与CRY1相互作用的转录因子蛋白的筛选  

Identification of CRY1-interactive transcription factor proteins

作　　者：何志敏[1] 冯盼盼[1] 荣朵艳 赵小英[1] 刘选明[1,2] 

机构地区：[1]湖南大学生物学院化学生物传感与计量学国家重点实验室,长沙410082 [2]湖南大学生物学院植物功能基因组学与发育调控湖南省重点实验室,长沙410082

出　　处：《生物学杂志》2016年第5期47-52,86,共7页Journal of Biology

基　　金：国家自然科学基金资助项目(31171176);湖南省自然科学基金资助项目(11JJA002);湖南省生物发育工程及新产品研发协同创新中心(20134486)

摘　　要：拟南芥CRY1(cryptochrome1)通过蛋白相互作用启动蓝光信号的传导,从而促进植物光形态建成。为深入研究CRY1介导蓝光信号传导的机制,以CRY1为诱饵蛋白,通过酵母双杂交系统的营养缺陷型试验、液体显色试验、分段相互作用试验方法,从拟南芥转录因子酵母库中筛选与其相互作用的转录因子蛋白。结果表明,通过营养缺陷型筛选试验以及以ONPG为底物的液体显色试验初步获得与CRY1相互作用的41个转录因子蛋白;选取与CRY1相互作用蓝光依赖程度较高或酶活性高的转录因子以CPRG为底物的液体显色试验进行进一步验证,发现选取的转录因子蛋白与CRY1的相互作用具有蓝光依赖性且随蓝光处理时间的延长相互作用强度增强;CRY1各分段与转录因子蛋白相互作用试验结果表明转录因子SPL3和IDD4主要与CRY1的N端相互作用,与C端的相互作用比较微弱,但是SPL3与CRY1 C端的相互作用具有蓝光特异性而IDD4与CRY1 C端的蓝光特异性相互作用不明显。Arabidopsis CRY1（ cryptochrome 1） protein initiates blue light signal transduction in a protein interaction manner,consequently causes the photomorphogenesis in plants. To further study the mechanism of CRY1 mediating blue light signal transduction,CRY1 was used as a bait,the methods of yeast two hybrid system,such as preliminary auxotrophy,liquid assays and domain interactions analyses in yeast cells were employed to screen the transcription factor proteins from the Arabidopsis transcription factor yeast library. The results showed that 41 transcription factor proteins were screened out by the preliminary auxotrophy and the liquid assays with ONPG as substrate; the transcription factor proteins which interacted with CRY1 with high blue light dependent or strong enzyme activity were picked out for a further validation using liquid assay with CPRG as substrate,and found that their interactions with CRY1 showed blue light dependent and the activity of interactions would increase with the time of blue light treatment; the interactions analyses results of CRY1 domains and transcription factor protein showed that the transcription factor proteins SPL3 and IDD4 interacted with the N terminus of CRY1 primarily,and relatively weak with the C terminus,however,the blue light dependent existed between the interaction of the C terminus of CRY1 and SPL3,but not so obvious with IDD4.

关 键 词：CRY1 酵母双杂交 转录因子 拟南芥 

分 类 号：Q71[生物学—分子生物学] Q943.2