Novel vector systems to accelerate functional analysis of transcription factors using chimeric repressor gene-silencing technology (CRES-T)

Abstract

Chimeric repressor gene-silencing technology (CRES-T) is a powerful tool that has recently been developed for the functional analysis of plant transcription factors and for the genetic manipulation of plant traits. For CRES-T, a transcription factor is converted to a strong repressor by fusion with an SRDX repression domain, which is then expressed in plants to induce a loss-of-function phenotype. However, the traditional CRES-T vectors are inconvenient for gene cloning and promoter exchange. In this study, we developed new CRES-T vectors that are efficient and convenient to use by employing the Gateway system, a new vector backbone and a terminator derived from the heat shock protein 18.2 (HSP) gene. Our test experiments revealed that the CRES-T vector containing the Gateway linker sequence within the transcribed region showed reduced efficiency of CRES-T when compared with the traditional CRES-T vector. However, the HSP terminator compensated for the negative effect of the Gateway sequence and improved the efficiency of CRES-T in all cases tested and resulted in the highest efficiency achieved to date. We found that the HSP terminator increased transcription efficiency or transcript stability; in contrast, these factors were negatively affected by the Gateway linker sequence in our vector system. We, therefore, propose that the appropriate CRES-T vector should be chosen depending on situations and purposes.