摘要
共生结瘤过程是根瘤菌与宿主植物交换信号分子的相互作用过程,结瘤信号通道蛋白基因NSP1和NSP2是结瘤因子诱导的共生信号转导途径的必要元件。LjNSP1和LjNSP2蛋白都包含植物所特有的GRAS结构域,推测为转录调节子,但缺乏明确的生化证据。为验证这一推测,本研究采用酵母双杂交系统,将两者分别融合到酵母GAL4转录因子的DNA结合结构域上,根据是否能激活报告基因的表达来验证它们的转录激活能力。结果表明LjNSP1和LjNSP2在酵母体内均具有转录激活的能力。为进一步确定转录激活的区域,对LjNSP1和LjNSP2构建了一系列的缺失突变,鉴别出转录激活区段均位于两者的N-末端的可变区域。为验证作为转录因子的LjNSP1和LjNSP2是否具有结合某种特定基因的启动子的能力,进行了酵母单杂交实验。其结果表明,LjNSP1和LjNSP2蛋白均能够结合根瘤起始基因NIN的启动子,但不能结合钙离子结合蛋白基因CBP1的启动子。
The symbiotic nodulation between legumes and rhizobacteria is an interactive process of exchange of signal molecules.Genes for nodulation signal pathway NSP1 and NSP2 are the essential components for nod-factor induced symbiotic signal pathway.Protein encoded by LjNSP1 and LjNSP2 includes the plant-specific GRAS domain,which are predicted to code transcription factor,but the unique biochemical evidence remains to be identified.To examine this inference,the above two genes were fused into DNA binding domain for yeast GAL4 transcription factor with yeast two hybrid.Whether or not activating expression of downstream reporter genes were utilized to test their function of transcriptional activation.Our result substantiated encoding products of these two genes possess such activation in yeast.To further determine the transcriptional activation region,a series of deletion mutants were constructed,which successfully identified the activation region belonging to the variable domain of N terminal of each protein.Yeast one-hybrid was performed to test whether LjNSP1 and LjNSP2 proteins,possess function of binding promoter for certain specified genes as transcription factor.The result showed that proteins encoded by LjNSP1 and LjNSP2 were capable of binding promoter of NIN（nodulation initiation）gene,but failed to bind that of CBP1（calcium binding protein）gene.This study demonstrates that LjNSP1 and LjNSP2 possess function of binding particular DNA and transcriptional activation,providing important significance for the elucidation of mechanism for nod-factor induced signaling.
出处
《华中农业大学学报》
CAS
CSCD
北大核心
2010年第5期582-587,共6页
Journal of Huazhong Agricultural University
基金
国家自然科学基金项目(30870186)资助
关键词
百脉根
共生信号转导
转录激活
GRAS蛋白
酵母单杂交
Lotus japonicus
symbiotic pathway
transcriptional activation
GRAS protein
yeast one-hybrid
