Isolation of plant transcription factors using a modified yeast one-hybrid system

Plant Methods
Sergiy LopatoPeter Langridge

Abstract

The preparation of expressional cDNA libraries for use in the yeast two-hybrid system is quick and efficient when using the dedicated Clontechtrade mark product, the MATCHMAKER Library Construction and Screening Kit 3. This kit employs SMART technology for the amplification of full-length cDNAs, in combination with cloning using homologous recombination.Unfortunately, such cDNA libraries prepared directly in yeast can not be used for the efficient recovery of purified plasmids and thus are incompatible with existing yeast one-hybrid systems, which use yeast transformation for the library screen. Here we propose an adaptation of the yeast one-hybrid system for identification and cloning of transcription factors using a MATCHMAKER cDNA library. The procedure is demonstrated using a cDNA library prepared from the liquid part of the multinucleate coenocyte of wheat endosperm. The method is a modification of a standard one-hybrid screening protocol, utilising a mating step to introduce the library construct and reporter construct into the same cell. Several novel full length transcription factors from the homeodomain, AP2 domain and E2F families of transcription factors were identified and isolated. In this paper we propose a method...Continue Reading

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Citations

Dec 12, 2012·Interface Focus·Christopher A Penfold, David L Wild
Jul 30, 2015·Journal of Experimental Botany·Dinesh YadavSergiy Lopato
Jul 21, 2010·Plant Biotechnology Journal·Sarah MorranSergiy Lopato
Jun 13, 2012·Bioinformatics·Christopher A PenfoldDavid L Wild
Mar 19, 2013·Plant Biotechnology Journal·Nataliya KovalchukSergiy Lopato
May 19, 2021·Biotechnology and Bioengineering·Hui ZhouBu-Jun Shi
Jul 25, 2021·International Journal of Molecular Sciences·Huihui WangHongying Duan

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Datasets Mentioned

BETA
DQ353852
DQ353853
DQ353854
DQ353855
DQ353856
DQ353857
DQ353858

Methods Mentioned

BETA
one-hybrid
Y1H
cDNA library
electrophoretic mobility
Y2H
two-hybrid
size fractionation
PCR
environmental stresses

Software Mentioned

AlignX ( Vector NTI Suite
Clustal W

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