Characterization of OsBI-1L-8 Promoter in Oryza sativa L.
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摘要:
目的 分析水稻OsBI-1L-8基因启动子的结构与功能,解析OsBI-1L-8基因的表达特性及生物学功能,进一步了解OsBI-1L家族在水稻生命进程中的作用机理。 方法 利用BDGP、Softberry及Promoter 2.0预测分析水稻OsBI-1L-8启动子大小,以日本晴基因DNA为模板扩增pOsBI-1L-8,PlantCARE分析序列中顺序调控元件。构建pOsBI-1L-8::GUS载体,导入水稻,组织化学染色法检测pOsBI-1L-8在水稻中的时空表达特征。 结果 通过预测分析并克隆pOsBI-1L-8长度为1 258 bp,该区域含有光信号、缺氧胁迫应答及激素调节等顺式作用元件。pOsBI-1L-8驱动的GUS报告基因仅表达于水稻抽穗期的茎结合部位、根茎过渡区和雄蕊。 结论 pOsBI-1L-8为组织特异型启动子,推测OsBI-1L-8可能在水稻生长及雄蕊发育中起重要作用。 Abstract:Objective Structure and function of OsBI-1L-8 promoter were studied to decipher the mechanism of OsBI-1L genetic family involved in the life of Oryza sativa L. Method Length of pOsBI-1L-8 was predicted using the online software BDGP, Softberry, and Promoter 2.0. The cis-acting regulatory elements of pOsBI-1L-8 isolated from the genomic DNA of Nipponbare were analyzed based on the PlantCARE database. The pOsBI-1L-8::GUS vector was then constructed and transformed into rice callus, and spatiotemporal expression characterization by histochemical staining. Result The length of pOsBI-1L-8 was determined to be 1 258 bp by the predictive analysis and PCR amplification. The region consisted of several cis elements associated with light signal, response to anaerobic stress, and hormone regulation. The GUS reporter gene driven by pOsBI-1L-8 was expressed only during the heading period at the stem-stem junctions, root-stem junctions, and stamens. Conclusion pOsBI-1L-8 was a tissue-specific promoter. OsBI-1L-8 gene was postulated to play an important role in the growth and stamen development of a rice plant. -
Key words:
- rice /
- OsBI-1L-8 /
- promoter /
- expression characterization /
- GUS activity
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图 3 pOsBI-1L-8::GUS载体酶切验证
注:Marker为λ-EcoT14 I digest DNA Marker,1为 Pst I/EcoR I酶切,片段大小为8 917 bp、3 349 bp;2为Pst I/Nco I酶切,片段大小为11 035 bp、1 265 bp;3为Hind III酶切,片段大小为11 734 bp、566 bp。
Figure 3. Enzymes digestion verification of pOsBI-1L-8::GUS vector
Note: Marker: λ-EcoT14 I digest DNA Marker; 1: Pst I/EcoR I enzyme-digested products, with fragment lengths of 8 917 bp and 3 349 bp; 2: Pst I/Nco I enzyme-digested products, with fragment lengths of 11 035 bp and 1 265 bp; 3: Hind III enzyme-digested products, with fragment lengths of 11 734 bp and 566 bp.
图 5 转基因植株GUS组织化学染色
注:a为根、b为茎、c为叶、d为叶夹角、e为内外稃、f为种子、g为根茎结合部、h为茎结合部、i为花穗、j为小花内部结构;1为阴性植株染色;2为转基因植株染色。
Figure 5. GUS histochemical staining of transgenic rice plant
Note:a: roots; b: stems; c: leaves; d: leaf angle; e: lemma and palea; f: seeds; g: root-stem junctions; h: stem-stem junctions; i: flower spikes; j: inner structure of floret; 1: specimens from plants of negative result; 2: specimens from transgenic plants.
表 1 OsBI-1L-8基因启动子序列顺式作用元件
Table 1. The cis-acting elements in promoter sequence of OsBI-1L-8
名称
Name序列
Sequence位置
Position/bp数量
Amount功能
FunctionTATA-box ATATAA 960 1 主要顺式调控元件
Common cis-acting elementsCAAT-box CAAT/CCAAT/CAAAT 9, 71, 111, 198, 268, 354, 877,
1 026, 1 040, 1 121, 1 12211 主要顺式调控元件
Common cis-acting elementsATCT-motif AATCTAATCC 199 1 光响应元件
Light response elementG-box TACGTG 670 1 光响应元件
Light response elementGC-motif CCCCCG 364 1 缺氧胁迫响应元件
Anaerobic stress response elementCGTCA-motif CGTCA 1 158 1 茉莉酸响应元件
MeJA response elementTGACG-motif TGACG 136, 490 2 茉莉酸响应元件
MeJA response elementABRE ACGTG/TACGTGTC/ACGTG 297, 670, 671 3 脱落酸响应元件
abscisic acid response elementABRE3a TACGTG 670 1 功能未知元件
Function unknown componentDRE core GCCGAC 369 1 功能未知元件
Function unknown componentMyb TAACTG 778 1 功能未知元件
Function unknown componentTCA TCATCTTCAT 1 101, 1 107 2 功能未知元件
Function unknown componentAT~ABRE TACGTGTC 670 1 功能未知元件
Function unknown componentUnnamed_4 CTCC 618, 621, 681, 758, 1 094, 1 097, 1 117,
1 167, 1 170,1 173, 1 176, 1 182, 1 18813 功能未知元件
Function unknown componentWRE3 CCACCT 479 1 功能未知元件
Function unknown componentas-1 TGACG 136, 490 2 功能未知元件
Function unknown component -
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