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

doi:10.11924/j.issn.1000-6850.2014-2290

Abstract

Abstract: 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.

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