A Three-Component Gene Expression System and Its Application for Inducible Flavonoid Overproduction in Transgenic Arabidopsis thaliana
نویسندگان
چکیده
Inducible gene expression is a powerful tool to study and engineer genes whose overexpression could be detrimental for the host organisms. However, only limited systems have been adopted in plant biotechnology. We have developed an osmotically inducible system using three components of plant origin, RD29a (Responsive to Dehydration 29A) promoter, CBF3 (C-repeat Binding Factor 3) transcription factor and cpl1-2 (CTD phosphatase-like 1) mutation. The osmotic stress responsible RD29a promoter contains the CBF3 binding sites and thus RD29A-CBF3 feedforward cassette enhances induction of RD29a promoter under stress. The cpl1-2 mutation in a host repressor CPL1 promotes stress responsible RD29a promoter expression. The efficacy of this system was tested using PAP1 (Production of Anthocyanin Pigment 1) transgene, a model transcription factor that regulates the anthocyanin pathway in Arabidopsis. While transgenic plants with only one or two of three components did not reproducibly accumulate anthocyanin pigments above the control level, transgenic cpl1 plants containing homozygous RD29a-PAP1 and RD29a-CBF3 transgenes produced 30-fold higher level of total anthocyanins than control plants upon cold treatment. Growth retardation and phytochemical production of transgenic plants were minimum under normal conditions. The flavonoid profile in cold-induced transgenic plants was determined by LC/MS/MS, which resembled that of previously reported pap1-D plants but enriched for kaempferol derivatives. These results establish the functionality of the inducible three-component gene expression system in plant metabolic engineering. Furthermore, we show that PAP1 and environmental signals synergistically regulate the flavonoid pathway to produce a unique flavonoid blend that has not been produced by PAP1 overexpression or cold treatment alone.
منابع مشابه
Analysis of SFL1 and SFL2 Promoter Region in Arabidipsis thaliana using Gateway Cloning System
SFL1 and SFl2 (SETH Four Like) genes are two members of SETH4 gene family in Arabidopsis thaliana expressed in saprophytic tissues. In this study, expression of SFL1 and SFL2 genes were studied using Gateway Cloning Technology. Primers were designed for PCR amplification of promoter region of SFL1 (900 bp) and SFL2 (930 bp) genes having attB1 recombination sites using Kod Hi Fi DNA polymerase e...
متن کاملIsolation of Brassica napus MYC2 gene and analysis of its expression in response to water deficit stress
Manipulation of stress related transcription factors to improve plant stress tolerance is a major goal of current biotechnology researches. MYC2 gene encodes a key stress-related transcription factor involved in Jasmonate (JA) and abscisic acid (ABA) signaling pathways in Arabidopsis. Brassica napus, as a globally important oilseed crop, is a close relative of Arabidopsis. In the present study...
متن کاملSelectable Marker Gene Removal and Expression of Transgene by Inducible Promoter Containing FFDD Cis-Acting elements in Transgenic plants
Abstract Background: Selectable marker gene (SMG) systems are critical for generation of transgenic crops. Transgenic crop production Background: Selectable marker gene (SMG) systems are critical for generation of transgenic crops. Transgenic crop production without using SMG is not economically feasible. However, SMGs are non-essential once an intact transgenic plant has been established. Eli...
متن کاملDifferential Expression of Arabidopsis thaliana Acid Phosphatases in Response to Abiotic Stresses
The objective of this research is to identify Arabidopsis thaliana genes encoding acid phosphatases induced by phosphate starvation. Multiple alignments of eukaryotic acid phosphatase amino acid sequences led to the classification of these proteins into four groups including purple acid phosphatases (PAPs). Specific primers were degenerated and designed based on conserved sequences of PAPs isol...
متن کاملNegative control of Strictisidine synthase like-7 gene on salt stress resistance in Arabidopsis thaliana
Strictosidine synthase-like (SSL) is a group of gene families in the Arabidopsis genome, which whose orthologues in other plants are key enzymes in mono-terpenoid indole-alkaloid biosynthesis pathway. The SSL7 is upregulated upon treatments of Arabidopsis plants with signaling molecules such as SA, methyl jasmonate and ethylene. To find the functional role of the gene, a T-DNA-mediated knockout...
متن کامل