| 摘要: |
| 【目的】克隆大豆天冬氨酸蛋白酶基因（soyAP1）启动子，并对其表达方式及活性进行分析。【方法】利用TAIL-PCR方法从大豆基因组DNA中克隆soyAP1基因启动子片段。利用启动子在线预测工具分析soyAP1基因启动子片段的启动子元件，并用克隆得到的SAP片段替换pCAMBIA1301中的CaMV35S 启动子，构建表达载体pCAM-SAP。通过农杆菌介导法，使其在大豆各组织中进行瞬时表达，用GUS组织化学染色法分析SAP在各组织中的表达活性。【结果】 克隆获得了大豆soyAP1基因的启动子片段，长1 650 bp，并将之命名为SAP，其转录起始位点可能是553 bp处的A；PLACE分析表明，SAP序列中含有多种典型的种子特异性表达元件；GUS组织化学染色显示，SAP驱动的GUS基因在大豆根、茎、叶中基本不表达，但在种子中有较高的表达活性，且表达强度与CaMV35S启动子相近。【结论】 大豆SAP启动子可能具有种子特异表达特性。 |
| 关键词: 大豆 天冬氨酸蛋白酶基因 启动子 瞬时表达 |
| DOI： |
| 分类号: |
| 基金项目:作物生物学国家重点实验室开放基金项目(2012KF13)；黑龙江省教育厅科学技术研究项目（12521611）；齐齐哈尔大学青年教师科研启动支持计划项目（2010K-M09） |
|
| Cloning and activity analysis of soybean soyAP1 promoter |
|
ZHAO Yan, SUN Tianguo, WANG Hui,etal
|
|
Qiqihar University
|
| Abstract: |
| 【Objective】The aim was cloning and analyzing the expression pattern and activity of soybean aspartic proteinase gene soyAP1 promoter.【Method】The 5′-flanking upstream sequence of soyAP1 gene was isolated from the genomic DNA of soybean by TAIL-PCR method.Promoter online tools were used to analyze the sequence.The binary expression vector pCAM-SAP was constructed by replacing CaMV35S promoter of pCAMBIA1301 with SAP fragment.Transient expression in soybean tissues was conducted by Agrobacterium tumefaciens mediated method,and the activity of SAP was characterized by histochemical GUS staining analysis.【Result】soyAP1 promoter,named SAP,was cloned with a length of 1 650 bp.The transcription start site might locate at 553 bp and the base was A.Sequence analysis by PLACE revealed that this fragment contained a series of motifs related to seed-specific promoters.The results of histochemical GUS analysis showed that there were little or no GUS activities in roots,stems and leaves,but there were higher activities in soybean seeds,which was similar to that driven by CaMV35S promoter.【Conclusion】It is inferred that SAP promoter might drive downstream gene expression exclusively in soybean seeds. |
| Key words: soybean soyAP1 promoter transient expression |
