龙眼<i>DlAGO4</i>和<i>DlAGO6</i>启动子的克隆及活性分析

陈荣珠; 赖钟雄

doi:10.19303/j.issn.1008-0384.2023.05.006

龙眼DlAGO4和DlAGO6启动子的克隆及活性分析

doi: 10.19303/j.issn.1008-0384.2023.05.006

陈荣珠^1,,
赖钟雄^2, ,

1.
漳州卫生职业学院，福建　漳州　363000
2.
福建农林大学园艺植物生物工程研究所，福建　福州　350002

基金项目: 福建省（山区）作物遗传改良与创新利用重点实验室开放课题资助项目（2022SKF04）；漳州卫生职业学院科技创新团队（Kjcx-5）；国家自然科学基金项目（31572088）

详细信息

作者简介:
陈荣珠（1986−），博士，讲师，研究方向：中药资源及生物技术（E-mail：349074689@qq.com）

通讯作者:
赖钟雄（1966−），博士，教授，主要从事园艺植物生物技术研究（E-mail：laizx01@163.com）

中图分类号: S667.2
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出版历程
- 收稿日期: 2023-02-01
- 录用日期: 2023-05-11
- 修回日期: 2023-04-30
- 网络出版日期: 2023-07-06
- 刊出日期: 2023-05-28

Cloning and Activity of DlAGO4 and DlAGO6 Promoters from Dimocarpus longan

CHEN Rongzhu^1
,,
LAI Zhongxiong^{2
, ,}

1.
Zhangzhou Health Vocational College, Zhangzhou, Fujian　363000, China
2.
Institute of Horticultural Biotechnology, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China

摘要

摘要: 目的 Argonaute（AGO）蛋白是RNA介导的沉默复合物RISC的核心成分，参与植物生长发育、组织形成、细胞增殖凋亡、病毒防御、逆境响应等多种生物过程。探究龙眼DlAGO4和DlAGO6基因启动子区域的顺式作用元件，以及重组表达载体在不同激素处理下的表达模式，可为进一步研究DlAGO4和DlAGO6基因的功能提供参考。方法使用常规PCR技术克隆龙眼DlAGO4和DlAGO6基因启动子序列，采用BPDG、PLACE和PlantCARE等生物信息学工具进行DlAGO4和DlAGO6基因启动子序列的生物信息学分析，并构建全长启动子与GUS基因融合表达载体，转化烟草叶片，进行瞬时表达，通过GUS组织化学染色分析启动子的活性。结果 DlAGO4基因启动子片段长度1514 bp，DlAGO6基因启动子片段长度1784 bp。两个启动子序列均含有TATA-box和CAAT-box核心元件、茉莉酸甲酯、脱落酸和光响应元件；DlAGO4启动子序列还含有水杨酸、赤霉素响应元件和厌氧应答调控元件；DlAGO6启动子序列还含有生长素及昼夜节律调控元件。2个启动子片段均可驱动GUS基因表达，表达强度弱于CaMV35S。MeJA和ABA处理可显著提高转DlAGO6启动子烟草叶片中GUS基因的相对表达水平，SA和MeJA处理可提高转DlAGO4启动子烟草叶片GUS基因的相对表达水平。结论成功克隆了龙眼DlAGO4和DlAGO6基因启动子，二者为激素诱导型启动子，具有驱动下游GUS表达的活性，可能参与了植物体胚发育以及对激素的响应。
- 龙眼 /
- DlAGO4 /
- DlAGO6 /
- 启动子 /
- GUS活性
Abstract: Objective Argonaute (AGO) proteins play the important roles in a wide varieties of plant biological processes. To explore the cis --acting elements of DlAGO4 and DlAGO6 promoters, the expression patterns of recombinant vector of DlAGO4 and DlAGO6 promoters under different hormones treatments in longan, DlAGO4 and DlAGO6 promoters in longan were cloned and analyzed. Method Promoters of DlAGO4 and DlAGO6 from Dimocarpus longan were cloned by PCR for a bioinformatic analysis. Fusion expression vectors of the full-length promoters and GUS gene were constructed. The Agrobacterium tumefaciens-mediated transformation was performed on protocorm of tobacco leaves to observe the transient expression of the genes, while promoter activity examined by GUS histochemical staining. Result The sequence of DlAGO4 promoter was 1 514 bp and the sequence of DlAGO6 promoter was 1 784 bp. Both had the core elements of TATA- and CAAT-box as well as ababolic acid (ABA), methyl jasmonate (MeJA), and photoresponsive elements. The DlAGO4 promoter also contained salicylic acid (SA), gibberellin response, and anaerobic response regulatory elements, while the DlAGO6 promoter had auxin and circadian rhythm regulatory elements. The promoters exhibited a GUS-driving activity that was weaker than CaMV35S. The relative expression of GUS of P_DlAGO6::GUS recombinant tobacco leaf was significantly elevated by the treatment of MeJA and ABA, whereas that of P_DlAGO4::GUS recombinant tobacco leaf by SA and MeJA. Conclusion The promoters of DlAGO4 and DlAGO6 from longan were successfully cloned. As hormone-induced promoters, they could activate the GUS expression and be involved in the somatic embryo development and hormone response of plants.
- Dimocarpus longan /
- DlAGO4 /
- DlAGO6 /
- promoter /
- GUS activity

HTML全文

图 1 龙眼DlAGO4、DlAGO6启动子克隆验证电泳图

（1）1-- DlAGO4 基因5＇端上游启动子序列；（2）2-- DlAGO6 基因5＇端上游启动子序列；（3）M-DL 2 000 bp。

Figure 1. Electrophoretograms of DlAGO4 and DlAGO6 promoters

(1)1: promoter of DlAGO4 ; (2)2: promoter of DlAGO6 ; (3)M-DL 2 000 bp .

下载: 全尺寸图片幻灯片

图 2 龙眼DlAGO4和DlAGO6基因启动子序列功能元件分析

Figure 2. Cis-acting elements of DlAGO4 and DlAGO6 promoters

下载: 全尺寸图片幻灯片

图 3 烟草叶片GUS组织化学染色

A：阴性对照；B：阳性对照；C：实验组（P_DlAGO4::GUS载体）；D：实验组（P_DlAGO6::GUS载体）

Figure 3. GUS histochemical staining of tobacco leaves

A: negative control; B: positive control; C: treatment group (P_DlAGO4:GUS recombinant tobacco leaf); D: treatment group (P_DlAGO6:GUS recombinant tobacco leaf).

下载: 全尺寸图片幻灯片

图 4 不同外源激素处理下瞬时转化烟草的GUS活性转录水平

*表示0.05水平显著性差异

Figure 4. GUS transcriptional level of transformed tobacco under exogenous hormones treatments

*indicates significant difference at P＜0.05.

下载: 全尺寸图片幻灯片

表 1 引物序列

Table 1. Information on primer sequences

引物名称 Primer name	引物序列（5′→3′） Primer sequence (5′→3′)	退火温度 Tm/℃
pDlAGO4-F	ACGAGAAGAGGGAGGCAAGA	59
pDlAGO4-R	AGACTGACACAACGCAGGAC	59
pDlAGO6-F	GTACACGCTCCAATTCAGACACG	62
pDlAGO6-R	TGCTGCAATCATCAATGTTCAATA	62
1300-pDlAGO4-F	CAGTGGTCTCACGAGAAGAGGGAGGCAAGA	58
1300-pDlAGO4-R	CAGTGGTCTCAGACTGACACAACGCAGGAC	58
1300-pDlAGO6-F	CAGTGGTCTCGTACACGCTCCAATTCAGACACG	59
1300-pDlAGO6-R	CAGTGGTCTCTGCTGCAATCATCAATGTTCAATA	59
加粗为保护碱基，下划线为Eco32I酶切位点 Bold CAGT are protective bases;underlined GGTCTC are restriction sites of Eco32I .

下载: 导出CSV

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