大豆紫色酸性磷酸酶基因GmPAP4启动子结构与活性分析

Construction and Activity Analysis of the Promoter of Purple Acid Phosphatase Gene GmPAP4 in Soybean

【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(P<0.05);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(P<0.05)。【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。

[ Objective ] GmPAP4 promoter （PAP4-pro） was cloned, and its expression character was analyzed, thus providing a basis for research of regulatory mechanism of GmPAP4. [Method] On the basis of GmPAP4 （GenBank No. HQ162477） cDNA sequence and blasting the soybean reference genome, GmPAP4 promoter sequence was cloned with specific primers. The promoter regulatory elements were predicted through the online database PLACE and PlantCARE. PAP4-pro-GUS was constructed and transformed into Agrobacterium turnefaciens GV3101, which subsequently was transformed into Arabidopsis thaliana with Floral dip method. The T3 transgenic plants were obtained by being screened with kanamycin （kan） and PCR amplification. [Result] GmPAP4 promoter was cloned, and its regulatory elements were analyzed using online databases PLACE and PlantCARE. The results showed that the promoter of GmPAP4 not only contained the core elements, but also contained the following elements： （1） tissue specific regulatory elements including asl （root specific expression element）, Skn-1 motif （endosperm specific expression element）; （2）： Response elements including TC-rich repeats （stress responsive elements）, Box-W3 （fungal response related regulatory element）; （3） binding sites including MBS （MYB binding sites of transcription factors）, etc. The results of GUS staining showed that GmPAP4 promoter was mainly expressed in roots of transgenic Arabidopsis. The expression and activity of GUS measurement showed that GUS expression and GUS activity in roots of transgenic plants under phytate condition was, respectively, 1.3-fold （P〈 0.05） and 1.9-fold （P〈0.05） than that under normal phosphorus condition.[ Conclusion]Soybean GmPAP4 promoter was obtained in this study. According to GUS staining in different tissues and activity analysis under different phosphorus conditions, it was found that the promoter of GmPAP4 was mainly expressed in roots and significantly induced by low phosphorus signal.