RNA-mediated gene silencing assay
Down-regulation of endogenous gene expression by post-transcriptional horizontal gene silencing (PTGS) is a key to characterize plant gene function. Many RNA level-based silencing mechanisms, such as post-transcriptional gene silencing, co-repression, gene repression and RNA interference (RNAi), have been identified in different species of plants, fungi and animals. Among them, RNAi is one of the most interesting discoveries.
Operation method
RNA-mediated gene silencing
Materials and Instruments
pGEM-T Vector DNA Template Move I. Parameters for screening target gene fragments For more product details, please visit Aladdin Scientific website.
dNTP mixture DNA polymerase (Sigma) and matching buffer Restriction enzymes
PCR purification kits or columns
1. Sequence
( 1 ) The construction of a designated RNA silencing vector starts with bioinformatics analysis. Primers are designed according to the known cDNA sequence corresponding to the target gene or the predicted gene sequence, and a portion of the sequence on the cDNA is amplified by reverse transcription-PCR ( RT-PCR). If the genomic sequence of the target gene is not available, the sequence of the gene in other species can be used to search for cDNAs with known sequences. if the target gene is a member of a multigene family, multiple comparisons with family members are needed to guide the design of PCR primers. The region of the gene to be amplified and its similarity to other genes determines whether the RNAi vector targets a single mRNA or the transcription products of multiple related genes.
( 2 ) Amplification of both coding and non-coding regions (UTR) results in good silencing. Since the silencing mechanism relies on sequence homology, it is possible that related mRNA sequences may be cross-silenced. If there is no special requirement, the sequence with lower homology with other sequences, such as 5' or 3' UTR, should be selected. to minimize the cross-silencing, avoid selecting the region with more than 20 bases identity with sequences other than the target gene to construct vectors.
( 3 ) Standard software is available to assist in the detection of sequence homology to accurately and systematically assess and minimize the occurrence of RNAi off-targeting between the siRNA sequence and the target gene [ 45 ] (see Note 6).
( 4 ) In addition to the coding region of a gene, truncated promoter-expressed dsRNAs can also induce gene repression. This approach induces transcriptional level gene silencing (TGS) [46, 47].
2. size
Gene fragments ranging from 50 to 1000 bp have been successfully used as targets for gene silencing. Two factors need to be taken into account when choosing the correct fragment length: the shorter the fragment, the lower the frequency of silencing; the longer the hairpin, the higher the probability of recombination in the bacterial host. In order to optimize the silencing efficiency, we recommend to use 300~600 bp fragment length.
RNAi
Generation of hairpin RNA carriers (hpRNA)
There are several ways to construct hpRNA. They can be constructed using standard plant transformation vectors, which require that the corresponding hairpin coding structure for each target gene be reconstructed into the vector. Alternatively, generic gene silencing vectors can be used, such as the pStarling and pStargate series of vectors for grain transformation developed by CSIRO ( Australia) (http ://www . pi. csiro. au/RNAi/vectors.htm ). The PCR derivatives of the target genes can be cloned into these vectors either by conventional cloning (pStarling) or by using the Gateway Targeted Recombination System (pStargate).
1. Cloning of cDNA fragments homologous to the target mRNAs
( 1 ) The primers used in the RT-PCR assay require the addition of BamH I and Bgl II restriction sites to amplify a fragment of 300-600 bp that is identical to the target gene sequence. This operation ensures that the gene fragment can be directionally cloned into the unique BamH l site of the PAHC7 vector [17].
( 2 ) The cloning strategy for the intron fragment is the same as that described above for the target gene.
( 3 ) PCR amplification was performed by denaturation at 94°C for 45 s, annealing at 62°C for 45 s, and extension at 72°C for 90 min, with a total of 35 cycles of amplification.
( 4 ) The amplified product of the cDNA fragment was digested with enzymes such as BamH I and Bgl II and cloned into the PAHC17 plasmid and the BamH I-specific restriction site (see Note 7 and Fig. 12.3).
( 5 ) After assembly of the reverse repeat sequence, it can be cloned into a suitable binary vector for Agrobacterium-mediated plant transformation.
2. pSTARLING vector
( 1 ) Silencing multiple target genes one by one would be a laborious task. The pSTAR-LING system was found to be very efficient in transformation and is suitable for silencing a small number of genes at the same time. 
( 2 ) This vector uses a maize ubiquitin promoter to drive the high level of constitutive expression of the hairpin RNAi product in monocotyledonous plants.
( 3 ) PCR fragments can be inserted into the vector using conventional restriction enzyme digestion and DNA ligation techniques, with the reverse fragment inserted into the BamH I Pad I Asc I polyclonal site and the forward fragment inserted into the SpeI SnaBI KpnI polyclonal site.
3. pSTARGATE vector
( 1 ) This vector can be used as an alternative to another high-throughput vector, pSTARLING, using the commercially available Gateway cloning system (http : // www . Invitrogen . com ) , which can silence a large number of genes (e.g., members of a gene family or a pathway).
( 2 ) The vector also enables directional cloning. The system contains a negative selection marker (ccdB) that screens out vectors that do not respond to recombination and efficiently obtains recombinant plasmids.
( 3 ) The pSTARGATE vector contains two recombination cassettes containing either attP1-ccdB-attP2 or attR1-ccdB-attR2 in reverse repeat mirrors , and thus can recombine with the vector to produce ihpRNA-encoded structures when the gene fragment flanks the appropriate att site.
4. Characterization of the constructed plasmid vector
( 1 ) Subclone all the amplified cDNA fragments into the pGEM-T vector.
( 2 ) Transform E.coli with the ligated product and screen for ampicillin-resistant clones. Extract the plasmid DNA with a small amount of bacterial culture medium, and identify the recombinant plasmid by restriction enzyme digestion profiles.
( 3 ) To make sure that the cloned DNA fragments are correct, each final vector needs to be DNA sequenced. This is because there is a potential problem in large projects where a large number of cDNA fragments can be cloned in parallel and mixed.
( 4 ) Each construct is finalized by sequencing the complete reverse sequence fragments and comparing the resulting sequence with the target gene sequence.
5. Detection of silent phenotype
( 1 ) Different hpRNA transgenic strains produce a range of defective phenotypes with varying degrees of interference [22 , 48 ].
( 2 ) Because of the widely varying (weak, moderate, strong) effects of RNAi interference, changes in the levels of target mRNAs can range from consistent with wild type to completely undetectable (see Note 8).
III. VIGS
1. VIGS inoculation technique
( 1 ) Each transcription product (wild-type or genetically modified) in the BSMV genome is mixed in a 1 : 1 : 1 ratio with inoculation buffer FES [49].
( 2 ) The mixture was frictionally inoculated into 7-day-old plants. After wearing gloves, the mixture was aspirated and placed between the thumb and forefinger which were held together.
( 3 ) Holding the base of the plant with the other hand, gently squeeze the first and second leaves with the gloved index finger and thumb.
( 4 ) After gently sliding the joined fingers from base to tip twice, the entire surface of the leaf is coated with the mixture.
2. Silencing of endogenous barley or wheat/genes with BSMV
( 1 ) Inoculate greenhouse-grown barley and wheat seedlings with a 1:1:1 mixture of in vitro transcription products from wild-type BSMV plasmids, βRNA plasmids, plasmids that do not carry plant sequences (BSMV: 00), or plasmids that contain fragments of γRNA derivatives.
( 2 ) The first and second leaves of seedlings grown for 7 days were friction inoculated with BSMV-PDS, and after 7 days, the third and fourth leaves of barley showed the first obvious symptoms of bleaching. In wheat, bleaching also occurred, but was not visible until after the 10th day of virus inoculation (see Notes 9 and 10).
3. Identification of genes required in the R-gene-mediated disease resistance pathway by the BSMV-VIGS method
VIGS is initiated after BSMV infestation, whereas the plant resistance system is initiated after pathogen infestation. Usually there is a necessary time interval between the two and the interval is usually 8 days.
Detection of mRNA down-regulation of target genes
1. real-time quantitative PCR
( 1 ) In order to determine whether the RNA-induced exogenous gene affects the mRNA level of the target gene, two pairs of primers are used for quantitative RT-PCR. One pair of primers is designed specifically for the mRNA of the target gene, and is used to detect the level of effective endogenous mRNA rather than the level of exogenous gene transcripts.
( 2 ) The second primer pair amplifies an endogenous gene that is not associated with target RNA silencing, such as the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH, AF251217), which is used as an endogenous reference.
( 3 ) Three replicates (three cDNAs) were performed for each RNA sample to verify the reproducibility of the assay.
( 4 ) Real-time PCR was performed using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems) and SYBR GreenPCR Assay Mix (Applied Biosystems). Add the cDNA template and appropriate primers to a final volume of 26 μl. Amplification conditions: 50°C, 2 min; 95°C, 10 min; 95°C, 15 s, 60°C, 1 min, 40 cycles.
2. Small interfering RNA detection
( 1 ) The housekeeping gene GAPDH (AF251217, glyceraldehyde-3-phosphate dehydrogenase) was used as a control for hybridization.
( 2 ) The relative intensity of hybridization signals in transgenic and wild-type plants can be detected using a phosphoimager (Cyclone gene array system, Perkin- Elmer, Boston).