Efficient virus-induced gene silencing in apple, pear and Japanese pear using Apple latent spherical virus vectors

Background

Virus-induced gene silencing (VIGS) is an effective technology for the analysis of gene functions in plants. Though there are many reports on virus vectors for VIGS in plants, no VIGS vectors available for Rosaceae fruit trees were reported so far. We present an effective VIGS system in apple, pear, and Japanese pear using Apple latent spherical virus (ALSV) vectors.

Results

Inoculation of ALSV vectors carrying a partial sequence of endogenous genes from apple [ribulose-1, 5-bisphosphate carboxylase small subunit (rbcS), alpha subunit of chloroplast chaperonin (CPN60a), elongation factor 1 alpha (EF-1a), or actin] to the cotyledons of seeds by a particle bombardment induced highly uniform knock-down phenotypes of each gene on the true leaves of seedlings from 2~3 weeks after inoculation. These silencing phenotypes continued for several months. Northern blot and RT-PCR analyses of leaves infected with ALSV containing a fragment of rbcS gene showed that the levels of rbcS-mRNA drastically decreased in the infected apple and pear leaves, and, in reverse, rbcS-siRNAs were generated in the infected leaves. In addition, some of apple seedlings inoculated with ALSV vector carrying a partial sequence of a TERMINAL FLOWER 1 gene of apple (MdTFL1) showed precocious flowering which is expected as a knock-down phenotype of the silencing of MdTFL1 gene.

Conclusions

The ALSV-based VIGS system developed have provides a valuable new addition to the tool box for functional genomics in apple, pear, and Japanese pear.     

siRNA targeting fas protects donor liver against cold preservation and ischemic reperfusion injury in rat liver transplantation

AIM:To investigate the silencing effect of fas siRNA to alleviate ischemic-reperfusion (I/R) injury in liver transplantation. METHODS:Three pairs of 21-nt synthesized fas siRNAs were transfected into BRL cells respectively for evaluation of silence efficacy, and the most effective fas siRNA was chosen in vivo for experiment. In cold preservation experiment, siRNA was transfected in vivo by hydrodynamics method. After 48 h, livers of fas siRNA group and control group were harvested and cold preserved, and cell apoptosis and fas expression was evaluated at 2, 4 and 6 h. Orthotopic liver transplantation was performed in fas siRNA group and blank control group. At 1, 3, 6, 12 and 24 h after transplantation, blood and liver samples were collected for evaluation of serum ALT levels, Fas protein and mRNA expression, and apoptosis by TUNEL staining. RESULTS:fas siRNA2, which began at nt 315, inhibited fas gene expression much more than other siRNAs. As to cold preservation, apoptosis index (AI) and fas expression in fas siRNA group was lower than that in control group at each checked point (P<0.01). At 1, 3, 6, 12, and 24 h after blood reperfusion of liver transplantation, the serum ALT level in fas siRNA group was much less than that in control group. The cell apoptosis in fas siRNA group was substantially decreased, and the expressions of fas mRNA and protein were dramatically reduced. CONCLUSION:fas-mediated apoptosis plays an important role in I/R injury of rat liver transplantation. Silencing fas by siRNA holds therapeutic promise to limit I/R injury.     

Effect of RICTOR expression on cell viability in rheumatoid arthritis fibroblast-like synoviocytes

AIM：To investigate the effects of RICTOR expression on the viability of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). METHODS：RA-FLS were obtained by tissue culture. Chemically synthesized double-stranded siRNAs targeting RICTOR gene were transfected into RA-FLS by cationic liposome. The nonspecific siRNA was also transfected into the negative control cells. The mRNA expression of RICTOR was detected by RT-qPCR after transfection for 24 h. Western blotting was used to evaluate the inhibitory effects of siRNAs on RICTOR expression after transfection for 48 h and 72 h. The cell viability was examined by MTT assay. RESULTS：Compared with control group, the mRNA expression of RICTOR significantly decreased by 78.36%±3.71% after the transfection of RICTOR siRNA for 24 h. The protein level of RICTOR was also obviously lower in RICTOR siRNA transfection group than that in control group after transfection for 48 h (decreased by 92.48%±6.14%) and 72 h (decreased by 98.57%±1.40%). Knock-down of RICTOR in RA-FLS for 72 h markedly decreased the cell viability. CONCLUSION：Transfection of RICTOR siRNA reduces the viability of RA-FLS, indicating that mTORC2 may be required for the survival of RA-FLS.     

Virus-induced gene silencing as a tool for functional analyses in the emerging model plant Aquilegia (columbine, Ranunculaceae)

Background  

There is considerable interest in rapid assays or screening systems for assigning gene function. However, analysis of gene function in the flowers of some species is restricted due to the difficulty of producing stably transformed transgenic plants. As a result, experimental approaches based on transient gene expression assays are frequently used. Biolistics has long been used for transient over-expression of genes of interest, but has not been exploited for gene silencing studies. Agrobacterium-infiltration has also been used, but the focus primarily has been on the transient transformation of leaf tissue.     

Whole genome sequencing of enriched chloroplast DNA using the Illumina GAII platform

Background  

Complete chloroplast genome sequences provide a valuable source of molecular markers for studies in molecular ecology and evolution of plants. To obtain complete genome sequences, recent studies have made use of the polymerase chain reaction to amplify overlapping fragments from conserved gene loci. However, this approach is time consuming and can be more difficult to implement where gene organisation differs among plants. An alternative approach is to first isolate chloroplasts and then use the capacity of high-throughput sequencing to obtain complete genome sequences. We report our findings from studies of the latter approach, which used a simple chloroplast isolation procedure, multiply-primed rolling circle amplification of chloroplast DNA, Illumina Genome Analyzer II sequencing, and de novo assembly of paired-end sequence reads.     

High throughput generation of promoter reporter (GFP) transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns

Background  

Although the complete genome sequence and annotation of Arabidopsis were released at the end of year 2000, it is still a great challenge to understand the function of each gene in the Arabidopsis genome. One way to understand the function of genes on a genome-wide scale is expression profiling by microarrays. However, the expression level of many genes in Arabidopsis genome cannot be detected by microarray experiments. In addition, there are many more novel genes that have been discovered by experiments or predicted by new gene prediction programs. Another way to understand the function of individual genes is to investigate their in vivo expression patterns by reporter constructs in transgenic plants which can provide basic information on the patterns of gene expression.     

In planta transient expression as a system for genetic and biochemical analyses of chlorophyll biosynthesis

Background  

Mg chelatase is a multi-subunit enzyme that catalyses the first committed step of chlorophyll biosynthesis. Studies in higher plants and algae indicate that the Mg chelatase reaction product, Mg-protoporphyrin IX plays an essential role in nuclear-plastid interactions. A number of Mg chelatase mutants have been isolated from higher plants, including semi-dominant alleles of ChlI, the gene encoding the I subunit of the enzyme. To investigate the function of higher plant CHLI, bacterial orthologues have been engineered to carry analogous amino acid substitutions to the higher plant mutations and the phenotypes examined through in vitro characterization of heterologously produced proteins. Here, we demonstrate the utility of a transient expression system in Nicotiana benthamiana for rapidly assaying mutant variants of the maize CHLI protein in vivo.     

High frequency, cell type-specific visualization of fluorescent-tagged genomic sites in interphase and mitotic cells of living Arabidopsis plants

Background  

Interphase chromosome organization and dynamics can be studied in living cells using fluorescent tagging techniques that exploit bacterial operator/repressor systems and auto-fluorescent proteins. A nuclear-localized Repressor Protein-Fluorescent Protein (RP-FP) fusion protein binds to operator repeats integrated as transgene arrays at defined locations in the genome. Under a fluorescence microscope, the tagged sites appear as bright fluorescent dots in living cells. This technique has been used successfully in plants, but is often hampered by low expression of genes encoding RP-FP fusion proteins, perhaps owing to one or more gene silencing mechanisms that are prevalent in plant cells.     

Protocol: A high-throughput DNA extraction system suitable for conifers

Background  

High throughput DNA isolation from plants is a major bottleneck for most studies requiring large sample sizes. A variety of protocols have been developed for DNA isolation from plants. However, many species, including conifers, have high contents of secondary metabolites that interfere with the extraction process or the subsequent analysis steps. Here, we describe a procedure for high-throughput DNA isolation from conifers.     

Computer aided data acquisition tool for high-throughput phenotyping of plant populations

Background  

The data generated during a course of a biological experiment/study can be sometimes be massive and its management becomes quite critical for the success of the investigation undertaken. The accumulation and analysis of such large datasets often becomes tedious for biologists and lab technicians. Most of the current phenotype data acquisition management systems do not cater to the specialized needs of large-scale data analysis. The successful application of genomic tools/strategies to introduce desired traits in plants requires extensive and precise phenotyping of plant populations or gene bank material, thus necessitating an efficient data acquisition system.     

Construction of human HSV-1 UL40 gene recombinant and its application in siRNA screening

AIM: To construct the recombinant of human herpesvirus Ⅰ(HSV-1) UL40 gene and to screen siRNAs of silencing efficiently human HSV-1 UL40 gene expression.METHODS: The recombinant UL40-EGFP plasmid (pEGFP-N1-UL40) was constructed by cloning the UL40 gene into pEGFP-N1.siRNA target UL40 gene and pEGFP-N1-UL40 were cotransfected into Vero cells.The effects of RNAi were detected by green fluorescence signals.RESULTS: siRNA3,siRNA4 reduced prominently the expression of UL40 gene.The silence efficiency was 76.99% and 84.00% respectively.CONCLUSION: We succeed in construction of the pEGFP-N1-UL40 recombinant,and screen out siRNA3 and siRNA4 of silencing efficiently human HSV-1 UL40 gene expression.     

ModuleFinder and CoReg: alternative tools for linking gene expression modules with promoter sequences motifs to uncover gene regulation mechanisms in plants

Background  

Uncovering the key sequence elements in gene promoters that regulate the expression of plant genomes is a huge task that will require a series of complementary methods for prediction, substantial innovations in experimental validation and a much greater understanding of the role of combinatorial control in the regulation of plant gene expression.     

PlantDB – a versatile database for managing plant research

Background  

Research in plant science laboratories often involves usage of many different species, cultivars, ecotypes, mutants, alleles or transgenic lines. This creates a great challenge to keep track of the identity of experimental plants and stored samples or seeds.     

A simple way to identify non-viable cells within living plant tissue using confocal microscopy

Background  

Plant cell death is a normal process during plant development. Mutant plants may exhibit misregulation of this process, which can lead to severe growth defects. Simple ways of visualising cell death in living plant tissues can aid the study of plant development and physiology.     

A simple and efficient CRISPR/Cas9 platform for induction of single and multiple,heritable mutations in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Background

Genome editing of monocot plants can be accomplished by using the components of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9) technology specifically optimized for these types of plants. Here, we present the development of RNA-guided Cas9 system for simplex and multiplex genome editing in barley.

Results

We developed a set of customizable RNA-guided Cas9 binary vectors and sgRNA modules for simplex and multiplex editing in barley. To facilitate the design of RNA-guided Cas9 constructs, the pBract derived binary vectors were adapted to Gateway cloning and only one restriction enzyme was required for construction of the sgRNA. We designed a synthetic, codon optimized Cas9 gene containing the N terminal SV40 nuclear localization signal and the UBQ10 Arabidopsis 1st intron. Two different sgRNAs were constructed for simplex editing and one polycistronic tRNA-gRNA construct (PTG) for multiplex editing using an endogenous tRNA processing system. The RNA-guided Cas9 constructs were validated in transgenic barley plants produced by Agrobacterium-mediated transformation. The highest mutation rate was observed in simplex editing of the cytokinin oxidase/dehydrogenase HvCKX1 gene, where mutations at the hvckx1 locus were detected in 88% of the screened T₀ plants. We also proved the efficacy of the PTG construct in the multiplex editing of two CKX genes by obtaining 9 plants (21% of all edited plants) with mutations induced in both HvCKX1 and HvCKX3. Analysis of the T₁ lines revealed that mutations in the HvCKX1 gene were transmitted to the next generation of plants. Among 220 screened T₁ plants we identified 85 heterozygous and 28 homozygous mutants, most of them bearing frameshift mutations in the HvCKX1 gene. We also observed independent segregation of mutations and the Cas9-sgRNA T-DNA insert in several T₁ plants. Moreover, the knockout mutations of the Nud gene generated phenotype mutants with naked grains, and the phenotypic changes were identifiable in T₀ plants.

Conclusions

We demonstrated the effectiveness of an optimized RNA-guided Cas9 system that can be used for generating homozygous knockout mutants in the progeny of transgenic barely plants. This is also the first report of successful multiplex editing in barley using a tRNA processing system.

     

Transient expression vectors for functional genomics,quantification of promoter activity and RNA silencing in plants

Background  

We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes.