This document describes a set of binary vectors for producing dominant negative RNAi mutants using a target sequence cloning strategy that is based on the inclusion of two restriction enzyme cleavage sites in each of two primers used to amplify gene-specific fragments from cDNA. This design minimizes the number of PCR primers and results in the placement of unique restriction enzyme recognition sites to allow for flexibility in future manipulations of the plasmid, e.g., moving the inverted repeat target sequence to a different vector. ChromDB’s RNAi vectors are based on pCAMBIA binary vectors, a set of plasmids developed by the Center for Application of Molecular Biology to International Agriculture (CAMBIA). Importantly, CAMBIA vectors contain two origins of replication, a wide-host-range origin for plasmid replication in Agrobacterium tumefaciens and the pBR322 origin for replication in Escherichia coli, following the design of the Maliga lab (Plant Mol Biol 1994; 25(6):989-94). Detailed descriptions of pCAMBIA vectors, including conditions for use and distribution, can be obtained at the CAMBIA web siteRNAi vectors are distributed by ABRC
Please do not request plasmids from ChromDB. The RNAi vectors are distributed by ABRC/TAIR. Table 1 (and "Order Vectors" link) provides a list of available vectors and includes the ABRC stock number (direct link to the vector page at the TAIR database), brief information about each vector and links to bring up plasmid maps and FASTA sequence files. For those researchers using Gene Construction Kit software, GCKs file of each sequence are included as links.
The following overview is specific for Arabidopsis vectors, but the method is applicable for the maize vector, pMCG161. Please see the section below for details on the maize vector pMCG161. We generally recommend using vector pFGC5941 (plasmid information can be accessed via Table 1), which has the following key features: a kanamycin resistance (kanR) gene for bacterial selection, a basta resistance (bar) gene for plant selection, a CaMV 35S promoter to drive the expression of the inverted repeat target sequence, and a 1,352-bp ChsA intron (from the petunia Chalcone synthase A gene) to stabilize the inverted repeat of the target gene fragment. Plasmid pFGC5941 was built from pCAMBIA1300. The complete nucleotide sequence for pFGC5941, as well as a restriction enzyme map, can be found using the links in Table 1. (Note: The TMV omega leader sequence happens to be present between the CaMV 35S promoter and the inverted repeat but is not expected to influence RNAi.)Figure 1 shows the basic design of the 5’ and 3’ primers used for cloning Arabidopsis gene fragments. Each primer introduces “inner” restriction enzyme sites, AscI or SwaI, and “outer” restriction enzyme sites, BamHI or Xba, at the ends of the resulting PCR fragment. These same sites flank the ChsA intron in pFGC5941. Two bases, indicated by Xs in Figure 1 , are included 5’ to the restriction enzyme sites to allow for maximum cleavage at the 5’-most restriction enzyme site. Eighteen bases of gene-specific sequence, indicated by Ns in Figure 1 , are included for annealing the primer to the target site.
The inverted repeat is assembled directly in the binary vector by a two-step cloning process (Figure 2 ) using the introduced restriction enzyme sites. In the first cloning step, the PCR product is cleaved at the inner restriction sites, AscI and SwaI, and ligated to cleaved AscI and SwaI sites in pFGC5941. We recommend sequencing cloned fragments to confirm their identity. For the second cloning step, the plasmid resulting from the first cloning step serves as a template for a second amplification using the original set of primers. The resulting PCR product is cleaved with BamHI and XbaI and inserted into the BamHI and XbaI-cleaved template plasmid. This second ligation inserts the PCR product in inverted orientation with respect to first cloned fragment, yielding an inverted repeat separated by the ChsA intron.Variations on this Theme
If the PCR template for the first cloning step is a plasmid containing a known insert, rather than a cDNA population, no further sequencing of the product of the first ligation step should be required. In this case, one PCR amplification is sufficient to perform both set of digestions and the sequential subcloning steps to create the inverted repeat.
It is useful to note that the restriction enzyme digestions for the second subcloning step remove only the outer-most restriction enzyme sites and leave the inner AscI and SwaI sites intact. The inclusion of these sites in the second subcloning step, as well as the inversion of the PCR fragment, results in AscI sites flanking the entire inverted repeat/stuffer sequence and SwaI sites flanking the stuffer sequence. Thus, the entire inverted repeat and stuffer can be removed intact and subcloned into a different plasmid, although the orientation of the AscI would have to be determined, if the direction of the stuffer sequence is important. As SwaI sites flank the stuffer sequence, this fragment can be swapped for a different stuffer sequence. The orientation of these sites can be seen in Figure 2
The restriction enzymes AscI and SwaI recognize eight base pair sites that occur infrequently, whereas BamHI and XbaI, which have six base pair recognition sites, are more likely to occur in target genes. In general, it is possible to identity an appropriate span of DNA lacking BamHI and XbaI sites in the target transcript. If BamHI and/or XbaI sites cannot be avoided in the target sequence, other enzyme cleavage sites have been included between the BamHI and XbaI sites for pFGC5941 and between BamHI and SpeI for other vectors (see individual plasmid maps). Another approach is to use enzymes with compatible cohesive ends (e.g. BglII ligated to BamHI) or to use blunt end ligation and screen for the correct orientation of the fragment.Maize Vector pMCG161
The maize binary vector, pMCG161, can be accessed through Table 1. pMCG161 was built using pCAMBIA 1200. The same two-step cloning strategy outline above applies, but the restriction enzymes incorporated into the PCR fragments are different. AvrII replaces SwaI as one of the “inner” restriction enzymes, and SpeI and SgfI are used as the “outer” restriction enzyme sites.Other ChromDB RNAi Vectors
The original RNAi plasmid, pFGC1008 is derived from pCAMBIA1200 and has a chloramphenicol resistance gene for bacterial selection and a hygromycin resistance gene as the plant selectable marker. In the course of our research, and by way of inquiries from pFGC1008 users, it has been observed that use of the antibiotic chloramphenicol can be problematic for selecting antibiotic resistant bacteria, both Escherichia coli as well as different strains of Agrobacterium tumefaciens. We have found that different commercial sources of chloramphenicol vary in potency, and it is necessary to titrate the antibiotic, not only for each commercial source of the antibiotic, but also for each bacterial strain. Additionally, the plant selectable marker hygromycin slows the growth of resistant Arabidopsis plants, when compared to untransformed, wild-type plants. Due to these considerations, plasmid pFGC5941 was constructed, as described above. pFGC1008 does not possess a restriction enzyme cleavage site between the MAS1’promoter (promoter for hygromycin resistance gene) and the CaMV35S:dsRNA cassette, whereas pFGC5941 contains a unique EcoRI site between the MAS2’ promoter and CaMV35S. This restriction site in combination with other unique sites allows for replacement of the CaMV35S promoter with other promoters in pFGC5941. While the same one-primer set/two-step cloning strategy is applicable, the GUS stuffer in pFGC1008 lacks the SpeI site in petunia ChsA intron contained in pFGC5941 and so SpeI can be used for the second ligation step. The nucleotide sequence of pFGC1008, as well as its restriction map, can be found in Table 1.
A set of six additional RNAi binary vectors was constructed by modifying pCAMBIA1200. Table 1 presents these plasmids, as well as pFGC1008 and pGC5941. This series of eight vectors allows for the following choices: bacterial antibiotic resistance genes (chloramphenicol or kanamycin), plant resistance genes (basta, hygromycin, kanamycin), different versions of the CaMV35S promoter (200, 200x3, 500, 1400 bp) for dsRNA transcript production, and different stuffer fragment to separate the inverted repeats. The 200x3 promoter is a series of three tandem duplications of the 200 bp fragment and is referred to as 3x CaMV35S.
The remaining four binary vectors listed in Table 1 contain a unique AscI restriction enzyme site and are used to transfer the inverted repeats flanked by AscI restriction enzyme sites. Each plasmid contains the 3X CaMV35S promoter, chloramphenicol as the bacterial selection, but each plasmid is different with respect to the plant selectable marker and a polyadenylation signal sequence. Plasmids pGSA1427, pGSA1561, and pGSA1783 have different polyadenylation signal sequences (ocs3', nos3', ssu3').