| 摘要: |
| 【目的】研究犬CREBZF启动子对4种氨基酸（谷氨酸、亮氨酸、赖氨酸和蛋氨酸）缺失的应答效应。【方法】通过PCR和双酶切方法，从犬全血基因组中扩增5′侧翼区1 767 bp的DNA片段，对其进行克隆测序分析后，构建全长及系列包含不同长度CREBZF启动子的表达载体，用Quikchange试剂盒构建了CREBZF启动子定点缺失载体，转染MDCK细胞24 h后，用双荧光素酶报告系统检测在氨基酸缺失条件下表达载体的双荧光素酶活性。【结果】扩增到了犬CREBZF 5′侧翼区1 767 bp的启动子序列，并成功构建了包含不同长度CREBZF启动子的荧光素酶报告基因表达载体pGL3-ZF-A,pGL3-ZF-B,pGL3-ZF-C,pGL3-ZF-D,pGL3-ZF-E,pGL3-ZF-F；其中pGL3-ZF-A（－1 767→＋1 bp）能够应答4种氨基酸缺失；定点缺失结果显示，当缺失亮氨酸时，－1 227→－1 219 bp序列(5′-ATTCACTCA-3′) 的缺失可使CREBZF的转录活性完全丧失，该序列与已知的其他氨基酸应答元件（Amino acid response element,AARE）的同源性达89%。【结论】5′-ATTCACTCA-3′是犬CREBZF启动子中应答氨基酸应激必不可少的调控元件。 |
| 关键词: CREBZF 氨基酸应答 转录活性 犬 |
| DOI： |
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| 基金项目:The Natural Sciences and Engineering Research Council of Canada to R.L.；国家建设高水平研究型大学奖学金项目 |
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| Response effect of dog CREBZF promoter on amino acid deprivation |
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| Abstract: |
| 【Objective】The response effect of dog CREBZF promoter on amino acid (glutamine,leucine,lysine,methionine) deprivation was studied.【Method】The 5′flanking region of dog CREBZF sequence(1 767 bp) was obtained from dog blood by PCR and double enzyme digestion,confirmed by sequence,then a series of luciferase expression vector including different length fragments of CREBZF promoter were constructed.Quikchange Kit was used to produce site-deletion vector of CREBZF,MDCK-cells were used for transfection 24 h,protein was harvested at 24 h post-transfection and dual luciferase assay was conducted to detect dual luciferase activity under amino acid deprivation.【Result】The 5′flanking region of dog CREBZF sequence(1 767 bp) was cloned and a series of luciferase reporter gene vectors including different length CREBZF promoters were constructed successfully,namely pGL3-ZF-A,pGL3-ZF-B,pGL3-ZF-C,pGL3-ZF-D,pGL3-ZF-E,pGL3-ZF-F,only pGL3-ZF-A(－1 767→＋1 bp )of dog CREBZF can respond to four amino acid deprivation.Site deletion results showed the deletion of －1 227→－1 219 bp (5′-ATTCACTCA-3′) of dog CREBZF led to the complete loss of transcriptional activation of CREBZF promoter.There was 89% homology between these sequences and known amino acid response element (AARE).【Conclusion】5′-ATTCACTCA-3′ was an indispensable regulation element during the amino acid deprivation for CREBZF promoter. |
| Key words: CREBZF amino acid response transcription activity dog |
