酵母单杂交文库构建及巨峰葡萄VvFT基因启动子上游调控因子筛选

Yeast one-hybrid library construction and screening of upstream regulators of VvFT promoter in kyoho grape

【目的】利用酵母单杂交文库筛选巨峰葡萄VvFT基因启动子上游调控因子,挖掘参与调控葡萄开花时间的因子,为揭示花期转变代谢通路和定向改良葡萄品种提供理论参考。【方法】利用PlantCARE分析预测VvFT基因启动子顺式作用元件,并以VvFT基因启动子为诱饵,利用酵母单杂交文库筛选技术,筛选作用在VvFT基因启动子上游的调控因子。【结果】葡萄VvFT基因启动子区域存在13种启动子顺式作用元件,包括A-box、CAAT-box和TATA-box组成型调控元件;光响应调控元件ATC-motif、Box 4、G-Box和GT1-motif;茉莉酸甲酯(MeJA)响应调控元件CGTCA-motif和TGACG-motif;干旱诱导的MYB结合位点元件MBS;玉米蛋白代谢必需的调控元件O2-site;参与生物钟控制的调控元件circadian;厌氧诱导必需调控元件ARE。以VvFT基因启动子为诱饵,筛选获得34条表达序列标签(EST),其中有21条为未知功能,有13条在植物生长、抗逆防御、信号转导、转录调控、蛋白酶等方面均有已知或预测的功能,包括转录抑制因子ft41、GRF1互作因子ft44、NAP1相关蛋白ft64及ft70分子伴侣DnaJ10。酵母单杂交点对点验证结果表明VvDnaJ10和VvFT基因启动子之间有相互作用。【结论】通过酵母单杂交文库筛选获取13条候选EST,虽然大部分EST功能预测分析结果与VvFT调控开花时间无明显的直接关系,但研究结果为进一步探索VvFT转录或表达水平控制葡萄开花时间的分子机制提供侯选基因。

【Objective】Screening of upstream regulatory factors of VvFT gene promoter in kyoho grape by yeast onehybrid library and exploring factors that involved in the regulation of flowering time of grape could help to reveal the metabolic pathway during flowering and provide a theoretical basis for the directional improvement of grape varieties.【Method】Potential cis-acting regulatory elements of the VvFT promoter were analyzed online in PlantCARE database.VvFT promoter sequence was used as a bait to search potential regulatory factors acting on VvFT promoter upstream by yeast one-hybrid library screening system in latent buds.【Result】There were 13 cis-acting elements in the VvFT promoter region of kyoho grape,including constitutive regulatory elements(A-box,CAAT-box and TATA-box),light responsive elements(ATC-motif,Box 4,G-box and GT1-motif),cis-acting regulatory elements involved in the MeJA-responsiveness(CGTCA-motif and TGACG-motif),MYB binding site involved in drought-inducibility,cis-acting regulatory element involved in zein metabolism regulation(O2-site),cis-acting regulatory element involved in circadian control(circadian)and cis-acting regulatory element essential for the anaerobic induction(ARE).Using VvFT promoter as a bait,a total of 34 ESTs were acquired in the yeast one-hybrid library.Functional classification results showed that 21 were unknown functional proteins,and 13 had known or predicted functions including plant growth,stress resistance,signal transduction,transcriptional regulation,proteases.Among them,there were transcriptional suppressor ft41,GRF1 interaction factor ft44,NAP1-related protein ft64 and ft70 molecular chaperone DnaJ10.The results of yeast single hybridization point to point verification showed that there was interaction between VvDnaJ10 and VvFT promoter.【Conclusion】In this study,13 candidate ESTs are obtained by yeast one-hybrid library screening system.Although most of the results of ESTs function prediction analysis have no obvious direct relationship with VvFT regulating flowering time,the results provide candidate gene resources for further exploration of the molecular mechanism of VvFT transcription or expression level controlling flowering time in grape.