《植物生理学报》 2017, 53(7): 1259-1266

桃ABA信号关键基因PpABI5酵母单杂交文库构建及其上游转录因子的筛选

郇蕾, 王旭旭, 陈修淼, 文滨滨, 谭秋平, 陈修德, 高东升, 李玲^*, 付喜玲^*

山东农业大学园艺科学与工程学院, 作物生物学国家重点实验室, 山东泰安271018

通信作者：李；E-mail: lilingsdau@163.com; xilingfu@sdau.edu.cn

摘　要：

脱落酸(ABA)在植物的生长发育中起着重要的作用, ABI5是响应ABA信号的关键基因, 研究调控ABI5基因表达的转录因子对进一步阐述ABA信号转导及调控具有重要意义。本研究中, 将3-AF1、Box I和Sp1三次重复串联后连接到pAbAi诱饵质粒上, 转化酵母细胞构建诱饵载体, 构建桃(Prunus persica)酵母单杂交cDNA文库, 再共转化诱饵菌株, 经同源重组筛选PpABI5启动子的上游转录调节因子。构建的cDNA文库库容为1×10⁷ CFU, 插入片段长度平均在1 500 bp左右。酵母单杂交筛选结果经测序和Blast同源性分析, 获得PpDAM3和PpDAM5两个转录因子。酵母单杂交进一步证实PpDAM3和PpDAM5能与PpABI5启动子结合。这些研究结果表明, PpDAM3和PpDAM5可能参与调控PpABI5的转录, 并为深入研究ABA信号转导通路奠定基础。

关键词：ABI5; 桃; 启动子; 酵母单杂交; 转录因子

收稿：2017-03-06   修定：2017-06-16

资助：国家自然科学基金(31672137)和山东省自然科学基金(ZR2014CM015)。

Constructing yeast one-hybrid library and screening the potential regulator of PpABI5 in peach (Prunus persica)

HUAN Lei, WANG Xu-Xu, CHEN Xiu-Miao, WEN Bin-Bin, TAN Qiu-Ping, CHEN Xiu-De, GAO Dong-Sheng, LI Ling^*, FU Xi-Ling^*

College of Horticulture Science and Engineering, Shandong Agricultural University, State Key Laboratory of Crop Biology, Tai’an, Shandong 271018, China

Corresponding author: LI Ling; E-mail: lilingsdau@163.com; xilingfu@sdau.edu.cn

Abstract:

Abscisic acid (ABA) plays an important role in plant growth and development. ABI5 is a key ABA-responsive gene, so it is important to find the transcription factors that regulate the expression of ABI5 to reveal the mechanism underlying ABA signaling. In this study, three copies of 3-AF1 binding site, Box I and Sp1 were linked into pAbAi vector to construct bait vector, meanwhile the one-hybrid cDNA library of peach (Prunus persica) was built. Then we used the cDNA and pGADT7-Rec vector to co-transform into the bait yeast strain to screen upstream transcription factors of the promoter region of PpABI5 gene via homologous recombination. The estimated cDNA library storage capacity is almost 1×10⁷ CFU and inserted PCR fragments sizes are about 1 500 bp. Several proteins interacting with bait vector are screened out by sequencing analysis and by Blast homology analysis, including PpDAM3, PpDAM5 and proteins of unknown function. Yeast one-hybrid further confirmed that PpDAM3 and PpDAM5 could bind to the promoter of PpABI5. These results indicate that PpDAM3 and PpDAM5 might regulate the transcription of PpABI5 and provide a basis for further study of signaling pathway of ABA.

Key words: ABI5; peach; promoter; yeast one-hybrid; transcription factor