木薯干旱胁迫下离区发育相关基因MeAP_(2-2)酵母单杂交文库构建及其上游调控基因的筛选

为了研究干旱胁迫下调节木薯叶片脱落相关的重要节点基因及其分子调控网络,以‘华南5号’木薯为实验材料,通过采用酵母单杂交筛选系统,将Me AP2-2启动子中激素与胁迫相关的元件串联后整合入酵母染色体,构建诱饵菌株;采用SMART技术进行离区c DNA文库的构建,将离区c DNA与p GADT7-Rec表达载体共同转化诱饵菌株,通过同源重组在酵母细胞内筛选Me AP2-2的上游转录调节因子;利用酵母菌落PCR法获得阳性克隆中的c DNA插入片段,测序后在JGI网站进行Blast分析。结果表明,构建的c DNA文库库容为1.5×106,插入片段大小为250~3000 bp。c DNA插入片段经测序和Blast同源性分析和定量PCR分析,筛选出1个metallothionein protein,1个core histone protein,1个heavymetal-associated protein与Me AP2-2相关的调节因子。实验结果证明酵母单杂交文库构建成功,初步筛选获得了调节Me AP2-2的上游调节因子。为研究木薯干旱胁迫下离区发育的信号转导通路奠定了基础。

Yeast One-hybrid Library Construction and Upstream Gene Analysis of MeAP2-2 in Cassava Under Drought Condition.

In order to study the key genes and gene regulatory network in the leaf abscission zone under drought condition in cassava, we used cassava SC5 as experimental material, Matchmaker Gold Yeast OneHybrid Library Screening System was employed in this study. Bait yeast strain was constructed by synthesizing oligonucleotides containing three tandem copies of core element sequences of phytohormone and stress and integrating it into the genome of yeast. The cDNA for abscission zone of cassava under drought stress was synthesized via SMART technology and co-transformed into bait yeast strain with pGADT7-Rec vector; onehybrid cDNA library was simultaneously constructed and screened directly in yeast as a result of in vivo plasmid recombination. cDNA inserts in positive clones was amplified by yeast colony PCR and analyzed through JGI database Blast after sequencing. The estimated cDNA library storage capacity is almost 1.5×106, and inserted PCR fragments sizes are 250-3000 bp. The positive yeast colonies are harvested and cultured for extracting the plasmid, followed by PCR and sequencing analysis. Several genes are screened out such as Metallothionein protein, Core histone protein, and Heavy-metal-associated protein. QPCR indicates that the candidate genes have the same expression patterns with the MeAP2- 2 in response to drought stress. This research has laid the fundament in applying yeast one hybrid method to study signal transduction pathway of abscission zone development in cassava.