摘要
【目的】利用酵母双杂交技术从小麦cDNA文库中筛选TaDREB6互作蛋白,进一步研究DREB介导的小麦抗逆机制。【方法】以小麦的cDNA为模板扩增得到TaDREB6,构建小麦cDNA文库和pGBKT7-TaDREB6诱饵载体,将诱饵载体pGBKT7-TaDREB6以及pGADT7和cDNA文库混匀转入AH109酵母感受态细胞,在SD/-Ade/-His/-Leu/-Trp营养缺陷型培养基上,30℃培养3—5 d,挑取平板上直径大于2 mm的菌落,进行显蓝筛选。【结果】对筛选到的102个候选阳性克隆进行测序,在NCBI上进行BLAST比对分析,获得与能量代谢、抗逆和防御、转运、转录和翻译、信号转导以及生长发育等相关的候选蛋白。【结论】筛选得到候选蛋白的功能预测表明TaDREB6可能参与了多条信号转导途径,在抗逆境调控中具有重要作用。
【Objective】TaDREB6 was used as a bait to screen the interaction proteins from wheat cDNA library by yeast-two hybrid system to further explore the DREB-mediated stress resistance mechanism in wheat.【Method】The TaDREB6 gene was amplified using wheat cDNA as the template.The wheat cDNA library and bait vector pGBKT7-TaDREB6 were constructed,respectively.The mixture of bait vectors pGBKT7-TaDREB6,pGADT7 and wheat cDNA library was introduced into yeast competent cells AH109 and cultured on SD/-Ade/-His/-Leu/-Trp plates for incubation at 30℃ for 3-5 days.Then the clones whose diameter was greater than 2 mm were selected for incubation on the plates containing X-α-gal to screen blue clones.【Result】A total of 102 candidate positive clones were sequenced and analyzed through homology analysis using the BLAST in NCBI.Homology analysis showed that those candidate proteins were related to energy metabolism,stress and defense,transport,transcription and translation,signal transduction,growth and development.【Conclusion】Function prediction of the candidate proteins suggested that TaDREB6 was possibly involved in several stress signal transduction pathways and play a important role in regulation stress resistance.
出处
《中国农业科学》
CAS
CSCD
北大核心
2011年第22期4740-4747,共8页
Scientia Agricultura Sinica
基金
转基因生物新品种培育科技重大专项(2009ZX08009-083B,2009ZX08002-008B)
关键词
普通小麦
酵母双杂交
DREB
互作蛋白
抗逆机制
common wheat
yeast two-hybrid
DREB
interaction protein
stress-resistant mechanism
