Construction of a genomewide RNAi mutant library in rice

Long hairpin RNA (hpRNA) transgenes are a powerful tool for gene function studies in plants, but a genomewide RNAi mutant library using hpRNA transgenes has not been reported for plants. Here, we report the construction of a hpRNA library for the genomewide identification of gene function in rice using an improved rolling circle amplification‐mediated hpRNA (RMHR) method. Transformation of rice with the library resulted in thousands of transgenic lines containing hpRNAs targeting genes of various function. The target mRNA was down‐regulated in the hpRNA lines, and this was correlated with the accumulation of siRNAs corresponding to the double‐stranded arms of the hpRNA. Multiple members of a gene family were simultaneously silenced by hpRNAs derived from a single member, but the degree of such cross‐silencing depended on the level of sequence homology between the members as well as the abundance of matching siRNAs. The silencing of key genes tended to cause a severe phenotype, but these transgenic lines usually survived in the field long enough for phenotypic and molecular analyses to be conducted. Deep sequencing analysis of small RNAs showed that the hpRNA‐derived siRNAs were characteristic of Argonaute‐binding small RNAs. Our results indicate that RNAi mutant library is a high‐efficient approach for genomewide gene identification in plants.     

Sequence analysis of rice rubi3 promoter gene expression cassettes for improved transgene expression

In a construct containing a GUS reporter gene driven by the 5′ regulatory elements from rubi3, expression was enhanced 4-fold when a 20-nucleotide (nt) GUS 5′ untranslated sequence was replaced with 9 nt sequences derived from rubi3′s second exon. The roles of the sequences immediately upstream from the GUS translation initiation codon, and their significance in gene expression, were investigated. Sequence analysis suggests that complementarity between sequences immediately 5′ of a translation initiation codon and the rice 17S rRNA may be responsible for the reduction in protein levels from constructs containing the GUS leader sequence. The results demonstrate an affect sequences immediately upstream from transgenic coding sequences have on expression, and when using the rubi3 5′ regulatory sequence in particular.     

pSAT RNA interference vectors: a modular series for multiple gene down-regulation in plants

RNA interference (RNAi) is a powerful tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target genes, enabling loss-of-function studies in living cells. Hairpin (hp) RNA expression cassettes are typically constructed on binary plasmids and delivered into plant cells by Agrobacterium-mediated genetic transformation. Realizing the importance of RNAi for basic plant research, various vectors have been developed for RNAi-mediated gene silencing, allowing the silencing of single target genes in plant cells. To further expand the collection of available tools for functional genomics in plant species, we constructed a set of modular vectors suitable for hpRNA expression under various constitutive promoters. Our system allows simple cloning of the target gene sequences into two distinct multicloning sites and its modular design provides a straightforward route for replacement of the expression cassette's regulatory elements. More importantly, our system was designed to facilitate the assembly of several hpRNA expression cassettes on a single plasmid, thereby enabling the simultaneous suppression of several target genes from a single vector. We tested the functionality of our new vector system by silencing overexpressed marker genes (green fluorescent protein, DsRed2, and nptII) in transgenic plants. Various combinations of hpRNA expression cassettes were assembled in binary plasmids; all showed strong down-regulation of the reporter genes in transgenic plants. Furthermore, assembly of all three hpRNA expression cassettes, combined with a fourth cassette for the expression of a selectable marker, resulted in down-regulation of all three different marker genes in transgenic plants. This vector system provides an important addition to the plant molecular biologist's toolbox, which will significantly facilitate the use of RNAi technology for analyses of multiple gene function in plant cells.     

Sugar-Dependent Expression of the CHS-A Gene for Chalcone Synthase from Petunia in Transgenic Arabidopsis

Transgenic Arabidopsis thaliana plants were constructed by introduction of a fusion of the gene for β-glucuronidase (GUS) to the CHS-A gene, which is one of the two genes for chalcone synthase that are actively expressed in the floral organs of petunia. The expression of the fusion gene CHS-A::GUS was low in transgenic Arabidopsis plantlets, but it was enhanced when plantlets or detached leaves were transferred to a medium that contained 0.3 molar sucrose, glucose, or fructose. No enhancement was observed when plantlets were transferred to a medium that contained 0.3 molar mannitol. Measurements of cellular levels of sugars revealed a tight linkage between the level of expression of the CHS-A::GUS gene and the level of accumulation of exogenously supplied sugars, in particular sucrose. The parallelism between the organ-specific accumulation of sugar and the organ-specific expression of the CHS-A::GUS gene was also observed in petunia and A. thaliana plants grown under normal conditions in soil. The consensus sequences for sugar responses, such as boxes II and III in members of the family of sporamin genes from the sweet potato, were found in the promoter region of the CHS-A gene that was used for fusion to the GUS gene. It is suggested that the expression of the CHS-A gene is regulated by sugars, as is the expression of other sugar-responsive genes, such as the genes for sporamin. A putative common mechanism for the control of expression of “sugar-related” genes, including the CHS-A gene, is discussed.     

Improvement of the Coprinopsis cinerea molecular toolkit using new construct design and additional marker genes

This paper describes the optimisation of an existing basidiomycete molecular toolkit through the development of new versatile vectors. These vectors enable the straightforward and rapid construction of gene expression and silencing cassettes by allowing the easy exchange of promoters, coding regions and terminator elements. The constructs contain multiple cloning sites (MCS) allowing any gene to be inserted using a range of restriction sites, with the option of a 5′ integral intron for efficient gene expression. We describe the testing of these vectors through marker gene expression in Coprinopsis cinerea. This work also extends the range of marker genes available for use in C. cinerea with the first report of DsRed and monomeric red fluorescent protein (mRFP) expression in C. cinerea and further demonstrates the requirement for an intron in the expression cassette for some marker genes. However, analysis of transformants containing either β-glucuronidase (GUS) or luciferase (LUC) genes, with and without an intron revealed no detectable marker gene expression. The inclusion of an intron does therefore not guarantee expression and other genetic factors may be involved.     

MicroRNA-based RNA Interference Vector for Gene Silencing in Plants

RNA interference (RNAi) is a powerful tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target genes. In this study a microRNA 159a-based binary vector was constructed which can be used for hpRNA expression. Hairpin (hp) RNA expression cassettes carrying the gene sequences are typically constructed on binary plasmid and delivered into plant cells by Agrobacterium-mediated genetic transformation. This system allows simple insertion of 21- nt target gene sequences into microRNA backbone, to facilitate the processing of microRNA hpRNA by the endogenous machinery of host, thereby producing artificial microRNA carrying the sequence of target gene(s). The functionality of new vector system was tested by silencing viral gene in transgenic plants. Strong down regulation of viral gene was observed in virus infected tobacco plants transformed with pAmiR₁₅₉ vector. The processing of amIRNA leading to viral-specific sIRNA was confirmed by northern blotting. This vector system provides an important addition to the plant molecular biologists’ toolbox, which will significantly facilitate the use of RNAi technology for analyses of various gene functions in plant cells.     

Constructs and methods for high-throughput gene silencing in plants

Gene silencing can be achieved by transformation of plants with constructs that express self-complementary (termed hairpin) RNA containing sequences homologous to the target genes. The DNA sequences encoding the self-complementary regions of hairpin (hp) RNA constructs form an inverted repeat. The inverted repeat can be stabilized in bacteria through separation of the self-complementary regions by a "spacer" region. When the spacer sequence encodes an intron, the efficiency of gene silencing is very high. There are at least three ways in which hpRNA constructs can be made. The construct may be generated from standard binary plant transformation vectors in which the hairpin-encoding region is generated de novo for each gene. Alternatively, generic gene-silencing vectors such as the pHANNIBAL and the pHELLSGATE series can be used. They simply require the insertion of PCR products, derived from the target gene, into the vectors by conventional cloning or by using the Gateway directed recombination system. In this article, we describe and evaluate the advantages of these vectors and then provide the protocols for their efficient use.