水稻<i>OsPLATZ14</i>基因启动子的克隆及表达分析

陈睿; 陈建民; 吴明基; 杨绍华; 胡昌泉

doi:10.19303/j.issn.1008-0384.2019.10.004

水稻OsPLATZ14基因启动子的克隆及表达分析

doi: 10.19303/j.issn.1008-0384.2019.10.004

陈睿^{1, 2,},
陈建民^{1, 2},
吴明基^{1, 2},
杨绍华^{1, 2},
胡昌泉^{1, 2}

1.
福建省农业科学院生物技术研究所，福建　福州　350003
2.
福建省农业遗传工程重点实验室，福建　福州　350003

基金项目: 福建省自然科学基金项目（2017J01055）；福建省科技计划公益类专项（2016R1018-8）

详细信息

作者简介:
陈睿（1980−），女，硕士，副研究员，研究方向：水稻遗传育种（E-mail：candy_chenrui@163.com）

中图分类号: S 511
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出版历程
- 收稿日期: 2019-09-18
- 修回日期: 2019-10-18
- 刊出日期: 2019-10-01

Cloning and Expression Analysis of OsPLATZ14 Promoter in Rice (Oryza sativa L.)

CHEN Rui^{1, 2
,},
CHEN Jian-ming^{1, 2},
WU Ming-ji^{1, 2},
YANG Shao-hua^{1, 2},
HU Chang-quan^{1, 2}

1.
Biotechnology Institute, Fujian Academy of Agricultural Sciences , Fuzhou , Fujian　350003 , China
2.
Fujian Provincial Key Laboratory of Genetic Engineering for Agriculture , Fuzhou , Fujian　350003 , China

摘要

摘要: 目的研究pOsPLATZ14的结构及时空表达模式，对深入研究OsPLATZ14基因功能、了解PLATZ转录因子在水稻生长发育进程的作用具有重要意义。方法利用BDGP、FPROM及Cister预测分析pOsPLATZ14大小，以水稻日本晴基因DNA为模板扩增pOsPLATZ14；应用PlantCARE分析序列中的顺序作用元件，构建pOsPLATZ14::GUS载体，转化水稻获得转基因植株；通过GUS组织化学染色法分析pOsPLATZ14在水稻中的时空表达特征。结果经PCR扩增获得pOsPLATZ14长度为1 899 bp，该区域含有光信号、逆境响应及激素应答等多种顺式作用元件。pOsPLATZ14驱动的GUS报告基因在水稻种子萌发期，苗期根、茎、叶，抽穗期的根、茎、叶、花穗、小花、叶夹角、茎结合部位、根茎过渡区及成熟期种子均有明显表达。结论 pOsPLATZ14为组成型启动子，其下游调控基因OsPLATZ14可能在水稻生长发育过程中起重要作用。
- 水稻 /
- PLATZ转录因子 /
- 启动子pOsPLATZ14 /
- 表达分析 /
- GUS活性
Abstract: Objective In order to further understand the function of OsPLATZ14 and the role of PLATZ transcription factor in rice growth and development, the structure and spatiotemporal expression pattern of pOsPLATZ14 were studied. Method The length of pOsPLATZ14 was predicted by using the online software of BDGP, FPROM, and Cister. The cis-acting regulatory elements of pOsPLATZ14 were analyzed based on the PlantCARE database. pOsPLATZ14 was isolated from the genomic DNA of Nipponbare, then pOsPLATZ14::GUS vector was constructed and applied for the rice callus transformation. Using GUS histochemical assay, the spatiotemporal expression characteristics of pOsPLATZ14 were revealed. Result The pOsPLATZ14 was determined to be 1 899 bp in length containing several cis-elements which were associated with the light signaling, stress and hormone response. The GUS reporter gene driven by the promoter was significantly expressed at the stages of seed germination, the seedling stage of roots, stems and leaves, in the heading period of roots, stems, leaves, flower spikes, florets, leaf angle, stem-stem junctions, root-stem junctions, as well as the seed maturation. Conclusion pOsPLATZ14 was the constitutive promoter and its downstream regulatory gene OsPLATZ14 might play an important role in the growth and development of rice.
- Rice /
- PLATZ transcription factor /
- promoter OsPLATZ14 /
- expression analysis /
- GUS activity

HTML全文

图 1 表达载体pOsPLATZ14::GUS示意图

Figure 1. Schematic diagram of pOsPLATZ14 :: GUS

下载: 全尺寸图片幻灯片

图 2 OsPLATZ14启动子扩增片段电泳图

注：Marker:DL5000。

Figure 2. Electrophoretogram of pOsPLATZ14 amplified fragment

Note:Marker:DL5000.

下载: 全尺寸图片幻灯片

图 3 重组质粒pOsPLATZ14::GUS酶切验证

注：Marker为λ-EcoT14 I digest DNA Marker，1为BamHI、NcoI双酶切pOsPLATZ14::GUS产物。

Figure 3. The recombinant plasmid digested by restriction enzymes

Note: Marker: λ-EcoT14 I digest DNA Marker; 1: pOsPLATZ14 :: GUS plasmid digested by BamHI and NcoI.

下载: 全尺寸图片幻灯片

图 4 转基因水稻T₀代PCR检测

注：Marker为DL5000，1-6为转基因阳性株，7为质粒pOsPLATZ14::GUS，8为转基因阴性株，9为日本晴。

Figure 4. PCR analysis on DNA in T₀ transgenic rice plant

Note: Marker: DL5000; 1-6: transgene positive line; 7: pOsPLATZ14 :: GUS ; 8: transgene negative line; 9: Nipponbare.

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图 5 pOsPLATZ14::GUS转基因水稻的组织化学染色

注：a-b为根、c-d为茎、e为茎结合部、f为叶、g为叶夹角、h为雄蕊、i为雌蕊、j为种子、k为小花、l为根茎结合部、m为萌发期种子、n为花穗。

Figure 5. Histochemical staining of pOsPLATZ14::GUS transgenic rice

Note：a-b: root; c-d: stem; e: stem-stem junction; f: leaf; g: leaf angle; h: stamen; i: pistil; j: seed; k: floret; l: root-stem junction; m: germinating seed; n: flower spike.

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表 1 OsPLATZ14基因启动子序列顺式作用元件

Table 1. The cis-acting elements in promoter sequence of OsPLATZ14

位点名称 Site name	信号序列 Signal Sequence	数量 Amount	位点功能 Function of Site
TATA-box	ATATAA/ TATA	2	主要顺式调控元件 Common cis-acting elements
CAAT-box	CAAT/CCAAT/ CAAAT	10	主要顺式调控元件 Common cis-acting elements
3-AF1 binding site	TAAGAGAGGAA	1	光响应元件 Light response element
GATA-motif	GATAGGA	1	光响应元件 Light response element
Sp1	GGGCGG	1	光响应元件 Light response element
MRE	AACCTAA	1	光响应元件 Light response element
ARE	AAACCA	2	缺氧胁迫响应元件 Anaerobic stress response element
MBS	CAACTG	1	干旱胁迫响应元件 Drought stress response element
CAT-box	GCCACT	1	分生组织特异性元件 Meristem expression element
ABRE	ACGTG	1	脱落酸响应元件 abscisic acid response element
P-box	CCTTTTG	1	赤霉素响应元件 Gibberellin response element
TCA-element	TCAGAAGAGG	1	水杨酸响应元件 Salicylic acid response element
TGA-element	AACGAC	1	生长素响应元件 Auxin response element
TGACG-motif	TGACG	1	茉莉酸响应元件 MeJA response element
CCAAT-box	CAACGG	1	MYB结合位点 MYB binding site
AAGAA-motif	GAAAGAA	3	功能未知元件 Function unknown component
ERE	ATTTCATA	1	功能未知元件 Function unknown component
MYB	CAACCA	1	功能未知元件 Function unknown component
MYC	CATTTG	3	功能未知元件 Function unknown component
Myb	CAACTG	1	功能未知元件 Function unknown component
STRE	AGGGG	2	功能未知元件 Function unknown component
TATA	TATAAAAT	1	功能未知元件 Function unknown component
TCA	TCATCTTCAT	1	功能未知元件 Function unknown component
Unnamed_1	CGTGG	1	功能未知元件 Function unknown component
Unnamed_4	CTCC	11	功能未知元件 Function unknown component
WUN-motif	AAATTACT	1	功能未知元件 Function unknown component
as-1	TGACG	1	功能未知元件 Function unknown component

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