桂花OfLCYB和OfLCYE启动子的克隆和活性分析

doi:10.13560/j.cnki.biotech.bull.1985.2017-0706

生物技术通报 ›› 2018, Vol. 34 ›› Issue (1): 137-143.doi: 10.13560/j.cnki.biotech.bull.1985.2017-0706

桂花OfLCYB和OfLCYE启动子的克隆和活性分析

沈子又¹, 张超¹, 董彬¹, 付建新¹, 胡绍庆², 赵宏波¹

1. 浙江农林大学风景园林与建筑学院,临安 311300;
2. 浙江理工大学建筑工程学院,杭州 310018

收稿日期:2017-08-17 出版日期:2018-01-26 发布日期:2018-01-22
作者简介:沈子又,女,硕士研究生,研究方向：园林植物遗传育种;E-mail：392504890@qq.com
基金资助:
国家自然科学基金项目（31501790）,浙江省自然科学基金项目（LQ15C160004,LQ16C160003）,浙江省农业新品种选育重大科技专项（2016C02056-12）,浙江农林大学科研发展基金人才启动项目（2016FR031）

Cloning and Expression Analysis of the Promoters of OfLCYB and OfLCYE in Osmanthus fragrans

SHEN Zi-you¹, ZHANG Chao¹, DONG Bin¹, FU Jian-xin¹, HU Shao-qing², ZHAO Hong-bo¹

1. School of Landscape Architecture,Zhejiang A&F University,Lin’an 311300;
2. College of Civil Engineering and Architecture,Zhejiang Sci-Tech University,Hangzhou 310018

Received:2017-08-17 Published:2018-01-26 Online:2018-01-22

摘要/Abstract

摘要： 番茄红素环化是类胡萝卜素合成过程中的重要分支点,植物中存在两种番茄红素环化酶LCYB和LCYE。前期研究发现,OfLCYB1、OfLCYE1等基因的差异表达决定了桂花不同品种中类胡萝卜素代谢分支下游产物的含量不同。以‘堰虹桂’基因组DNA为模板,通过染色体步移的方法（Genome walking）克隆出OfLCYB和OfLCYE启动子分别为1 028 bp和904 bp。通过生物信息学分析发现两个启动子序列包含TATA-box、CAAT-box等基本元件,同时都含有水杨酸响应元件TCA-element,以及一些光响应元件如Sp1、ACE、Box4、G-box等;OfLCYB启动子还包括脱落酸响应元件ABRE,赤霉素响应元件P-box,OfLCYE启动子包括赤霉素响应元件GARE-motif及热击响应元件HSE。将克隆到的启动子片段与pBI121载体进行重组,构建植物表达载体并转入农杆菌,采用叶盘法侵染烟草叶片进行瞬时表达。结果表明,OfLCYB和OfLCYE启动子片段均具有驱动下游报告基因表达的功能。

关键词: 桂花, 启动子, LCYB, LCYE, 瞬时表达

Abstract: Lycopene cyclization is an important branching point in the synthesis of carotenoids. There are two kinds of lycopene cyclase LCYB and LCYE in plants. Previous studies have found that the differential expressions of OfLCYB1,OfLCYE1 and other genes determine the contents of downstream products of carotenoid metabolism in different varieties of Osmanthus fragrans. In this study,1028 bp and 904 bp of the OfLCYB and OfLCYE promoters were cloned by genome walking with the genomic DNA of cultivar ‘Yanhong Gui’ as the template. The bioinformatics analysis discovered two promoter sequences that contained TATA-box and CAAT-box,also the TCA-element of salicylic acid,and some photoresponsive elements such as Sp1,ACE,Box4,and G-box. The OfLCYB promoter also includes abscisic acid response element ABRE,gibberellin response element P-box. OfLCYE promoter includes gibberellin response element GARE-motif and thermal response element HSE. The cloned promoter fragment was recombined with pBI121 vector,and the plant expression vector was constructed and transferred into Agrobacterium tumefaciens. The tobacco leaf was used for transient expression. The results proved that OfLCYB and OfLCYE promoter fragments all had the function of driving downstream reporter gene expression.

Key words: Osmanthus fragrans, promoter, LCYB, LCYE, transient expression

沈子又, 张超, 董彬, 付建新, 胡绍庆, 赵宏波. 桂花OfLCYB和OfLCYE启动子的克隆和活性分析[J]. 生物技术通报, 2018, 34(1): 137-143.

SHEN Zi-you, ZHANG Chao, DONG Bin, FU Jian-xin, HU Shao-qing, ZHAO Hong-bo. Cloning and Expression Analysis of the Promoters of OfLCYB and OfLCYE in Osmanthus fragrans[J]. Biotechnology Bulletin, 2018, 34(1): 137-143.

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桂花OfLCYB和OfLCYE启动子的克隆和活性分析

Cloning and Expression Analysis of the Promoters of OfLCYB and OfLCYE in Osmanthus fragrans

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