酵母单杂交技术验证叶用莴苣LsHPB互作启动子  

Validation of LsHPB interaction promoter of leaf lettuce by yeast one hybrid

作　　者：曹佳琰 王惠玉 韩莹琰[1] CAO Jiayan;WANG Huiyu;HAN Yingyan(Beijing Key Laboratory of New Technology in Agricultural Application/National Demonstration Center for Experimental Plant Production Education/College of Plant Science and Technology,Beijing University of Agriculture,Beijing 102206,China)

机构地区：[1]农业应用新技术北京市重点试验室/植物生产国家级实验教学示范中心/北京农学院植物科学技术学院,北京102206

出　　处：《北京农学院学报》2025年第1期37-43,共7页Journal of Beijing University of Agriculture

基　　金：国家自然科学基金资助项目“LsFUL上游调控因子响应高温启动叶用莴苣抽薹开花的作用机制研究”(32472769)。

摘　　要：【目的】AG和SOC1都属于MADS-box家族基因,AG参与调控多种植物花器官中雄蕊和雌蕊的发育和成熟;SOC1在多种植物中参与整合许多开花途径。以叶用莴苣SOC1-like基因LsHPB基因为研究对象,挖掘出LsHPB基因下游互作蛋白之一LsAG,解析LsHPB参与叶用莴苣抽薹的分子机理,进一步完善MADS-box家族基因调控植物响应高温诱导后抽薹的模型。【方法】通过酵母单杂试验验证转录因子LsHPB与候选基因启动子proLsAG之间的关系,构建双荧光素酶载体,注射烟草并比较检测荧光信号强度和LUC和REN两种酶活性的比值,验证转录因子LsHPB能否激活LsAG启动子。【结果】酵母单杂交试验显示酵母菌在0 ng/mL AbA的SD/-Leu培养基上正常生长,但在含有200 ng/mL AbA抑制浓度的SD/-Leu固体培养基上只有同时含有Prey和Bait的酵母菌株才能生长。随着酵母菌液稀释程度加倍,酵母菌斑的个数明显减少。双荧光素酶试验显示了烟草叶片荧光表达情况,pGreenII62-SK-LsHPB+pGreenII0800-LUC-ProLsAG的组合菌液注射部分荧光信号强度最高,此外此组合LUC和REN两种酶活性的比值高于其他组合。由此可验证LsAG为LsHPB基因下游调控因子,最终确定LsHPB蛋白结合LsAG启动子的互作关系。【结论】初步解析了LsHPB参与叶用莴苣抽薹的分子机理,为完善MADS-box家族基因调控植物响应高温诱导后抽薹的模型奠定基础。[Objective] AG and SOC1 belong to the MADS-box family.AG is involved in regulating the development and maturation of stamen and pistil and SOC1 is involved in the integration of many flowering pathways.In this study,LsAG was identified as one of the downstream binding gene of SOC1-like gene LsHPB of leaf lettuce.Explore the molecular mechanism of LsHPB in leaf lettuce bolting will further improve the understanding of the MADS-box family in response to high temperature-induced bolting.[Methods] The interaction between LsHPB and the candidate promoter LsAG was analyzed by Yeast-one-hybrid.Constructed vectors and coexpressed in tobacco leaves to analyze the ratio of LUC and REN activity.[Result] Yeast-one-hybrid showed that yeast grew normally on SD/-Leu medium with 0 ng/mL AbA,but only yeast strains containing both Prey and Bait could grow on SD/-Leu solid medium with 200 ng/mL AbA.Dual-LUC assays showed the combination of pGreenII62-SK-LsHPB+pGreenII0800-LUC-ProLsAG had the highest fluorescence signal intensity and LUC and REN ratio in the injected part.These results verified the interaction between LsHPB protein and LsAG promoter.[Conclusion]In this study,the molecular mechanism of LsHPB involved in bolting of leaf lettuce was analyzed,which laid a foundation of the MADS-box family in the regulation of plant bolting in response to high temperature.

关 键 词：叶用莴苣 酵母单杂 LsAG LsHPB MADS-box家族基因 

分 类 号：S636.2[农业科学—蔬菜学]