甜菜M14品系乙二醛酶I基因的转录因子参与响应盐胁迫的调控机制研究

Sugar beet monosomic addition line M14 was obtained from the intercross between Beta vulgaris L. and Beta corolliflora Zoss., and it contains the B. vulgaris L.genome with the addition of chromosome 9 of B. corolliflora Zoss. .The M14 has retained good characteristics including apomixis and tolerance to drought, cold and salt from B. corolliflora. Sugar beet M14 is a good material to study the resistance mechanisms to abiotic stresses. Based on the previous work of the gene and promoter function of BvM14-glyoxalase I , regulatory mechanisms of transcription factor in regulating BvM14-glyoxalase I gene (BvM14-glyoxalase I-TF) involved in the response to salt stress will be investigated in this project. First, using yeast one-hybrid technology, the coding sequence of BvM14-glyoxalase I-TF gene in the response to salt stress will be obtained. Then using gene overexpression and RNAi techniques, the function of BvM14-glyoxalase I-TF gene responsive to salt stress will be revealed. Combining co- immunoprecipitation (co-IP) and LC-MS/MS, other regulatory proteins including key protein kinase associated with the transcription factor will be obtained. The interactions between transcription factor and key protein kinase in tobacco and sugar beet M14 line are determined by two way co-IP and bimolecular fluorescence complementation (BiFC) technologies. By establishing the relationship between the key regulatory protein kinase, BvM14-glyoxalase I-TF and BvM14-glyoxalase I gene, regulatory mechanisms of BvM14-glyoxalase I-TF transcription factor responsive to salt stress will be established.

甜菜M14品系是二倍体栽培甜菜附加野生白花甜菜第9号染色体的单体附加系，具有耐盐、抗旱等优良性状，是研究甜菜抗逆机制的难得材料。本项目以对甜菜M14品系乙二醛酶I基因（BvM14-glyoxalase I）功能的研究及启动子核心序列的研究作为前期基础，研究甜菜M14品系乙二醛酶I基因转录因子参与响应盐胁迫的调控机制。首先利用酵母单杂交技术获得响应盐胁迫的乙二醛酶I基因转录因子（BvM14-glyoxalase I-TF）的编码基因。然后利用转基因技术研究该基因参与响应盐胁迫的功能。利用免疫共沉淀和质谱技术获得与BvM14-glyoxalase I-TF转录因子结合的蛋白激酶。通过建立关键蛋白激酶，BvM14-glyoxalase I-TF及BvM14-glyoxalase I基因三者之间的调控关系，阐明甜菜M14品系BvM14-glyoxalase I-TF参与响应盐胁迫的调控机制。

甜菜M14品系是二倍体栽培甜菜附加野生白花甜菜第9号染色体的单体附加系，具有耐盐、抗旱等优良性状，是研究甜菜抗逆机制的难得材料。本研究鉴定了BvM14-glyoxalase I基因响应盐胁迫的最强启动子片段，利用酵母单杂交技术获得盐胁迫下与BvM14-glyoxalase I基因启动子特异互作的转录因子；对其中关键的3个转录因子BvM14-GT-3b-2、BvM14-Dof3.4和BvM14-GAI参与响应盐胁迫功能研究表明，BvM14-GT-3b-2基因、BvM14-Dof3.4基因和BvM14-GAI基因能够提高转基因拟南芥植株的耐盐性；初步建立了上述转录因子在转录水平上调节 BvM14-glyoxalase I基因耐盐的分子网络。