不结球白菜BrCDF1的功能研究

[目的]本文旨在研究不结球白菜BrCDF1基因的功能，验证其与CO启动子之间的调控关系，以及其在植物抽薹开花中所起的作用。[方法]以不结球白菜‘苏州青’为材料，采用酵母单杂交技术，验证BrCDF1转录因子与CO启动子是否能够结合；构建过表达载体pEarlyGate101-BrCDF1，利用农杆菌介导法将过表达载体导入拟南芥中，筛选阳性苗，同时对阳性苗进行Western blot检测，观察转基因植株的表型变化；采用RT-qPCR技术，检测BrCDF1、CO、FT基因在转基因和野生型植株中不同时间点的相对表达量。[结果]酵母单杂交和β-半乳糖苷酶试验分析结果显示，BrCDF1转录因子和CO启动子之间具有较高的结合活性。Western blot检测结果表明：转基因拟南芥植株中的BrCDF1蛋白成功表达。RT-qPCR结果表明，BrCDF1基因在转基因植株中的相对表达量比野生型植株高；CO、FT基因在转基因植株中的相对表达量比野生型低，同时BrCDF1过表达植株较野生型开花晚。[结论]BrCDF1转录因子能与CO启动子结合，从而抑制CO、FT基因的表达，进而影响不结球白菜光周期开花途径。
[Objectives]The paper aimed to study the function of BrCDF1 gene in non-heading Chinese cabbage,and to verify its regulatory relationship with CO promoter and its role in bolting and flowering of plants.[Methods]The yeast single hybridization technique was used to verify whether BrCDF1 transcription factor could bind to CO promoter by using the non-heading cabbage ‘Suzhouqing’. Then the over-expression vector pEarlyGate101-BrCDF1 was constructed and introduced into Arabidopsis thaliana by Agrobacterium-mediated method. The positive seedlings were detected by Western blot. The phenotypes of transgenic plants were observed. The expression of BrCDF1,CO and FT genes in transgenic and wild type plants at different time points was detected by RT-qPCR.[Results]The results of yeast single hybridization and β-galactosidase(ONPG)assay showed that BrCDF1 transcription factor and CO promoter had strong binding activity. Western blot analysis showed that the expression of BrCDF1 protein in transgenic Arabidopsis plants was successfully expressed. The results of RT-qPCR indicated that the relative expression level of BrCDF1 gene in transgenic plants was higher than that in wild type plants. The relative expression level of CO and FT genes in transgenic plants was lower than that in wild type plants,and BrCDF1 overexpression plants showed later flowering than wild type plants.[Conclusions]The BrCDF1 transcription factor could bind to the CO promoter,thereby inhibiting the expression of CO and FT genes and thus affecting the photoperiodic pathway regulating flowering in non-heading Chinese cabbage