大豆紫色酸性磷酸酶基因GmPAP4启动子结构与活性分析

发布时间：2018-08-27 17:34

【摘要】：【目的】克隆GmPAP4启动子(PAP4-pro),并分析其表达特性,为进一步研究其作用机制奠定基础。【方法】依据GmPAP4 c DNA序列(Gen Bank No.HQ162477),通过比对大豆参考基因组,设计特异引物,克隆GmPAP4启动子序列,通过PLACE与Plant CARE在线生物信息学数据库预测该启动子相关调控元件。构建GmPAP4启动子驱动GUS表达载体(PAP4-pro-GUS)并转化根癌农杆菌GV3101;通过Floral dip法将PAP4-pro-GUS转化拟南芥,利用卡那霉素(Kan)抗性筛选和特异引物的PCR鉴定,最终获得T3转基因拟南芥。通过对T_3转基因拟南芥不同组织GUS染色,分析启动子的组织表达特性,将T3转基因拟南芥通过适磷和植酸磷处理,20 d后,取其根部进行GUS活性和表达分析,研究启动子对不同磷环境的响应。【结果】克隆了GmPAP4上游启动子序列,通过PLACE与Plant CARE在线生物信息学数据库预测显示,GmPAP4启动子除含有启动子核心的调控元件外,还含有(1)组织特异调控元件:as1(根系特异表达调控元件)和Skn-1_motif(胚乳特异表达调控元件);(2)应答元件:TC-rich repeats(逆境胁迫反应调控元件)和Box-W3(真菌应答相关调控元件);(3)结合位点:MBS(MYB转录因子的结合位点)等。不同组织GUS染色结果显示,转基因拟南芥整个根系GUS染色较深,茎、叶中仅微管组织有较明显GUS染色,花瓣微管组织中也能观察到微弱GUS染色。定量PCR结果显示,植酸磷处理条件下转基因拟南芥根系GUS表达比适磷处理提高了1.3倍(P0.05);同时GUS活性测定显示,与适磷处理相比,植酸磷处理条件下转基因拟南芥根系GUS活性提高了1.9倍(P0.05)。【结论】获得大豆GmPAP4启动子,通过不同组织GUS染色和不同磷环境GUS表达分析显示该启动子主要在根部且受低磷信号诱导表达,为诱导型启动子。
[Abstract]:[objective] to clone GmPAP4 promoter (PAP4-pro) and analyze its expression characteristics, so as to lay a foundation for further study of its mechanism. [methods] according to the GmPAP4 c DNA sequence (Gen Bank No.HQ162477), a specific primer was designed by comparing the reference genome of soybean. GmPAP4 promoter sequence was cloned and predicted by PLACE and Plant CARE online bioinformatics database. GmPAP4 promoter driven GUS expression vector (PAP4-pro-GUS) was constructed and transformed into Agrobacterium tumefaciens GV3101; by Floral dip method to transform PAP4-pro-GUS into Arabidopsis thaliana. Finally, T3 transgenic Arabidopsis thaliana was obtained by screening kanamycin (Kan) resistance and PCR identification of specific primers. The tissue expression characteristics of T 3 transgenic Arabidopsis thaliana were analyzed by GUS staining. The GUS activity and expression of T 3 transgenic Arabidopsis thaliana were analyzed by GUS activity and expression analysis in the roots of T 3 transgenic Arabidopsis thaliana treated with phosphorus and phytate for 20 days. [results] the upstream promoter sequence of GmPAP4 was cloned and predicted by PLACE and Plant CARE online bioinformatics database. It also contains (1) the binding sites of the tissue specific regulatory element: as1 (root specific expression regulator) and Skn-1_motif (endosperm specific expression regulatory element); (2) response element: TC-rich repeats (stress regulatory element) and Box-W3 (fungal response-related regulatory element); (3). Points: MBS (MYB transcription factor binding sites) and so on. The results of GUS staining in different tissues showed that the GUS staining of the whole root system of transgenic Arabidopsis thaliana was deep, only microtubule tissue was stained by GUS in stem and leaf, and weak GUS staining was also observed in petal microtubule tissue. The results of quantitative PCR showed that the expression of GUS in transgenic Arabidopsis thaliana root system was 1.3-fold higher than that of the control (P0.05), and the GUS activity of transgenic Arabidopsis thaliana was higher than that of the control (P0.05). The GUS activity of transgenic Arabidopsis thaliana roots was increased by 1.9 times (P0.05). [conclusion] Soybean GmPAP4 promoter was obtained. The results of GUS staining in different tissues and GUS expression in different phosphorus environments showed that the promoter was mainly expressed in the root and was induced by low phosphorus signal.
【作者单位】：河北农业大学农学院/教育部华北作物种质资源研究与利用重点实验室;
【基金】：转基因生物新品种培育科技重大专项(2014ZX0800404B) 河北省自然科学基金(C2014204035)
【分类号】：S565.1;Q943.2

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