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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2019, Vol. 45 Issue (1): 23-29    DOI: 10.3785/j.issn.1008-9209.2017.09.042
园艺学     
桂花扩展蛋白基因OfEXPA2、OfEXPA4和OfEXLA1启动子克隆及活性分析
高晓月1(),董彬1,张超1,付建新1,胡绍庆2,赵宏波1(),梁立军1
1. 浙江农林大学风景园林与建筑学院，杭州 311300
2. 浙江理工大学建筑工程学院，杭州 310018
Cloning and activity analysis of promoters of expansin genes OfEXPA2, OfEXPA4 and OfEXLA1 from Osmanthus fragrans
Xiaoyue GAO1(),Bin DONG1,Chao ZHANG1,Jianxin FU1,Shaoqing HU2,Hongbo ZHAO1(),Lijun LIANG1
1. School of Landscape Architecture, Zhejiang A & F University, Hangzhou 311300, China
2. College of Civil Engineering and Architecture, Zhejiang Sci-Tech University, Hangzhou 310018, China
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摘要：

为研究扩展蛋白基因在桂花花开放过程中的作用机制，利用染色体步移法，从桂花（Osmanthus fragrans）中克隆得到花开放相关基因OfEXPA2、OfEXPA4和OfEXLA1起始密码子上游1 108、808、945 bp的启动子序列。利用在线数据库Plantcare预测启动子中的顺式作用元件，发现这3个启动子序列中均存在基本元件TATA盒和CAAT盒，还含有脱落酸、生长素、水杨酸等激素诱导元件及响应干旱、高温等多个与植物非生物胁迫相关的元件，如MBS、HSE。分别将这3个启动子与β-葡萄糖苷酸酶（β-glucuronidase, GUS）报告基因融合进行瞬时表达，结果显示：OfEXPA2、OfEXPA4和OfEXLA1的启动子均能够驱动GUS基因在转基因烟草叶片中的表达。
关键词： 桂花;  扩展蛋白基因;  启动子;  β-葡萄糖苷酸酶基因    
Abstract:

Osmanthus fragrans is not only one of the top ten traditional famous flowers of our country, but also one of the famous greening and perfume plants in China. In order to study the flower opening mechanism of O. fragrans, expansin genes were identified and selected from the obtained transcriptome of O. fragrans. Three expansin genes, OfEXPA2, OfEXPA4 and OfEXLA1, were performed, which contributed to flower opening mechanism of O. fragrans. Promoters of the three genes were cloned by the method of Genome Walker. The lengths of 1 108, 808 and 945 bp (OfEXPA2, OfEXPA4 and OfEXLA1) were obtained from O. fragrans, respectively. Online database ‘Plantcare' analysis revealed that the sequences of the three promoters contained basic cis-elements, such as TATA-box and CAAT-box. In addition, the promoter of OfEXPA2 contained hormone inducing elements ABRE, and the promoter of OfEXLA1 had AuxRR and TCA, indicating that the two genes would be regulated by corresponding hormones. Besides, there were many other elements involved in the plant abiotic stress. MBS, a cis-acting element in the promoter of OfEXPA2, was the MYB binding site, which responded to drought stress; there was a cis-acting element that responded to high temperature, HSE, in the promoter of OfEXLA1. In addition, the recombinant vectors were constructed and named OfEXPA2::GUS, OfEXPA4::GUS and OfEXLA1::GUS, respectively. The result indicated that the promoters of OfEXPA2, OfEXPA4 and OfEXLA1 could drive the GUS gene exclusively to express in leaves of transgenic tobacco.
Key words: Osmanthus fragrans    expansin genes    promoter    β-glucuronidase gene
收稿日期: 2017-09-04 出版日期: 2019-03-28
CLC:  S 685.13  
基金资助: 国家自然科学基金(31501790);浙江省自然科学基金(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)
通讯作者: 赵宏波     E-mail: 417971876@qq.com;zhaohb@zafu.edu.cn
作者简介: 高晓月（https://orcid.org/0000-0003-4584-2239）|赵宏波（https://orcid.org/0000-0003-4714-8240）
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引用本文:

高晓月,董彬,张超,付建新,胡绍庆,赵宏波,梁立军. 桂花扩展蛋白基因OfEXPA2、OfEXPA4和OfEXLA1启动子克隆及活性分析[J]. 浙江大学学报（农业与生命科学版）, 2019, 45(1): 23-29.

Xiaoyue GAO,Bin DONG,Chao ZHANG,Jianxin FU,Shaoqing HU,Hongbo ZHAO,Lijun LIANG. Cloning and activity analysis of promoters of expansin genes OfEXPA2, OfEXPA4 and OfEXLA1 from Osmanthus fragrans. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(1): 23-29.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2017.09.042    或    http://www.zjujournals.com/agr/CN/Y2019/V45/I1/23

引物名称

Primer name

序列（5′→3′）

Sequence (5′→3′)

用途

Function

OfEXPA2-GSP1CGCAGTGTTAGTGCCATACCCTTGAC

启动子扩增

Amplification of promoters

OfEXPA2-GSP2GAACGGTAGGAGGTCTTAGTGGTATC
OfEXPA4-GSP1TGAGTCCAACCAGAAGCAGTAAAGGC
OfEXPA4-GSP2AGTGGGGTTATTTCAGTTGATGGCTC
OfEXLA1-GSP1CAAGAACCATAGCCACAAGCACCAGAT
OfEXLA1-GSP2GTTGAGAAAGAAAGGAAGTGAAGAAGC
AP1GTAATACGACTCACTATAGGGC
AP2ACTATAGGGCACGCGTGGT
OfEXPA2-FTGATTACGCCAAGCTATCGACAAATTGTGTGATTATGTATGAACC

载体构建片段扩增

Amplification of construction of the vector

OfEXPA2-RCCGGGGATCCTCTAGCATTAAGAATATAAGAATGTATATGGATGG
OfEXPA4-FTGATTACGCCAAGCTCTGGTCTTGCCATTAAGCC
OfEXPA4-RCCGGGGATCCTCTAGCATTTTTCTCAATGAGAAGAAAGAT
OfEXLA1-FTGATTACGCCAAGCTATCTTCAAAAAACTTTTTAAACATGAAATGCTC
OfEXLA1-RCCGGGGATCCTCTAGCATTTTTTGTTGAGGAATGTGTGAATATT
表1  引物序列及用途
图1  桂花OfEXPA2、OfEXPA4和OfEXLA1启动子扩增
图2  OfEXPA2启动子序列及顺式作用元件
图3  OfEXPA4启动子序列及顺式作用元件
图4  OfEXLA1启动子序列及顺式作用元件
图5  瞬时表达检测结果
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