Expressional Characterization Of Arabidopsis RDR1 Gene Promoter

Gene silencing, as a common mechanism of gene expression, widely utilizes small RNAs and protein factors to repress gene transcription or arouse mRNA degradation in a sequence specific manner. Among those protein factors involved in gene silencing, RNA-dependent RNA polymerase(RDR) plays a big role in catalyzing synthesis of the RNA strand complementary to a given single-stranded RNA template, producing double-stranded RNA. The resulting double-stranded RNAs are processed by cellular RNase III-like DCLs into 20~24nt small interfering RNAs(siRNA) transcriptionally or post-transcriptionally repressing target gene expression. Depending on gene silencing pathway, RDR participates in multiple biological processes, such as plant developmental regulation, stress response and epigenetic modification. In plants, different RDRs with varied biological functions proved coexist in the genome. Previous study of forward genetics showed us that AtRDR1 mainly participates in anti-viral process, but AtRDR6 possesses versatile functions including virus defense, stress response and developmental regulation. Additionally, there are several endogenous signals and stress treatments able to affect their expression. To further investigate into anti-viral mechanism of AtRDR1 and explore its biological functions, it is of great significance to deeply characterize its expression.Here, the full-length promoter of Arabidopsis AtRDR1 was cloned and then used to get five truncated promoters with varied deletions. All of the six promoters substituted CaMV 35S promoter in pCAMBIA 1303 plant binary vector for controlling the expression of GUS-GFP fusion gene. Different transgenic lines of each construct obtained were subjected to analysis of expression profile and its response to endogenous signals and stresses. Finally, under the guidance of our experimental results, we pointedly observed the phenotype of rdr1 mutant. Results we have obtained are listed below.1. Expression of AtRDR1 was mainly detected in the phloem of vascular tissue system and mature silique, but not in apical meristem and young tissues such as root tip, shoot, young true leaf, young bud and young silique.2. AtRDR1 promoter was able to response to treatments with NAA, JA, GA3 and cold which can induce expression of marker gene driven by AtRDR1 promoter. Contrarily, other treatments, including NaCl, SA, ABA and PEG6000, repressed its expression. Among these treatments, the broadest fluctuations of expression level were found after NAA, SA and NaCl treatments.6. Compared with deleted promoters, full-length AtRDR1 promoter showed the maximal expression ability. Furtherly, results showed that DNA fragments localized in -1088~-690 region and 5'UTR intron positioned in +112~+199 region exerted a great influence on expressional strength of the promoter; and -434~ +215 DNA fragment controls minimal expression level. Investigation into 5'UTR intron indicated that its existence benefited cellular accumulation of GUS-GFP transcripts, finally increasing gene expression.3. Cis-elements positively responding to plant auxin and negatively responding to dehydration were concurrently positioned in -1088~-690 region. Cis-element positively responding to low temperature was found in -690~-434 region. Additionally, 5'UTR intron carries negative element involved in the JA responsiveness.4. In Arabidopsis, AtRDR1 has been proved to play a role in silique development. Its mutation decreases silique length and seed number, but does not affect seed germination, root growth, leaf phenotype, flowering time and seed size.The above results richened our understanding to expressional regulation mechanism of AtRDR1, uncovered its new biological function, and also laid a foundation for further illustrating its anti-viral mechanism in plant.