Bone marrow mesenchymal stem cells with Nogo-66 receptor gene silencing for repair of spinal cord injury

We hypothesized that RNA interference to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells before transplantation might further improve neurological function in rats with spinal cord transection injury. After 2 weeks, the number of neurons and BrdU-positive cells in the Nogo-66 receptor gene silencing group was higher than in the bone marrow mesenchymal stem cell group, and significantly greater compared with the model group. After 4 weeks, behavioral performance was signiifcantly enhanced in the model group. Af-ter 8 weeks, the number of horseradish peroxidase-labeled nerve ifbers was higher in the Nogo-66 receptor gene silencing group than in the bone marrow mesenchymal stem cell group, and signiifcantly higher than in the model group. The newly formed nerve ifbers and myelinated ner ve ifbers were detectable in the central transverse plane section in the bone marrow mesenchymal stem cell group and in the Nogo-66 receptor gene silencing group.

Establishment and Verification of An Efficient Virus-induced Gene Silencing System in Forsythia

To understand the functional identification of large-scale genomic sequences in Forsythia,tobacco rattle virus(TRV)-mediated virus-induced gene silencing(VIGS),suitable for the plant,was explored in this study.The results showed that the TRV-mediated VIGS system could be successfully used in Forsythia for silencing the reporter gene FsPDS(Forsythia phytoene desaturase)using stem infiltration and leaf infiltrationmethods.All the treated plants were pruned below the injection site after 7–15 d infection;the FsPDS was silenced and typical photobleaching symptoms were observed in newly sprouted leaves at the whole-plant level.Meanwhile,this system has been successfully tested and verified through virus detection and qRT-PCR analysis.After the optimization,Forsythia magnesium chelatase subunit H(FsChlH)was silenced successfully in Forsythia using this system,resulting in yellow leaveswith decreased chlorophyll content.The system was stable,highly efficient and had greater rapidity and convenience,which made it suitable to study the function of genes related to physiological pathways such as growth and development,and metabolic regulation in Forsythia.

Targeted Silencing of Heparanase Gene by Small Interfering RNA Inhibits Invasiveness and Metastasis of Osteosarcoma Cells

The effects of targeted silencing of heparanase gene by small interfering RNA（siRNA） on invasiveness and metastasis of osteosarcoma cells（MG63 cells） were investigated in the present study.Two complementary oligonucleotide strands were synthesized and inserted into pGenesil-1 vector based on the mRNA sequence of heparanase gene.The expression vector containing short hairpin RNA（pGenesil-shRNA） was constructed successfully.MG63 cells were randomly allocated into 3 groups：blank group,empty vector（pGenesil） transfected group and expression vector（pGenesil-shRNA） transfected group.Under the induction of Lipofectamine 2000,the recombinants were transfected into MG63 cells.Heparanase gene expression level was detected by RT-PCR and Western blotting.Cell prolifera-tion was measured by MTT assay.Cell invasiveness and metastasis were examined by cell adhesion and Transwell-ECM assays.HUVECs migration assay was applied for the detection of angiogenesis.As compared with negative controls,the mRNA and protein expression levels of heparanase were down-regulated by 76.1%（P0.01） and 75.3%（P0.01） respectively in the pGenesil-shRNA transfected group.Meanwhile,the proliferation,adhesiveness,invasiveness and angiogenesis properties of MG63 cells were all significantly inhibited.It was suggested that targeted silencing of heparanase gene by siRNA could dramatically inhibit the invasiveness and metastasis of osteosarcoma cells.

Silencing hepatitis B virus covalently closed circular DNA: The potential of an epigenetic therapy approach

Global prophylactic vaccination programmes have helped to curb new hepatitis B virus(HBV)infections.However,it is estimated that nearly 300 million people are chronically infected and have a high risk of developing hepatocellular carcinoma.As such,HBV remains a serious health priority and the development of novel curative therapeutics is urgently needed.Chronic HBV infection has been attributed to the persistence of the covalently closed circular DNA(cccDNA)which establishes itself as a minichromosome in the nucleus of hepatocytes.As the viral transcription intermediate,the cccDNA is responsible for producing new virions and perpetuating infection.HBV is dependent on various host factors for cccDNA formation and the minichromosome is amenable to epigenetic modifications.Two HBV proteins,X(HBx)and core(HBc)promote viral replication by modulating the cccDNA epigenome and regulating host cell responses.This includes viral and host gene expression,chromatin remodeling,DNA methylation,the antiviral immune response,apoptosis,and ubiquitination.Elimination of the cccDNA minichromosome would result in a sterilizing cure;however,this may be difficult to achieve.Epigenetic therapies could permanently silence the cccDNA minichromosome and promote a functional cure.This review explores the cccDNA epigenome,how host and viral factors influence transcription,and the recent epigenetic therapies and epigenome engineering approaches that have been described.

Aquaporin-4 gene silencing protects injured neurons after early cerebral infarction

Aquaporin-4 regulates water molecule channels and is important in tissue regulation and water transportation in the brain. Upregulation of aquaporin-4 expression is closely related to cellular edema after early cerebral infarction. Cellular edema and aquaporin-4 expression can be determined by measuring cerebral infarct area and apparent diffusion coefficient using diffusion-weighted imaging（DWI）. We examined the effects of silencing aquaporin-4 on cerebral infarction. Rat models of cerebral infarction were established by occlusion of the right middle cerebral artery and si RNA-aquaporin-4 was immediately injected via the right basal ganglia. In control animals, the area of high signal intensity and relative apparent diffusion coefficient value on T2-weighted imaging（T2WI） and DWI gradually increased within 0.5–6 hours after cerebral infarction. After aquaporin-4 gene silencing, the area of high signal intensity on T2 WI and DWI reduced, relative apparent diffusion coefficient value was increased, and cellular edema was obviously alleviated. At 6 hours after cerebral infarction, the apparent diffusion coefficient value was similar between treatment and model groups, but angioedema was still obvious in the treatment group. These results indicate that aquaporin-4 gene silencing can effectively relieve cellular edema after early cerebral infarction; and when conducted accurately and on time, the diffusion coefficient value and the area of high signal intensity on T2 WI and DWI can reflect therapeutic effects of aquaporin-4 gene silencing on cellular edema.

Piwi like RNA-mediated gene silencing 1 gene as a possible major player in gastric cancer

AIM To establish a permanent piwi like RNA-mediated genesilencing 1(PIWIL1) gene knockout in AGP01 gastric cancer cell line using CRISPR-Cas9 system and analyze phenotypic modifications as well as gene expression alterations.METHODS CRISPR-Cas9 system used was purchased from Dharmacon GE Life Sciences(Lafayette, CO, United States) and permanent knockout was performed according to manufacturer's recommendations. Woundhealing assay was performed to investigate the effect of PIWIL1 knockout on migration capability of cells and Boyden chamber invasion assay was performed to investigate the effect on invasion capability. For the gene expression analysis, a one-color microarray-based gene expression analysis kit(Agilent Technologies, Santa Clara, CA, United States) was used according to the protocol provided by the manufacturer. RESULTS PIWIL1 gene knockout caused a significant decrease in AGP01 migration capacity as well as a significant decrease in cell invasiveness. Moreover, functional analysis based on grouping of all differentially expressed m RNAs identified a total of 35 genes(5 up-regulated and 30 down-regulated) encoding proteins involved in cellular invasion and migration. According to current literature, 9 of these 35 genes(DOCK2, ZNF503, PDE4 D, ABL1, ABL2, LPAR1, SMAD2, WASF3 and DACH1) are possibly related to the mechanisms used by PIWIL1 to promote carcinogenic effects related to migration and invasion, since their functions are consistent with the changes observed(being up-or down-regulated after knockout). CONCLUSION Taken together, these data reinforce the idea that PIWIL1 plays a crucial role in the signaling pathway of gastric cancer, regulating several genes involved in migration and invasion processes; therefore, its use as a therapeutic target may generate promising results in the treatment of gastric cancer.

Insect resistance management in Bacillus thuringiensis cotton by MGPS(multiple genes pyramiding and silencing)

The introduction of Bacillus thuringiensis(Bt)cotton has reduced the burden of pests without harming the environment and human health.However,the efficacy of Bt cotton has decreased due to field-evolved resistance in insect pests over time.In this review,we have discussed various factors that facilitate the evolution of resistance in cotton pests.Currently,different strategies like pyramided cotton expressing two or more distinct Bt toxin genes,refuge strategy,releasing of sterile insects,and gene silencing by RNAi are being used to control insect pests.Pyramided cotton has shown resistance against different cotton pests.The multiple genes pyramiding and silencing(MGPS)approach has been proposed for the management of cotton pests.The genome information of cotton pests is necessary for the development of MGPS-based cotton.The expression cassettes against various essential genes involved in defense,detoxification,digestion,and development of cotton pests will successfully obtain favorable agronomic characters for crop protection and production.The MGPS involves the construction of transformable artificial chromosomes,that can express multiple distinct Bt toxins and RNAi to knockdown various essential target genes to control pests.The evolution of resistance in cotton pests will be delayed or blocked by the synergistic action of high dose of Bt toxins and RNAi as well as compliance of refuge requirement.

Nanotechnology-based gene therapy as a credible tool in the treatment of Alzheimer’s disease

Toxic aggregated amyloid-βaccumulation is a key pathogenic event in Alzheimer’s disease.Treatment approaches have focused on the suppression,deferral,or dispersion of amyloid-βfibers and plaques.Gene therapy has evolved as a potential therapeutic option for treating Alzheimer’s disease,owing to its rapid advancement over the recent decade.Small interfering ribonucleic acid has recently garnered considerable attention in gene therapy owing to its ability to down-regulate genes with high sequence specificity and an almost limitless number of therapeutic targets,including those that were once considered undruggable.However,lackluster cellular uptake and the destabilization of small interfering ribonucleic acid in its biological environment restrict its therapeutic application,necessitating the development of a vector that can safeguard the genetic material from early destruction within the bloodstream while effectively delivering therapeutic genes across the bloodbrain barrier.Nanotechnology has emerged as a possible solution,and several delivery systems utilizing nanoparticles have been shown to bypass key challenges regarding small interfering ribonucleic acid delivery.By reducing the enzymatic breakdown of genetic components,nanomaterials as gene carriers have considerably enhanced the efficiency of gene therapy.Liposomes,polymeric nanoparticles,magnetic nanoparticles,dendrimers,and micelles are examples of nanocarriers that have been designed,and each has its own set of features.Furthermore,recent advances in the specific delivery of neurotrophic compounds via gene therapy have provided promising results in relation to augmenting cognitive abilities.In this paper,we highlight the use of different nanocarriers in targeted gene delivery and small interfering ribonucleic acid-mediated gene silencing as a potential platform for treating Alzheimer’s disease.

Cloning and Function Identification of a Phytoene Desaturase Gene from Eucommia ulmoides

The phytoene desaturase(PDS)encodes a crucial enzyme in the carotenoid biosynthesis pathway.Silencing or inhibiting PDS expression leads to the appearance of mottled,chlorosis,or albino leaves.In this study,the CDS sequence of EuPDS(Eucommia ulmoides Phytoene Desaturase)was first cloned and then PDS was silenced in Nicotiana benthamiana.Result showed the expression level of EuPDS in leaves was higher than that in the roots and stems.In N.benthamiana leaves,which were treated by Agrobacterium for 24 h,photo-bleaching was shown on the fresh leaves one week after injection and the transcript level of PDS was down-regulated during the period of emersion.This suggested that EuPDS could silence PDS of N.benthamiana,so as to cause the phenotype of leaf whitening.PDS is the main reporter gene involved in virus-induced gene silencing(VIGS).This study offered molecular evidence for identifying PDS gene involved in Carotenoid’s biosynthesis pathway and the regulation networks in E.ulmides.It also laid a useful foundation for study on leaf discoloration mechanism of other woody plants.

Gene interference regulates aquaporin-4 expression in swollen tissue of rats with cerebral ischemic edema Correlation with variation in apparent diffusion coefficient

To investigate the effects of mRNA interference on aquaporin-4 expression in swollen tissue of rats with ischemic cerebral edema, and diagnose the significance of diffusion-weighted MRI, we injected 5 pL shRNA- aquaporin-4 （control group） or siRNA- aquaporin-4 solution （1：800） （RNA interference group） into the rat right basal ganglia immediately before occlusion of the middle cerebral artery. At 0.25 hours after occlusion of the middle cerebral artery, diffusion-weighted MRI displayed a high signal; within 2 hours, the relative apparent diffusion coefficient decreased markedly, aquaporin-4 expression increased rapidly, and intracellular edema was obviously aggravated; at 4 and 6 hours, the relative apparent diffusion coefficient slowly returned to control levels, aquaporin-4 expression slightly increased, and angioedema was observed. In the RNA interference group, during 0.25- 6 hours after injection of siRNA- aquaporin-4 solution, the relative apparent diffusion coefficient slightly fluctuated and aquaporin-4 expression was upregulated; during 0.5 4 hours, the relative apparent diffusion coefficient was significantly higher, while aquaporin-4 expression was significantly lower when compared with the control group, and intracellular edema was markedly reduced; at 0.25 and 6 hours, the relative apparent diffusion coefficient and aquaporin-4 expression were similar when compared with the control group; obvious angioedema remained at 6 hours. Pearson＇s correlation test results showed that aquaporin-4 expression was negatively correlated with the apparent diffusion coefficient （r = -0.806, P 〈 0.01）. These findings suggest that upregulated aquaporin-4 expression is likely to be the main molecular mechanism of intracellular edema and may be the molecular basis for decreased relative apparent diffusion coefficient. Aquaporin-4 gene interference can effectively inhibit the upregulation of aquaporin-4 expression during the stage of intracelfular edema with time-effectiveness. Moreover, diffusion-weighted MRI can accurately detect intracellular edema.

Transplantation of Nogo-66 receptor gene-silenced cells in a poly(D,L-lactic-co-glycolic acid) scaffold for the treatment of spinal cord injury

Inhibition of neurite growth, which is in large part mediated by the Nogo-66 receptor, affects neural regeneration following bone marrow mesenchymal stem cell transplantation. The tissue engineering scaffold poly（D,L-lactide-co-glycolic acid） has good histocompatibility and can promote the growth of regenerating nerve fibers. The present study used small interfering RNA to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells and Schwann cells, which were subsequently transplanted with poly（D,L-lactide-co-glycolic acid） into the spinal cord lesion regions in rats. Simultaneously, rats treated with scaffold only were taken as the control group. Hematoxylin-eosin staining and immunohistochemistry revealed that at 4 weeks after transplantation, rats had good motor function of the hind limb after treatment with Nogo-66 receptor gene-silenced ceils prus the poly（O,L-lactide-co-glycolic acid） scaffold compared with rats treated with scaffold only, and the number of bone marrow mesenchymal stem cells and neuron-like cells was also increased. At 8 weeks after transplantation, horseradish peroxidase tracing and transmission electron microscopy showed a large number of unmyelinated and myelinated nerve fibers, as well as intact regenerating axonal myelin sheath following spinal cord hemisection injury. These experimental findings indicate that transplantation of Nogo-66 receptor gene-silenced bone marrow mesenchymal stem cells and Schwann cells plus a poly（D,L-lactide-co-glycolic acid） scaffold can significantly enhance axonal regeneration of spinal cord neurons and improve motor function of the extremities in rats following spinal cord injury.

A MADS-box gene is involved in soybean resistance to multiple Soybean mosaic virus strains

Soybean mosaic virus(SMV)is a member of the genus Potyvirus that extensively impairs global soybean production.The full-length coding sequence of the MADS-box transcription factor Gm CAL was cloned from the SMV-resistant soybean cultivar Kefeng 1.SMV-induced expression analysis indicated that Gm CAL responded quickly to SMV-SC8 infection in Kefeng 1 but not in NN1138-2.Gm CAL was expressed at high levels in flowers and pods but at lower levels in leaves.The gene was localized to the nucleus by subcellular localization assay.Virus-induced gene silencing did not increase the accumulation of SMV in Gm CAL-silenced Kefeng 1 plants(with silencing efficiency~80%)after SC8 inoculation.Gm CAL-silencing plants still conferred resistance to SC8 that might be owing to incomplete silencing of genes with lower expression.SMV content decreased significantly in Gm CAL-overexpressing NN1138-2 plants after SMVSC3,SMV-SC7,and SMV-SC8 inoculation in comparison with a vector control,showing that overexpression of Gm CAL conferred broad-spectrum resistance to multiple SMV strains.These results confirm that Gm CAL,a key regulator but not a specific SC8 resistance gene(Rsc8),is a positive regulatory transcription factor involved in soybean resistance to SMV.

Construction of RNAi Expression Vector against Riboflavin Synthase Gene

[Objective] This study aimed to construct RNAi expression vector against r/boflavin synthase （RS） gene. [Method] By using the primers designed based on RS gone coding sequence that was screened from Arabidopsis cDNA library, the 476 bp cDNA fragment of RS was amplified from pGADTT-RS recombinant plasmid, and then cloned into pUCm-T vector to obtain pUCm-RS. Two RS fragments （476 bp） were obtained through digesting pUCm-RS with restriction enzymes PstI/BamHI and PstI / Xhol, and then respectively connected into vector pBSSK-in to form pBSSK-RS-in-RS, in which the two RS fragments were inverted re- peats. Finally, the transform unit RS-intron-RS, got by digesting vector pBSSK-RS-in-RS with Sac I and Kpn I, was ligated into expression vector pCAMBIA1301 to obtain the RS gene silencing vector. [ Result] The restriction enzyme digestion sequencing analysis proved that the RS gene silencing vector was successfully con- structed. [ Conclusion] This study may provide a basic material for further studies on the bio-function of RS gene and the mechanism of signal transduction induced by HpaGxoo in plant.

Isolation and Functional Characterization of a B3 Transcription Factor Gene <i>FUSCA3</i>Involved in Pre-Harvest Sprouting Resistance in Wheat

Pre-harvest sprouting (PHS) reduces yields and grain quality, resulting in seriously economic losses in wheat. It has been showed that PHS is significantly correlated to seed dormancy levels. FUSCA3 (FUS3) gene is considered to be the key regulator of seed dormancy. However, little information is available about the function of FUS3 gene (TaFUS3) in wheat. In this study, three homologous genes were identified in wheat grain, and their functions were investigated by gene silencing. Three full-length DNA (3477, 3534 and 3501 bp) and cDNA (1015, 1012 and 1015 bp) sequences encoding a B3 transcription factor, designated TaFUS3-3A, TaFUS3-3B and TaFUS3-3D, were first isolated from common wheat. The transcription of three TaFUS3 genes in seed development and germination process was detected. TaFUS3-3B and TaFUS3-3D had similar expression profiles, and high levels of gene transcripts were detected in seeds at 25 DAP (days after pollination) and after 24 h of imbibition. However, the transcription of TaFUS3-3A was not detected. Silencing of TaFUS3 in common wheat spikes resulted in increased seed germination and PHS. Compared with wild-type, the TaFUS3-silenced plants showed increased expression of genes related to GA biosynthesis and ABA metabolism, and decreased expression of genes associated with ABA biosynthesis. Moreover, silencing of TaFUS3 in wheat plants led to a decrease in embryo sensitivity to ABA and changed the expression of genes involved in ABA signal transduction. The results of gene silencing indicated that TaFUS3 plays a positive role in wheat seed dormancy and PHS-resistance, which might be associated with ABA, GA level and signal transduction.

A small knottin-like peptide negatively regulates in wheat to stripe rust resistance during early infection of wheat

Although Blufensins(Bln)have important functions in the response of plants to biotic stress the precise functioning of Bln in wheat remains largely unknown.Here we isolated a Bln gene(TaBln4)from Suwon 11 infected by Puccinia striiformis f.sp.tritici(Pst).Expression of TaBln4 increased in host plants at the early stage of infection with a virulent Pst race(CYR31)but was unchanged in response to infection by an avirulent race(CYR23).Transcription levels of TaBln4 were also regulated by hormone and abiotic stresses.Expression of TaBln4 in tobacco leaves suppressed Bax-induced programmed cell death.Knockdown of TaBln4 by virus-induced gene silencing inhibited colonization of race CYR31 by increasing the accumulation of H2O2 and formation of hypersensitive responses(HR).Transient overexpression of TaBln4 by a transient overexpression system(BSMV-VOX)increased the susceptibility of wheat to CYR31.Results from bimolecular fluorescence complementation and pull-down assays demonstrated that TaBLN4 interacted with calmodulin.Taken together,our results suggest that TaBln4 negatively regulates resistance in wheat to Pst in a reactive oxygen species(ROS)-and HR-dependent manner.

Effects of Transparent Testa8(TT8) gene and Homeobox12(HB12) gene silencing in alfalfa(Medicago sativa L.) on molecular structure spectral profile in relation to energy,degradation,and fermentation characteristics in ruminant systems

Alfalfa(Medicago sativa L.) is a legume forage that is widely cultivated owing to its high biomass yield and favorable nutrient values. However, alfalfa contains relatively high lignin, which limits its utilization.Downregulation of two transcriptional factors, Transparent Testa8(TT8) and Homeobox12(HB12), has been proposed to reduce lignin content in alfalfa. Therefore, silencing of TT8(TT8i) and HB12(HB12i) in alfalfa was achieved by RNAi technology. The objective of this project was to determine effect of gene modification through silencing of TT8 and HB12 genes in alfalfa plants on lignin and phenolic content,bioenergic value, nutrient supply from rumen degradable and undegradable fractions, and in vitro ammonia production in response to the silencing of TT8 and HB12 genes in alfalfa. All gene silenced alfalfa plants(5 TT8i and 11 HB12i) were grown under greenhouse conditions with wild type as a control.Samples were analyzed for bioactive compounds, degradation fractions, truly digestible nutrients, energetic values and in vitro ammonia productions in ruminant systems. Furthermore, relationships between physiochemical, metabolic and fermentation characteristics and molecular spectral parameters were determined using vibrational molecular spectroscopy. Results showed that the HB12i had higher lignin, while TT8i had higher phenolics. Both silenced genotypes had higher rumen slowly degraded carbohydrate fractions and truly digestible neutral detergent fiber, but lower rumen degradable protein fractions. Moreover, the HB12i had lower truly digestible crude protein, energetic values and ammonia production compared with other silenced genotypes. In addition, in relation to the nutritive values of alfalfa, structural carbohydrate parameters were negatively correlated, whereas alpha/beta ratio in protein structure was positively correlated. Furthermore, good predictions were obtained for degradation of protein and carbohydrate fractions and energy values from molecular spectral parameters. In conclusion, silencing of the TT8 and HB12 genes decreased protein availability and increased fiber availability. Silencing of the HB12 gene also increased lignin and decreased energy and rumen ammonia production. Moreover, nutritional alterations were closely correlated with molecular spectral parameters. Therefore, gene modification through silencing the TT8 and HB12 genes in alfalfa influenced physiochemical, metabolic and fermentation characteristics.

陆地棉NF-YA基因家族的全基因组鉴定与功能分析

【目的】通过对陆地棉NF-YA基因家族进行全基因组鉴定,分析其表达特性,鉴定其中与棉花开花相关的基因。【方法】利用生物信息学方法系统分析了其理化性质、基因结构、共线性、Ka/Ks、顺式作用元件和表达模式,实时荧光定量聚合酶链反应(Polymerase chain reaction,PCR)分析表达特征,采用病毒诱导基因沉默技术验证基因功能。【结果】陆地棉中鉴定到29个NF-YA基因家族成员,分为5个亚组,定位在18条染色体上;全基因组复制和片段复制是GhNF-YA基因家族扩张的主要动力;启动子区含有大量光反应的顺式作用元件。GhNF-YA基因家族基因在茎、叶中高表达;12个GhNF-YA基因在早熟品种鲁棉研19号和晚熟品种鲁棉研37号中均在第三至六叶时期表达量较高,并且大部分基因在品种间表达差异显著。沉默GhNF-YA18基因的鲁棉研37号植株比对照提前11 d现蕾,并且该基因在白化期和现蕾期的表达量均低于未沉默对照棉株。【结论】本研究对陆地棉NF-YA基因家族进行了鉴定和表达分析,为进一步研究棉花开花调控的分子机制奠定了基础。

Transgenerational Inheritance and Resetting of Stress, Induced Loss of Epigenetic Gene Silencing in Arabidopsis

Plants, as sessile organisms, need to sense and adapt to heterogeneous environments and have developed sophisticated responses by changing their cellular physiology, gene regulation, and genome stability. Recent work dem- onstrated heritable stress effects on the control of genome stability in plants--a phenomenon that was suggested to be of epigenetic nature. Here, we show that temperature and UV-B stress cause immediate and heritable changes in the epi- genetic control of a silent reporter gene in Arabidopsis. This stress-mediated release of gene silencing correlated with pronounced alterations in histone occupancy and in histone H3 acetylation but did not involve adjustments in DNA meth- ylation. We observed transmission of stress effects on reporter gene silencing to non-stressed progeny, but this effect was restricted to areas consisting of a small number of cells and limited to a few non-stressed progeny generations. Further- more, stress-induced release of gene silencing was antagonized and reset during seed aging. The transient nature of this phenomenon highlights the ability of plants to restrict stress-induced relaxation of epigenetic control mechanisms, which likely contributes to safeguarding genome integrity.

Virus-induced Gene Silencing in Eggplant(Solanum melongena)

Eggplant （Solanum melongena） is an economically important vegetable requiring investigation into its various genomic functions. The current limitation in the investigation of genomic function in eggplant is the lack of effective tools available for conducting functional assays. Virus-induced gene silencing （VIGS） has played a critical role in the functional genetic analyses. In this paper, TRV-mediated VIGS was successfully elicited in eggplant. We first cloned the CDS sequence of PDS （PHYTOENE DESATURASE） in eggplant and then silenced the PDS gene. Photo-bleaching was shown on the newly-developed leaves four weeks after agroinoculation, indicating that VIGS can be used to silence genes in eggplant. To further illustrate the reliability of VIGS in eggplant, we selected Chl H, Su and CLA1 as reporters to elicit VIGS using the high-pressure spray method. Suppression of Chl H and Su led to yellow leaves, while the depletion of CLA1 resulted in albino. In conclusion, four genes, PDS, Chl H, Su （Sulfur）, CLA1, were down-regulated significantly by VIGS, indicating that the VIGS system can be successfully applied in eggplant and is a reliable tool for the study of gene function.

Graft-accelerated virus-induced gene silencing facilitates functional genomics in rose flowers

Rose has emerged as a model ornamental plant for studies of flower development, senescence, and morphology, as well as the metabolism of floral fragrances and colors.Virus-induced gene silencing(VIGS) has long been used in functional genomics studies of rose by vacuum infiltration of cuttings or seedlings with an Agrobacterium suspension carrying TRV-derived vectors. However, VIGS in rose flowers remains a challenge because of its low efficiency and long time to establish silencing. Here we present a novel and rapid VIGS method that can be used to analyze gene function in rose,called ‘graft-accelerated VIGS’, where axil ary sprouts are cut from the rose plant and vacuum infiltrated with Agrobacterium. The inoculated scions are then grafted back onto the plants to flower and silencing phenotypes can be observed within 5 weeks, post-infiltration. Using this new method, we successfully silenced expression of the RhDFR, RhA G, and RhNUDXin rose flowers, and affected their color, petal number, as well as fragrance, respectively. This grafting method will facilitate high-throughput functional analysis of genes in rose flowers. Importantly, it may also be applied to other woody species that are not currently amenable to VIGS by conventional leaf or plantlet/seedling infiltration methods.