Researchers commonly use cyclization recombination enzyme/locus of X-over P1(Cre/loxP)technology-based conditional gene knockouts of model mice to investigate the functional roles of genes of interest in Sertoli and L...Researchers commonly use cyclization recombination enzyme/locus of X-over P1(Cre/loxP)technology-based conditional gene knockouts of model mice to investigate the functional roles of genes of interest in Sertoli and Leydig cells within the testis.However,the shortcomings of these genetic tools include high costs,lengthy experimental periods,and limited accessibility for researchers.Therefore,exploring alternative gene silencing techniques is of great practical value.In this study,we employed adeno-associated virus(AAV)as a vector for gene silencing in Sertoli and Leydig cells.Our findings demonstrated that AAV serotypes 1,8,and 9 exhibited high infection efficiency in both types of testis cells.Importantly,we discovered that all three AAV serotypes exhibited exquisite specificity in targeting Sertoli cells via tubular injection while demonstrating remarkable selectivity in targeting Leydig cells via interstitial injection.We achieved cell-specific knockouts of the steroidogenic acute regulatory(Star)and luteinizing hormone/human chorionic gonadotropin receptor(Lhcgr)genes in Leydig cells,but not in Sertoli cells,using AAV9-single guide RNA(sgRNA)-mediated gene editing in Rosa26-LSL-Cas9mice.Knockdown of androgen receptor(Ar)gene expression in Sertoli cells of wild-type mice was achieved via tubular injection of AAV9-short hairpinRNA(shRNA)-mediated targeting.Our findings offer technical approaches for investigating gene function in Sertoli and Leydig cells through AAV9-mediated gene silencing.展开更多
The occurrence,development,and metastasis of tumors often entail abnormal expression of genetic substances.Monitoring and regulating changes in intracellular nucleic acid substances hold promise for achieving accurate...The occurrence,development,and metastasis of tumors often entail abnormal expression of genetic substances.Monitoring and regulating changes in intracellular nucleic acid substances hold promise for achieving accurate tumor diagnosis and effective treatment.However,the effectiveness of integrated tumor diagnosis and treatment based on functional nucleic acids still needs to be improved.In this study,we engineered a multifunctional nucleic acid delivery system grounded in a cationic covalent organic framework carrier.This system not only showcases effective gene silencing but also boasts high sensitivity in detecting miR21 levels within tumor cells,enabling real-time monitoring of tumor gene therapy efficacy.The construction of this integrated functional nucleic acid delivery platform provides new ideas for precise tumor detection and effective tumor treatment.展开更多
In the article“Silencing of the long non-coding RNA LINC00265 triggers autophagy and apoptosis in lung cancer by reducing protein stability of SIN3A oncogene”(Oncology Research.2024,Vol.32,No.7,pp.1185–1195.doi:10....In the article“Silencing of the long non-coding RNA LINC00265 triggers autophagy and apoptosis in lung cancer by reducing protein stability of SIN3A oncogene”(Oncology Research.2024,Vol.32,No.7,pp.1185–1195.doi:10.32604/or.2023.030771,https://www.techscience.com/or/v32n7/57163),an inadvertent error occurred during the compilation of Fig.3H.This needed corrections to ensure the accuracy and integrity of the data presented.展开更多
Since transgene silencing was found in transgenic plants,many scholars have studied it extensively and considered that it has three functional mechanisms:post dependent gene silencing,transcriptional gene silencing,p...Since transgene silencing was found in transgenic plants,many scholars have studied it extensively and considered that it has three functional mechanisms:post dependent gene silencing,transcriptional gene silencing,post transcriptional gene silencing.At the moment,people have mainly focused on the study of post transcriptional gene silencing and found its features:extensivity,conduction and peculiarity,also put forward some hypothesis for its mechanisms,for example,RNA threshold model,aberrant RNA model,inter or intra molecular base pairing model and so on.Furthermore,post transcriptional gene silencing is being applied in gene engineering of plants.Recently the people have found that post transcriptional gene silencing has bearing on capacity plants resisting virus.Many researchers have studied post transcriptional gene silencing,but there are some questions which need be solved in the future.This article summarizes progresses in features,mechanisms,applies of post transcriptional gene silencing about transgenic plants.展开更多
Rice blast disease is one of the most devastating diseases in rice production,which severely affects the high and stable yield of rice.The formation of appressorium plays a key role in the pathogenesis of Magnaporthe ...Rice blast disease is one of the most devastating diseases in rice production,which severely affects the high and stable yield of rice.The formation of appressorium plays a key role in the pathogenesis of Magnaporthe grisea in rice.It has been confirmed that a P-type ATPase （P-ATPase） is involved in the formation of appressorium.A number of small molecular substances are able to enter the pathogen from the host during the interactions between pathogens and hosts,thus resisting the infection of pathogens.In this study,a 232 bp DNA sequence with good specificity from the first exon of P-ATPase gene MgAPT2 was used as an interference fragment and was inserted into interference vector forward and reversely.The interfering vector was then transformed into rice blast-susceptible rice variety Nipponbare via Agrobacterium-mediated transformation.Identification of rice plants inoculated with M.grisea at the seedling stage and detection of the expression level of P-ATPase gene MgAPT2 showed that the expression level of MgAPT2 gene in transgenic plants was reduced and the rice blast resistance was improved.This study provided a new way for the innovation of rice germplasm resources resistant to rice blast disease.展开更多
Virus-induced gene silencing (VIGS) is a recently developed technique for characterizing the function of plant genes by gene transcript suppression and is increasingly used to generate transient loss-of-function assay...Virus-induced gene silencing (VIGS) is a recently developed technique for characterizing the function of plant genes by gene transcript suppression and is increasingly used to generate transient loss-of-function assays. Here we report that the 2mDNA1, a geminivirus satellite vector, can induce efficient gene silencing in Nicotiana tabacum with Tobacco curly shoot virus. We have successfully silenced the β-glucuronidase (GUS) gene in GUS transgenic N. tabacum plants and the sulphur desaturase (Su) gene in five different N. tabacum cultivars. These pronounced and severe silencing phenotypes are persistent and ubiquitous. Once initiated in seedlings, the silencing phenotype lasted for the entire life span of the plants and silencing could be induced in a variety of tissues and organs including leaf, shoot, stem, root, and flower, and achieved at any growth stage. This system works well between 18-32°C. We also silenced the NtEDS1 gene and demonstrated that NtEDS1 is essential for N gene mediated resistance against Tobacco mosaic virus in N. tabacum. The above results indicate that this system has great potential as a versatile VIGS system for routine functional analysis of genes in N. tabacum.展开更多
The sea cucumber, Apostichopusjaponicus, undergoes aestivation to improve survival during periods of high-temperature. During aestivation, the metabolic rate is depressed to reduce the consumption of reserved energy. ...The sea cucumber, Apostichopusjaponicus, undergoes aestivation to improve survival during periods of high-temperature. During aestivation, the metabolic rate is depressed to reduce the consumption of reserved energy. We evaluated the role of epigenetic modification on global gene silencing during metabolic rate depression in the sea cucumber. We compared the expression of epigenetic modifiers in active and aestivating sea cucumbers. The expression of three genes involved in DNA methylation and chromatin remodeling （DNA （cytosine-5）-methyltransferase l, Methyl-CpG-binding domain protein 2）, and Chromodomain-helicase-DNA-binding protein 5） was significantly higher during aestivation （Days 20 and 40）. Similarly, we observed an increase in the expression of genes involved in histone acetylation （Histone deacetylase 3） and Histone-binding protein RBBP4） during the early （Days 5 and 10） and late phases （Days 20 and 40） of aestivation. There was no change in the expression of KAT2B, a histone acetyltransferase. However, the expression of histone methylation associated modifiers （Histone-arginine methyltransferase CARMER and Histone-lysine N-methyltransferase MLL5） was significantly higher after 5 d in the aestivating group. The results suggest that the expression of epigenetic modifiers involved in DNA methylation, chromatin remodeling, histone acetylation, and histone methylation is upregulated during aestivation. We hypothesize that these changes regulate global gene silencing during aestivation in A. japonicus.展开更多
Phytic acid is the principal storage form of phosphorus in plant seeds and an essential signalling molecule in several regulatory processes of plant development.However,it is known as an anti-nutrient compound owing t...Phytic acid is the principal storage form of phosphorus in plant seeds and an essential signalling molecule in several regulatory processes of plant development.However,it is known as an anti-nutrient compound owing to its potent chelating property.Thus,reducing the phytic acid content in crops is desirable.Studies involving regulation of MIPS and IPK1 genes to generate low phytate rice have been reported earlier.However,the functional significance of OsITPK and the effect of its down-regulation on phytic acid content and the associated pleiotropic effects on rice have not yet been investigated.In this study,tissue specific RNA interference(RNAi)-mediated down-regulation of a major ITPK homolog(OsITP5/6K-1)resulted in 46.2%decrease in phytic acid content of T2 transgenic seeds with a subsequent 3-fold enhancement in the inorganic phosphorus content.Silencing of OsITP5/6K-1 altered the transcript levels of essential phytic acid pathway genes,without significantly affecting the transcript levels of other OsITPK homologs.Furthermore,the mapping of elements through X-ray microfluorescence analysis revealed significant changes in the spatial distribution pattern and translocation of elements in low phytate seeds.Additionally,low phytate polished seeds exhibited 1.3-fold and 1.6-fold enhancement in iron and zinc content in the grain endosperm,respectively.Silencing of OsITP5/6K-1 also altered the amino acid and myo-inositol content of the transgenic seeds.Our results successfully established that RNAi-mediated silencing of OsITP5/6K-1 gene significantly reduced the phytate levels in seeds without hampering the germination potential of seeds and plant growth.The present study provided an insight into the mechanism of phytic acid biosynthesis pathway.展开更多
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 res...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.展开更多
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 trans...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.展开更多
Invasive nucleic acids such as transposons and viruses usually exhibit aberrant characteristics,e.g.,unpaired DNA or abnormal doublestranded RNA.Organisms employ a variety of strategies to defend themselves by disting...Invasive nucleic acids such as transposons and viruses usually exhibit aberrant characteristics,e.g.,unpaired DNA or abnormal doublestranded RNA.Organisms employ a variety of strategies to defend themselves by distinguishing self and nonself substances and disabling these invasive nucleic acids.Furthermore,they have developed ways to remember this exposure to invaders and transmit the experience to their descendants.The mechanism underlying this inheritance has remained elusive.Recent research has shed light on the initiation and maintenance of RNA-mediated inherited gene silencing.Small regulatory RNAs play a variety of crucial roles in organisms,including gene regulation,developmental timing,antiviral defense,and genome integrity,via a process termed as RNA interference（RNAi）.Recent research has revealed that small RNAs and the RNAi machinery are engaged in establishing and promoting transgenerational gene silencing.Small RNAs direct the RNAi and chromatin modification machinery to the cognate nucleic acids to regulate gene expression and epigenetic alterations.Notably,these acquired small RNAs and epigenetic changes persist and are transmitted from parents to offspring for multiple generations.Thus,RNAi is a vital determinant of the inheritance of gene silencing and acts as a driving force of evolution.展开更多
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 he...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.展开更多
This paper reports results of the authors’ studies on the virtual design method used in the development of low noise intake system of I.C. engine. The resulting high pass-by noise at level above the legislative targe...This paper reports results of the authors’ studies on the virtual design method used in the development of low noise intake system of I.C. engine. The resulting high pass-by noise at level above the legislative target at full throttle when engine speed was around 5200 r/min necessitated a BEM-aided redesign task, following the typical process of design and development of an intake system. During the initial design, based on the acoustic theory and the requirements (1. The air flux of the redesigned should equal to or exceed the value of the original flux; 2. The filtering area must not be degraded), and considering the constraint of space in the engine compartment, total volume and rough internal dimensions were determined. During the detailed design, the exact internal dimensions of the air cleaner were determined, and an effective method was applied to improve the acoustic performance at low frequency. The predicted sound power of the intake system indicated that the objective of reducing the overall engine noise by minimizing intake system noise was achieved.展开更多
RNA silencing(RNAi)is a nucleotide sequence-specific process that results in blockage of gene expression(Baulcombe,2004,2005;Gunter Meister,2004;Vaucheret,2006;Chinnusamy and Zhu,2009;Heo and Kim,2009;Matzke et al.,20...RNA silencing(RNAi)is a nucleotide sequence-specific process that results in blockage of gene expression(Baulcombe,2004,2005;Gunter Meister,2004;Vaucheret,2006;Chinnusamy and Zhu,2009;Heo and Kim,2009;Matzke et al.,2009;Simon and Meyers,2011).In plants,post-transcriptional gene silencing(PTGS)occurs in the cytoplasm that is induced by small RNAs(sRNAs),which are the products of double-stranded RNAs(dsRNAs)processed by Dicer-like ribonucleases and achieve specificity through base pairing with targeted RNA sequences(Carmell and Hannon,2004;Gunter Meister,2004;Gasciolli et al.,2005).In an alternative pathway occurring in the nucleus,sRNAs corresponding to promoter sequences direct the silencing machinery to block the transcription of homologous promoters(transcriptional gene silencing,TGS).This process requires 24-nt sRNAs for de novo DNA methylation,a process known as RNA-directed DNA methylation(RdDM)(James P.Jackson and Jacobsen,2002;Matzke and Birchler,2005;Matzke et al.,2007;Zilberman et al.,2007;Wierzbicki et al.,200&Zilberman,2008).Histone modifications also play an important role in the establishment and maintenance of DNA methylation(Zuzana Jasencakova,2003;Ooi et al.,2007;Cedar and Bergman,2009;Law and Jacobsen,2010).In plants,it has been shown that the transcription of exogenous transgene transcribing inverted-repeat(exo-/R)sequences produces dsRNAs,triggering exo-//?PTGS that is negatively autoregulated through methylation spreading/transitive silencing.This transitive silencing「einforces the self-silencing of exo-//?and leads to reduced exo-//?PTGS and exo-/R-derived sRNA production.exo-/R-derived sRNAs function as mobile signals to trigger sRNA-mediated non-cell autonomous silencing of an endogenous homologous target gene(endo-gene)(Dong et al.,2011).It remains unknown whether histone modifications play a role in the exo-IR-triggered endo-gene silencing.展开更多
Rice sheath blight, caused by Rhizoctonia solani AG1-IA, is a major disease in rice-growing areas worldwide. Effectors of phytopathogenic fungi play important roles during the infection process of fungal pathogens ont...Rice sheath blight, caused by Rhizoctonia solani AG1-IA, is a major disease in rice-growing areas worldwide. Effectors of phytopathogenic fungi play important roles during the infection process of fungal pathogens onto their host plants. However, the molecular mechanisms by which R. solani effectors regulate rice immunity are not well understood. Through prediction, 78 candidate effector molecules were identified. Using the tobacco rattle virus-host induced gene silencing(TRV-HIGS) system, 45 RNAi constructs of effector genes were infiltrated into Nicotiana benthamiana leaves. The results revealed that eight of these constructs resulted in a significant reduction in necrosis caused by infection with the AG1-IA strain GD-118. Additionally, stable rice transformants carrying the double-stranded RNA construct for one of the effector genes, AGLIP1, were generated to further verify the function of this gene. The suppression of the AGLIP1 gene increased the resistance of both N. benthamiana and rice against GD-118, and also affected the growth rate of GD-118, indicating that AGLIP1 is a key pathogenic factor. Small RNA sequencing showed that the HIGS vectors were processed into si RNAs within the plants and then translocated to the fungi, leading to the silencing of the target genes. As a result, AGLIP1 might be an excellent candidate for HIGS, thereby enhancing crop resistance against the pathogen and contributing to the control of R. solani infection.展开更多
The recent development of gene transfer approaches in plants and animals has revealed that transgene can undergo silencing after integration in the genome. Host genes can also be silenced as a consequence of the prese...The recent development of gene transfer approaches in plants and animals has revealed that transgene can undergo silencing after integration in the genome. Host genes can also be silenced as a consequence of the presence of a homologous transgene. More and more investigations have demonstrated that double- stranded RNA can silence genes by triggering degradation of homologous RNA in the cytoplasm and by directing methylation of homologous nuclear DNA sequences. Analyses of Arabidopsis mutants and plant viral suppressors of silencing are unraveling RNA-silencing mechanisms and are assessing the role of methy- lation in transcriptional and posttranscriptional gene silencing. This review will focus on double-stranded RNA mediated mRNA degradation and gene inactivation in plants.展开更多
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 oligon...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.展开更多
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 cerebr...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.展开更多
Objective:To determine the effect of gene silencing of cyclophilin B(CypB)on growth and proliferation of gastric cancer cells.Methods:CypB siRNA lentivirus(LV-CypB-si)and control lentivirus(LV-si-con)were produced.Cyp...Objective:To determine the effect of gene silencing of cyclophilin B(CypB)on growth and proliferation of gastric cancer cells.Methods:CypB siRNA lentivirus(LV-CypB-si)and control lentivirus(LV-si-con)were produced.CypB expression in gastric cancer cell lines was detected by Western blot.BGC823 and SGC7901 cells were chosen to be infected with LV-sicon and LV-CypB-si,and stable transfectants were isolated.The cell groups transfected with LV-CypB-siRNA,LV-siRNA-con and transfected no carrier were served as the experimental group,the implicit control group and the blank control group respectively.MTT and colony formation assays were used to examine the effect of CypB on the cell growth and proliferation in vitro.Cell cycle was analyzed with flow cytometry.The expression of VEGFR of BGC823-si and SGC7901-si was detected by Western blot.Results:Gene silencing of CypB can inhibit gastric cancer cell growth,proliferation,cell cycle progress and tumorigenesis.CypB expression level was obviously higher in SGC7901 and BGC823 than MKN28 and GES.These two cell lines were infected with LV-si-con and LV-CypB-si respectively.MTT and cloney formation assays showed a significantly decreased rate of cell proliferation from the forth day or the fifth day in cells transfected with LV-CypB-si(P<0.05).Down-regulation of CypB resulted in slightly decreased percentage of S phase and increased percentage of G_1(P<0.05).These findings indicated that CypB could promote the G_1-S transition of gastric cancer cell.In addition,the expression of VEGF of BGC823 and SGC7901 transfected with CypB siRNA was reduced in comparison with the implicit control group and the blank control group.Conclusions:Gene silencing of CypB decreases gastric cancer cells proliferation and in vivo tumorigenesis.These findings indiccate CypB could be a potential biomarker and therapeutic target for gastric cancer.展开更多
Transgenic tobacco(Nicotiana tabacum L.cv.Xanthi)plants which express coat protein(CP)gene(cp)of potato virus X(PVX)were generated via Agrobacterium tumefaciens-mediated gene transfer.Northern blotting analysis indica...Transgenic tobacco(Nicotiana tabacum L.cv.Xanthi)plants which express coat protein(CP)gene(cp)of potato virus X(PVX)were generated via Agrobacterium tumefaciens-mediated gene transfer.Northern blotting analysis indicated that cp silencing happened in three transformants,and Run on assay showed that the cp silencing was at post-transcriptional level.The result of cp methylation assay showed different degree of methylation existed in coat protein gene coding sequences.cp methylation was also analyzed during virus-induced gene silencing(VIGS),and results suggested that methylation pre-existed in cp non-silenced transgenic plants,while virus infection increase the methylation degree,which indicated DNA methylation in VIGS was not a de novo DNA methylation process.展开更多
基金supported by the National Natural Science Foundation of China(No.82070872 and No.82370854 to JXL)Innovative and Entrepreneurial Team of Jiangsu Province(No.JSSCTD2021 to JXL)+1 种基金China Postdoctoral Science Foundation(2023M741790 to JP)Jiangsu Funding Program for Excellent Postdoctoral Talent(2023ZB558 to JP).
文摘Researchers commonly use cyclization recombination enzyme/locus of X-over P1(Cre/loxP)technology-based conditional gene knockouts of model mice to investigate the functional roles of genes of interest in Sertoli and Leydig cells within the testis.However,the shortcomings of these genetic tools include high costs,lengthy experimental periods,and limited accessibility for researchers.Therefore,exploring alternative gene silencing techniques is of great practical value.In this study,we employed adeno-associated virus(AAV)as a vector for gene silencing in Sertoli and Leydig cells.Our findings demonstrated that AAV serotypes 1,8,and 9 exhibited high infection efficiency in both types of testis cells.Importantly,we discovered that all three AAV serotypes exhibited exquisite specificity in targeting Sertoli cells via tubular injection while demonstrating remarkable selectivity in targeting Leydig cells via interstitial injection.We achieved cell-specific knockouts of the steroidogenic acute regulatory(Star)and luteinizing hormone/human chorionic gonadotropin receptor(Lhcgr)genes in Leydig cells,but not in Sertoli cells,using AAV9-single guide RNA(sgRNA)-mediated gene editing in Rosa26-LSL-Cas9mice.Knockdown of androgen receptor(Ar)gene expression in Sertoli cells of wild-type mice was achieved via tubular injection of AAV9-short hairpinRNA(shRNA)-mediated targeting.Our findings offer technical approaches for investigating gene function in Sertoli and Leydig cells through AAV9-mediated gene silencing.
基金the National Key Research and Development Program of China(No.2021YFB3800900)National Natural Science Foundation of China(No.51925305)the talent cultivation project Funds for the Innovation Laboratory for Sciences and Technologies of Energy Materials of Fujian Province(No.HRTP[2022]52)。
文摘The occurrence,development,and metastasis of tumors often entail abnormal expression of genetic substances.Monitoring and regulating changes in intracellular nucleic acid substances hold promise for achieving accurate tumor diagnosis and effective treatment.However,the effectiveness of integrated tumor diagnosis and treatment based on functional nucleic acids still needs to be improved.In this study,we engineered a multifunctional nucleic acid delivery system grounded in a cationic covalent organic framework carrier.This system not only showcases effective gene silencing but also boasts high sensitivity in detecting miR21 levels within tumor cells,enabling real-time monitoring of tumor gene therapy efficacy.The construction of this integrated functional nucleic acid delivery platform provides new ideas for precise tumor detection and effective tumor treatment.
文摘In the article“Silencing of the long non-coding RNA LINC00265 triggers autophagy and apoptosis in lung cancer by reducing protein stability of SIN3A oncogene”(Oncology Research.2024,Vol.32,No.7,pp.1185–1195.doi:10.32604/or.2023.030771,https://www.techscience.com/or/v32n7/57163),an inadvertent error occurred during the compilation of Fig.3H.This needed corrections to ensure the accuracy and integrity of the data presented.
文摘Since transgene silencing was found in transgenic plants,many scholars have studied it extensively and considered that it has three functional mechanisms:post dependent gene silencing,transcriptional gene silencing,post transcriptional gene silencing.At the moment,people have mainly focused on the study of post transcriptional gene silencing and found its features:extensivity,conduction and peculiarity,also put forward some hypothesis for its mechanisms,for example,RNA threshold model,aberrant RNA model,inter or intra molecular base pairing model and so on.Furthermore,post transcriptional gene silencing is being applied in gene engineering of plants.Recently the people have found that post transcriptional gene silencing has bearing on capacity plants resisting virus.Many researchers have studied post transcriptional gene silencing,but there are some questions which need be solved in the future.This article summarizes progresses in features,mechanisms,applies of post transcriptional gene silencing about transgenic plants.
基金Supported by International Cooperation Project of Jiangsu Province(BZ2011039)Agricultural Technology Independent Innovation Fund of Jiangsu Province[CX(12)2024]~~
文摘Rice blast disease is one of the most devastating diseases in rice production,which severely affects the high and stable yield of rice.The formation of appressorium plays a key role in the pathogenesis of Magnaporthe grisea in rice.It has been confirmed that a P-type ATPase （P-ATPase） is involved in the formation of appressorium.A number of small molecular substances are able to enter the pathogen from the host during the interactions between pathogens and hosts,thus resisting the infection of pathogens.In this study,a 232 bp DNA sequence with good specificity from the first exon of P-ATPase gene MgAPT2 was used as an interference fragment and was inserted into interference vector forward and reversely.The interfering vector was then transformed into rice blast-susceptible rice variety Nipponbare via Agrobacterium-mediated transformation.Identification of rice plants inoculated with M.grisea at the seedling stage and detection of the expression level of P-ATPase gene MgAPT2 showed that the expression level of MgAPT2 gene in transgenic plants was reduced and the rice blast resistance was improved.This study provided a new way for the innovation of rice germplasm resources resistant to rice blast disease.
基金supported by the National Science and Technology Major Projects of China (No. 2009ZX08009-026B)the China Postdoctoral Science Foundation (No. 20090461375)the National Basic Research Program (973) of China (No. 2006CB101903)
文摘Virus-induced gene silencing (VIGS) is a recently developed technique for characterizing the function of plant genes by gene transcript suppression and is increasingly used to generate transient loss-of-function assays. Here we report that the 2mDNA1, a geminivirus satellite vector, can induce efficient gene silencing in Nicotiana tabacum with Tobacco curly shoot virus. We have successfully silenced the β-glucuronidase (GUS) gene in GUS transgenic N. tabacum plants and the sulphur desaturase (Su) gene in five different N. tabacum cultivars. These pronounced and severe silencing phenotypes are persistent and ubiquitous. Once initiated in seedlings, the silencing phenotype lasted for the entire life span of the plants and silencing could be induced in a variety of tissues and organs including leaf, shoot, stem, root, and flower, and achieved at any growth stage. This system works well between 18-32°C. We also silenced the NtEDS1 gene and demonstrated that NtEDS1 is essential for N gene mediated resistance against Tobacco mosaic virus in N. tabacum. The above results indicate that this system has great potential as a versatile VIGS system for routine functional analysis of genes in N. tabacum.
基金Supported by the National Key Technology Research and Development Program(No.2006AA10A411)the Agricultural Seed Project of Shandong Province
文摘The sea cucumber, Apostichopusjaponicus, undergoes aestivation to improve survival during periods of high-temperature. During aestivation, the metabolic rate is depressed to reduce the consumption of reserved energy. We evaluated the role of epigenetic modification on global gene silencing during metabolic rate depression in the sea cucumber. We compared the expression of epigenetic modifiers in active and aestivating sea cucumbers. The expression of three genes involved in DNA methylation and chromatin remodeling （DNA （cytosine-5）-methyltransferase l, Methyl-CpG-binding domain protein 2）, and Chromodomain-helicase-DNA-binding protein 5） was significantly higher during aestivation （Days 20 and 40）. Similarly, we observed an increase in the expression of genes involved in histone acetylation （Histone deacetylase 3） and Histone-binding protein RBBP4） during the early （Days 5 and 10） and late phases （Days 20 and 40） of aestivation. There was no change in the expression of KAT2B, a histone acetyltransferase. However, the expression of histone methylation associated modifiers （Histone-arginine methyltransferase CARMER and Histone-lysine N-methyltransferase MLL5） was significantly higher after 5 d in the aestivating group. The results suggest that the expression of epigenetic modifiers involved in DNA methylation, chromatin remodeling, histone acetylation, and histone methylation is upregulated during aestivation. We hypothesize that these changes regulate global gene silencing during aestivation in A. japonicus.
文摘Phytic acid is the principal storage form of phosphorus in plant seeds and an essential signalling molecule in several regulatory processes of plant development.However,it is known as an anti-nutrient compound owing to its potent chelating property.Thus,reducing the phytic acid content in crops is desirable.Studies involving regulation of MIPS and IPK1 genes to generate low phytate rice have been reported earlier.However,the functional significance of OsITPK and the effect of its down-regulation on phytic acid content and the associated pleiotropic effects on rice have not yet been investigated.In this study,tissue specific RNA interference(RNAi)-mediated down-regulation of a major ITPK homolog(OsITP5/6K-1)resulted in 46.2%decrease in phytic acid content of T2 transgenic seeds with a subsequent 3-fold enhancement in the inorganic phosphorus content.Silencing of OsITP5/6K-1 altered the transcript levels of essential phytic acid pathway genes,without significantly affecting the transcript levels of other OsITPK homologs.Furthermore,the mapping of elements through X-ray microfluorescence analysis revealed significant changes in the spatial distribution pattern and translocation of elements in low phytate seeds.Additionally,low phytate polished seeds exhibited 1.3-fold and 1.6-fold enhancement in iron and zinc content in the grain endosperm,respectively.Silencing of OsITP5/6K-1 also altered the amino acid and myo-inositol content of the transgenic seeds.Our results successfully established that RNAi-mediated silencing of OsITP5/6K-1 gene significantly reduced the phytate levels in seeds without hampering the germination potential of seeds and plant growth.The present study provided an insight into the mechanism of phytic acid biosynthesis pathway.
基金Thanks for the technical support of Dr.Daqi Fu and Dr.Lanhuan Meng of China Agricultural University.This work was supported by Beijing Municipal Science and Technology Project(Grant No.Z181100002418006)the Fundamental Research Fund for the Central University(Grant No.2015ZCQ-YL-03)the World-Class Discipline Construction and Characteristic Development Guidance Funds for Beijing Forestry University(Grant No.2019XKJS0323).
文摘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.
文摘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.
基金supported by the grants from the State Key Development Program for Basic Research of China (No.2011 CBA01103)the National Natural Science Foundation of China(No.31171254)the Fundamental Research Funds for Central Universities(No.WK 2060190018)
文摘Invasive nucleic acids such as transposons and viruses usually exhibit aberrant characteristics,e.g.,unpaired DNA or abnormal doublestranded RNA.Organisms employ a variety of strategies to defend themselves by distinguishing self and nonself substances and disabling these invasive nucleic acids.Furthermore,they have developed ways to remember this exposure to invaders and transmit the experience to their descendants.The mechanism underlying this inheritance has remained elusive.Recent research has shed light on the initiation and maintenance of RNA-mediated inherited gene silencing.Small regulatory RNAs play a variety of crucial roles in organisms,including gene regulation,developmental timing,antiviral defense,and genome integrity,via a process termed as RNA interference（RNAi）.Recent research has revealed that small RNAs and the RNAi machinery are engaged in establishing and promoting transgenerational gene silencing.Small RNAs direct the RNAi and chromatin modification machinery to the cognate nucleic acids to regulate gene expression and epigenetic alterations.Notably,these acquired small RNAs and epigenetic changes persist and are transmitted from parents to offspring for multiple generations.Thus,RNAi is a vital determinant of the inheritance of gene silencing and acts as a driving force of evolution.
文摘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.
文摘This paper reports results of the authors’ studies on the virtual design method used in the development of low noise intake system of I.C. engine. The resulting high pass-by noise at level above the legislative target at full throttle when engine speed was around 5200 r/min necessitated a BEM-aided redesign task, following the typical process of design and development of an intake system. During the initial design, based on the acoustic theory and the requirements (1. The air flux of the redesigned should equal to or exceed the value of the original flux; 2. The filtering area must not be degraded), and considering the constraint of space in the engine compartment, total volume and rough internal dimensions were determined. During the detailed design, the exact internal dimensions of the air cleaner were determined, and an effective method was applied to improve the acoustic performance at low frequency. The predicted sound power of the intake system indicated that the objective of reducing the overall engine noise by minimizing intake system noise was achieved.
基金financially supported by the National Natural Science Foundation of China (31730078 to H.S.G., and 31600124 to J.H.Z.)
文摘RNA silencing(RNAi)is a nucleotide sequence-specific process that results in blockage of gene expression(Baulcombe,2004,2005;Gunter Meister,2004;Vaucheret,2006;Chinnusamy and Zhu,2009;Heo and Kim,2009;Matzke et al.,2009;Simon and Meyers,2011).In plants,post-transcriptional gene silencing(PTGS)occurs in the cytoplasm that is induced by small RNAs(sRNAs),which are the products of double-stranded RNAs(dsRNAs)processed by Dicer-like ribonucleases and achieve specificity through base pairing with targeted RNA sequences(Carmell and Hannon,2004;Gunter Meister,2004;Gasciolli et al.,2005).In an alternative pathway occurring in the nucleus,sRNAs corresponding to promoter sequences direct the silencing machinery to block the transcription of homologous promoters(transcriptional gene silencing,TGS).This process requires 24-nt sRNAs for de novo DNA methylation,a process known as RNA-directed DNA methylation(RdDM)(James P.Jackson and Jacobsen,2002;Matzke and Birchler,2005;Matzke et al.,2007;Zilberman et al.,2007;Wierzbicki et al.,200&Zilberman,2008).Histone modifications also play an important role in the establishment and maintenance of DNA methylation(Zuzana Jasencakova,2003;Ooi et al.,2007;Cedar and Bergman,2009;Law and Jacobsen,2010).In plants,it has been shown that the transcription of exogenous transgene transcribing inverted-repeat(exo-/R)sequences produces dsRNAs,triggering exo-//?PTGS that is negatively autoregulated through methylation spreading/transitive silencing.This transitive silencing「einforces the self-silencing of exo-//?and leads to reduced exo-//?PTGS and exo-/R-derived sRNA production.exo-/R-derived sRNAs function as mobile signals to trigger sRNA-mediated non-cell autonomous silencing of an endogenous homologous target gene(endo-gene)(Dong et al.,2011).It remains unknown whether histone modifications play a role in the exo-IR-triggered endo-gene silencing.
基金supported by the Henan Province Science and Technology Research Project, China (Grant No. 242102110232)the National Natural Science Foundation of China (Grant No. 31801677)the Major Program of Guangdong Basic and Applied Basic Research, China (Grant No. 2019B030302006)。
文摘Rice sheath blight, caused by Rhizoctonia solani AG1-IA, is a major disease in rice-growing areas worldwide. Effectors of phytopathogenic fungi play important roles during the infection process of fungal pathogens onto their host plants. However, the molecular mechanisms by which R. solani effectors regulate rice immunity are not well understood. Through prediction, 78 candidate effector molecules were identified. Using the tobacco rattle virus-host induced gene silencing(TRV-HIGS) system, 45 RNAi constructs of effector genes were infiltrated into Nicotiana benthamiana leaves. The results revealed that eight of these constructs resulted in a significant reduction in necrosis caused by infection with the AG1-IA strain GD-118. Additionally, stable rice transformants carrying the double-stranded RNA construct for one of the effector genes, AGLIP1, were generated to further verify the function of this gene. The suppression of the AGLIP1 gene increased the resistance of both N. benthamiana and rice against GD-118, and also affected the growth rate of GD-118, indicating that AGLIP1 is a key pathogenic factor. Small RNA sequencing showed that the HIGS vectors were processed into si RNAs within the plants and then translocated to the fungi, leading to the silencing of the target genes. As a result, AGLIP1 might be an excellent candidate for HIGS, thereby enhancing crop resistance against the pathogen and contributing to the control of R. solani infection.
文摘The recent development of gene transfer approaches in plants and animals has revealed that transgene can undergo silencing after integration in the genome. Host genes can also be silenced as a consequence of the presence of a homologous transgene. More and more investigations have demonstrated that double- stranded RNA can silence genes by triggering degradation of homologous RNA in the cytoplasm and by directing methylation of homologous nuclear DNA sequences. Analyses of Arabidopsis mutants and plant viral suppressors of silencing are unraveling RNA-silencing mechanisms and are assessing the role of methy- lation in transcriptional and posttranscriptional gene silencing. This review will focus on double-stranded RNA mediated mRNA degradation and gene inactivation in plants.
文摘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.
基金supported by the National Natural Science Foundation of China,No.30960399a grant from Hainan Provincial International Cooperation Project of China,No.Qiongke(2012)65a grant from Hainan Provincial Health Department Project of China,No.2011-SWK-10-136/Qiongwei2011-65
文摘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.
基金supported by Natural Science Fundation of Heilongjiang Province.China(Grant no.H2013114)
文摘Objective:To determine the effect of gene silencing of cyclophilin B(CypB)on growth and proliferation of gastric cancer cells.Methods:CypB siRNA lentivirus(LV-CypB-si)and control lentivirus(LV-si-con)were produced.CypB expression in gastric cancer cell lines was detected by Western blot.BGC823 and SGC7901 cells were chosen to be infected with LV-sicon and LV-CypB-si,and stable transfectants were isolated.The cell groups transfected with LV-CypB-siRNA,LV-siRNA-con and transfected no carrier were served as the experimental group,the implicit control group and the blank control group respectively.MTT and colony formation assays were used to examine the effect of CypB on the cell growth and proliferation in vitro.Cell cycle was analyzed with flow cytometry.The expression of VEGFR of BGC823-si and SGC7901-si was detected by Western blot.Results:Gene silencing of CypB can inhibit gastric cancer cell growth,proliferation,cell cycle progress and tumorigenesis.CypB expression level was obviously higher in SGC7901 and BGC823 than MKN28 and GES.These two cell lines were infected with LV-si-con and LV-CypB-si respectively.MTT and cloney formation assays showed a significantly decreased rate of cell proliferation from the forth day or the fifth day in cells transfected with LV-CypB-si(P<0.05).Down-regulation of CypB resulted in slightly decreased percentage of S phase and increased percentage of G_1(P<0.05).These findings indicated that CypB could promote the G_1-S transition of gastric cancer cell.In addition,the expression of VEGF of BGC823 and SGC7901 transfected with CypB siRNA was reduced in comparison with the implicit control group and the blank control group.Conclusions:Gene silencing of CypB decreases gastric cancer cells proliferation and in vivo tumorigenesis.These findings indiccate CypB could be a potential biomarker and therapeutic target for gastric cancer.
文摘Transgenic tobacco(Nicotiana tabacum L.cv.Xanthi)plants which express coat protein(CP)gene(cp)of potato virus X(PVX)were generated via Agrobacterium tumefaciens-mediated gene transfer.Northern blotting analysis indicated that cp silencing happened in three transformants,and Run on assay showed that the cp silencing was at post-transcriptional level.The result of cp methylation assay showed different degree of methylation existed in coat protein gene coding sequences.cp methylation was also analyzed during virus-induced gene silencing(VIGS),and results suggested that methylation pre-existed in cp non-silenced transgenic plants,while virus infection increase the methylation degree,which indicated DNA methylation in VIGS was not a de novo DNA methylation process.
