毛白杨酵母单杂交体系的建立  

Establishment of a yeast one-hybrid library from Populus tomentos Carr.

作　　者：刘蕾[1] 胡旭东[1] 张强[1] 蒋璐瑶 李丽红[1] 要笑云 陆海[1] 

机构地区：[1]北京林业大学生物科学与技术学院,北京100083

出　　处：《南方农业学报》2015年第3期370-375,共6页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31170574)

摘　　要：【目的】通过建立酵母单杂交文库筛选出与毛白杨特异性调控元件结合的候选蛋白,为研究毛白杨木材合成相关基因奠定基础。【方法】采用模板基因PCR方法,分别将ABRE-box、AC-box及-317-Box三次重复序列连接至p Ab Ai诱饵质粒上,再将诱饵载体转入酵母中;使用磁珠法获得m RNA,采用SMART技术合成c DNA,以c DNA为模板进行LD-PCR并将产物纯化后与p GADT7-Rec载体共转化诱饵酵母,同时进行文库的构建与筛选。【结果】以p BI121质粒为模板扩增获得的ABRE/AC/-317-GUS片段约为650 bp。以KpnⅠ对重组质粒p Ab Ai-ABRE/AC/-317-GUS进行单酶切,获得约5000 bp的大片段与650 bp的小片段;测序结果表明,插入序列与ABRE/AC/-317序列一致且方向正确。在SD/Ura培养基中,当Ab A为100 ng/m L时,酵母菌落数为0,建库时Ab A为100 ng/m L。c DNA与p GADT7载体共转化的酵母Y1HGold(p AC-Ab Ai)细胞稀释100倍后,在SD/-Leu培养基上长出89个克隆,含AC-box的诱饵酵母所建库容量为1.3×106,且无r RNA污染。酵母转化产物在SD/-Leu/Ab A(100 ng/m L)培养基上长出18个克隆,大于250 bp的有13个克隆;测序和同源分析结果表明,成功筛选到5个候选蛋白,主要是蛋白激酶、核小体蛋白及功能未知蛋白,这些蛋白可作为与AC元件结合的候选蛋白。【结论】建立的毛白杨酵母单杂体系可用于进一步筛选与木材合成相关的基因。[Objective]The present study was conducted to screen candidate binding proteins of specific regulatory elements from Populustomentos by constructing a yeast one-hybrid system in order to provide references for researching its related genes of timber synthesis. 【Method】Three copies of ABRE-box, AC-box and-317-box were linked into p Ab Ai vector by template-gene PCR, followed by transforming the bait vector into the yeast to generate bait yeast strains. By using Poly ATtract m RNA Isolation Systems, the m RNA was obtained and utilized to synthesize c DNA via SMART Technique. The purified c DNA was used as the template to synthesize dsc DNA by LD-PCR. The purified dsc DNA was co-transformed into bait yeast strain with p GADT7-Rec vector to simultaneously construct and screen yeast one-hybrid li-brary. 【 Result 】 The results showed that the amplified ABRE / AC /- 317- GUS fragment by using p BI121 plasmid as template was 650 bp in length. A large fragment of 5000 bp and a small fragment of 650 bp were obtained from recombinant plasmid p Ab Ai-ABRE/AC/-317-GUS digested by KpnⅠ. The sequencing results indicated that the inserted sequence was coincided with that of ABRE/AC/-317 in the right direction. In SD/Ura culture, the yeast colony was 0when Ab A was 100 ng/m L, so 100 ng/m L of Ab A was used for constructing the library. After yeast Y1HGold（p AC-Ab Ai）co-transformed with c DNA and p GADT7 vector was diluted 100 times, 89 clones were obtained in SD/-Leu culture, the constructed library of bait yeast with AC-box was 1.3 ×10^6without pollution of r RNA. The yeast transformed products grew out 18 clones in SD/-Leu/Ab A（100 ng/m L） culture, out of which, 13 clones was above 250 bp. The sequencing and alignment analysis results indicated that, the 5 screened candidate proteins mainly included protein kinase, nucleosome protein and proteins of unknown function, which could be used as candidate proteins combining with AC elements.【Conclusion 】The constructed yeast one-hybrid system of Populus toment

关 键 词：毛白杨 酵母单杂交体系 载体构建 文库构建 

分 类 号：S792.117[农业科学—林木遗传育种]