活性最高。因此我们判断WY172及其上游2K序列上4个不同长度缺失片段均具有启动子活性,其中以WY172Q3启动子片段的表达活性最强。

关键词: 橡胶树白粉菌内源启动子WY172, GUS染色, 缺失突变, GUS酶活性

Abstract: The aim of this study is to clone 4 different-length deletion fragments of Oidium heveae promoter WY172 and its upstream 2K sequence in order to analyze the expression activity of each fragment of the promoter. Based on the prior research in our laboratory,the 2K sequence upstream of WY172 was used as the research object to conduct the gradual deletion mutation,and 4 fragments in different lengths which might have promoter activity were obtained. In combination with WY172,the pBI121 vector was used as the backbone,and the CaMV35S promoter before the GUS gene was replaced,then the recombinant expression vector was constructed respectively,and the Agrobacterium was transformed by ATMT method. The enzyme activities of the WY172 promoter and fragments of different lengths were analyzed by GUS staining and enzyme activity assay. Five recombinant plant expression vectors including pBI121-WY172,pBI121-WY172Q,pBI121-WY172Q1,pBI121-WY172Q2 and pBI121-WY172Q3 were constructed. GUS gene was expressed in all of the recombinant plant expression vectors,and its expression（blue occurred）was stronger than the positive control CaMV35S promoter. Moreover,the transient expression of pBI121-WY172Q3 recombinant vector was the most intense. The results of GUS enzyme activity assay showed that all deletion mutant fragments presented promoter activity regulating gene expression,and the activation activity was stronger than that of CaMV35S promoter. Among them,the GUS enzyme activity regulated by WY172Q3 was the highest. Therefore,we infer that WY172 and its four 2K-length deletion fragments on the upstream 2K sequence have promoter activity,and WY172Q3 promoter fragment has the strongest expression activity.

Key words: endogenous promoter WY172 of Oidium heveae, GUS staining, deletion mutation, GUS activity

殷金瑶, 王义, 徐良向, 朱利, 王晨, 刘文波, 缪卫国. 橡胶树白粉菌（HO-73）启动子WY172不同长度片段的克隆及表达活性分析[J]. 生物技术通报, 2020, 36(1): 29-36.

YIN Jin-yao, WANG Yi, XU Liang-xiang, ZHU Li, WANG Chen, LIU Wen-bo, MIAO Wei-guo. Cloning and Expression Analysis of Different-length Fragments of Oidium heveae（HO-73）Promoter WY172[J]. Biotechnology Bulletin, 2020, 36(1): 29-36.

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橡胶树白粉菌（HO-73）启动子WY172不同长度片段的克隆及表达活性分析

Cloning and Expression Analysis of Different-length Fragments of Oidium heveae（HO-73）Promoter WY172

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