海岛棉纤维发育相关基因GbPDF2酵母单杂交文库构建及其上游基因解析

Yeast One-hybrid Library Construction and Upstream Gene Analysis of GbPDF2 in Gossypium barbadence L.

为研究棉纤维起始发育的分子调控网络,以海岛棉(Gossypium barbadence L.)品种"海渝"开花当天(0 DPA)胚珠为实验材料,利用酵母单杂交技术来筛选棉纤维起始发育相关基因GbPDF2的上游调控转录因子。首先,克隆GbPDF2的启动子(Accession:KJ475468);构建了该启动子的诱饵融合载体(GbPDF2-PRO)-pAbAi,转入酵母细胞中重组为Y1HGold[Bait-pAbAi]菌株。之后,应用Matchmaker Gold Yeast One-Hybrid Library Screening System系统构建酵母单杂交cDNA文库,转化重组酵母菌株,通过同源重组筛选GbPDF2的上游转录调节因子。构建的cDNA文库库容为2.23×106,重组率为95.8%,插入片段大小为300 bp^2000 bp,其中大于1000bp的阳性克隆为109个。cDNA插入片段经测序和Blast同源性分析,筛选出2个生长素响应因子,1个WRKY7转录因子,1个WD-repeatprotein 40等与纤维发育相关的转录调节因子,为研究棉纤维起始发育的信号转导通路奠定了基础。

In order to study the gene regulatory network underlining the cotton fiber initiation,a yeast one hybrid cDNA library of the Gossypium barbadence L.ovule at the stage of flowering（0 DPA,0 days post anthesis） was constructed and used to screen the potential upstream genes of GbPDF2,an important gene related to fiber initiation,using the Matchmaker Gold Yeast One-Hybrid Library Screening System developed by the Clonetech company.The promoter region of GbPDF2 was cloned and inserted into the vector pAbAi.The recombination bait vector and the cDNA library within the pGADT7-Rec fusion expression vectors were co-transformed into the yeast Y1HGold.The estimated cDNA library storage capacity is almost 2.23 × 106,recombinant rate is 95.8 ％ and inserted PCR fragments sizes are 300 bp～ 2000 bp,of which the fragment length of 109 positive insertion clones are larger than 1000 bp.The positive yeast colonies were harvested and cultured for extracting the plasmid,followed by PCR and sequencing analysis.Several transcription factors are screened out such as WRKY7 transcription factor,WD-repeat protein 40,IAA responsible transcription factors.This research has laid the fundament in applying yeast one hybrid method to study signal transduction pathway of fiber initiation in cotton.