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Journal of Zhejiang University (Agriculture and Life Sciences)  2019, Vol. 45 Issue (1): 23-29    DOI: 10.3785/j.issn.1008-9209.2017.09.042
Horticulture     
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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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 wordsOsmanthus fragrans      expansin genes      promoter      β-glucuronidase gene     
Received: 04 September 2017      Published: 28 March 2019
CLC:  S 685.13  
Corresponding Authors: Hongbo ZHAO     E-mail: 417971876@qq.com;zhaohb@zafu.edu.cn
Cite this article:

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.

URL:

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


桂花扩展蛋白基因OfEXPA2OfEXPA4OfEXLA1启动子克隆及活性分析

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OfEXPA2OfEXPA4OfEXLA1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:OfEXPA2OfEXPA4OfEXLA1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 

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
Table 1 Primer sequences and their function
Fig.1 Amplification of OfEXPA2, OfEXPA4 and OfEXLA1 promoters from O. fragrans
Fig. 2 Sequences and cis-elements of OfEXPA2 promoter
Fig. 3 Sequences and cis-elements of OfEXPA4 promoter
Fig. 4 Sequences and cis-elements of OfEXLA1 promoter
Fig. 5 Detection result of transient expression
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