Antisense Fragment Research Articles

Functional characterization of chalcone reductase gene CHR3 through RNAi

In this experiment, the soybean genome "Jilin 30" was used as a template to clone the target gene CHR3 using specific primers. The sense fragment, antisense fragment, and intron of the target gene were ligated into pCAMBIA3300 through double enzyme digestion to form a stem loop structure. We then transferred the constructed RNAi vector pCAMBIA3301-CHR3 RNAi into the recipient soybean genome via Agrobacterium mediated method, specifically reducing the expression of the target gene CHR3. By genetic transformation, positive plants were obtained, and molecular biology methods such as Southern blotting, real-time quantitative PCR, isoliquiritigenin analysis, and identification of resistance to Phytophthora root rot were used to detect the expression level of the CHR3 gene in the positive plants and study its function.The RNAi expression vector pCAMBIA3300-CHR3-RNAi was successfully constructed using the sense and antisense fragments of 428 bp CHR3 gene and an intron of 110 bp was inserted to form the stem-loop structure of the RNAi vector. PCR was used to detect 6 lines of the T1 generation and 15 lines of the T2 generation. The results of Southern analysis confirmed the integration of CHR3 and marker genes into the soybean genome. The expression CHR3 gene in the T1 genotypes decreased by 13.1 to 58.3%; the expression in the root decreased by 0.9 to 47.4%; the expression in the leaf decreased by 20 to 44.2%; and the expression in the stem decreased by 7.6 to 23%. The content of isoliquiritigenin decreased by 26.7% as transgenic shoots had a lower content of isoliquiritigenin and reduced resistance to Phytophthora sojae. Bangladesh J. Bot. 53(3): 627-633, 2024 (September) Special

Generation of Asynaptic Mutants in Potato by Disrupting StDMC1 Gene Using RNA Interference Approach.

Fixing the genomic composition and multiplication through true potato seed (TPS) is an important challenge in autotetraploid potato. Disrupted meiotic cDNA (DMC1) is a meiotic gene that plays a central role in DNA recombination through crossing over in meiosis. Using the Arabidopsis DMC1 (AtDMC1) gene sequence, we retrieved Solanum tuberosum DMC1(StDMC1) from the diploid potato genome, and subsequently, sense and antisense regions of the StDMC1 gene were amplified in potato cv. Kufri Jyoti. The sense and antisense fragments were confirmed by Sanger-sequencing and cloned in the pRI101 vector. Agrobacterium-mediated transformation of the RNAi construct resulted in 44% transformation efficiency, and a total of 137 mutant lines were obtained. These mutant lines were further validated through pollen viability testing, and selected lines were used for gene expression analysis. The acetocarmine-based pollen staining showed reduced pollen viability ranging from 14 to 21% in four DMC1 mutant lines (DMC4-37, DMC4-41, DMC6-20, and DMC6-21), as compared to the Kufri Jyoti control plants, which on average exhibited 78% pollen viability. The phenotypic data was supported by the reduced expression of the StDMC1 gene in these four mutant lines compared to the control Kufri Jyoti. The results confirmed the generation of StDMC1 knockdown lines. This is the first report of StDMC1 mutant line generation in tetraploid potatoes and will be a step forward in generating non-recombinant mutants through sexual reproduction in potatoes.

Knock-down of amh transcription by antisense RNA reduces FSH and increases follicular atresia in female Oreochromis niloticus

Anti-Müllerian hormone (Amh) plays an important role in regulating gonad development in teleosts. However, little is known about the effects of Amh on follicle development. In this study, we transfected the vector containing antisense RNA fragments of the amh gene to produce Nile tilapia, Oreochromis niloticus, with knocked-down Amh function in vivo. The results confirmed that the antisense RNA effectively inhibited amh transcription and Amh protein expression in female tilapia ovarian tissue. At 180 days of age, compared with control fish, female tilapia with knocked-down Amh function showed significantly increased growth and significantly decreased ovary weight and gonadosomatic index (P < 0.05). Female fish in the control group had ruddy-colored external genitalia, eggs extruded from the abdomen when gently squeezed, and most oocytes were developmental stage V. In contrast, the external genitalia of female fish with knocked-down Amh function did not have the ruddy color, no eggs extruded from the abdomen when squeezed, most oocytes were at developmental stages II and III, and considerable follicular atresia was apparent. At 180 days of age, the transcript levels of amhrII, cyp19a1a, foxl2 and sox9b in ovarian tissue, and the titers of luteinizing hormone, follicle stimulating hormone, and estradiol in the serum, were significantly lower in fish with knocked-down Amh function than in control fish (P < 0.05). We concluded that decreased serum hormone levels and an abnormal AMH signal delayed development and caused follicular degeneration in Nile tilapia with knocked-down Amh function. These findings show that antisense RNA is a feasible approach for gene silencing in fish, and represents an accurate and effective strategy to study gene function.

An innovative method of &lt;i&gt;Diaspis echinocacti&lt;/i&gt; Bouche control using DNA insecticide on&lt;i&gt;Opuntia ficus-indica&lt;/i&gt; (L.) Mill. in the Nikitsky Botanical Garden, Crimea

Aim. Investigation of the effectiveness of a DNA insecticide with a high level of environmental friendliness on Diaspis echinocacti Bouche in the greenhouse of the Nikitsky Botanical Garden.Materials and Methods. The object of the study was the insect pest Diaspis echinocacti Bouche. The number of D. echinocacti larvae was identified on segments of Opuntia ficus-indica L. (Mill.) using a Nikon SMZ 745T microscope and computer microphotography. In the experiment, the effectiveness of the DNA insecticide "Cactus-NBG" on D. echinocacti was studied, the preparation "Tanrek VK", VRK, from the class of neonicotinoids being used as a standard insecticide.Results. It was revealed that the treatment with the DNA insecticide "Cactus-NBG" against D. echinocacti larvae had a significant insecticidal effect. The biological effectiveness of the preparation was 82.0%. The mortality of larvae after treatment significantly increased in comparison with the control (p&lt;0.05) and was measured at 43.2±5.0%, 53.2±2.3%, and 84.2±2.2% on the 3rd, 7th, and 14th day after treatment respectively.Conclusion. As a result of treatment of O. ficus-indica against D. echinocacti with the contact DNA insecticide "Cactus-NBG", a significant insecticidal effect was found. On the 14th day after treatment, the mortality of D. echinocacti in "Cactus-NBG" was 84.2±2.2%, in the "Tanrek" group - 86.0±1.4% and in the control group treated with water -11.2±1.2%. The biological effectiveness of "Cactus-NBG" on the 14th day was 82.0%. Thus, the preparation "Cactus-NBG", based on the antisense fragment of the D. echinocacti genome, caused a significant mortality of the target insect pest and can compete with modern chemical preparations.

Knock-down of glucose transporter and sucrose non-fermenting gene in the hemibiotrophic fungus Colletotrichum falcatum causing sugarcane red rot.

Red rot caused by Colletotrichum falcatum, is one of the economically important disease of sugarcane and breeding for resistant varieties is considered to be the major solution to manage the disease. However, breakdown of red rot resistance become usual phenomenon due to development of newer races by culture adaptation on newly released varieties. Hence it is needed to characterize the genes responsible for pathogen virulence in order to take care of host resistance or to manage the disease by other methods. The transcript studies gave foundation to characterize the huge number of pathogenicity determinants and their role in pathogenesis. Here we studied role of two important genes viz., Glucose Transporter (GT) and Sucrose Non-Fermenting1 (SNF1) during pathogenesis of C. falcatum, which said to be involved in carbon source metabolism. Sugar metabolism has a vital role in disease progression of C. falcatum by regulating their cell growth, metabolism and development of the pathogen during various stages of infection. The present study was aimed to find out the role of GT and SNF1 genes in response to pathogenicity by RNA silencing (RNAi) approach. Knock-down of the target pathogenicity gene homologs in standard C. falcatum isolate Cf671 was carried out by amplifying sense and antisense fragments of targets individually using pSilent-1 vector. The expression cassette was cloned into the binary vector pCAMBIA1300 followed by fungal transformation through Agarobacterium mediated transformation. Resulted mutants of both the genes showed less virulence compared to wild type isolate. Simultaneously, both the mutants did not produce spores. Moreover, the molecular confirmation of the mutants displayed the expression of hygromycin gene with reduced expression of the target gene during host-pathogen interaction. Knockdown of the pathogenicity related genes (GT and SNF1) by RNAi approach corroborate the possible role of the genes in causing the disease.

Fragmentation and Matching of Human MicroRNA Sequences in 3'utr.

Definition of sense and antisense microRNA matches in 3'utr. Matches of mature microRNAs (m-miRs) in human 3'utr could be traced to mutations producing fragments of original m-miR sequences without physical separation. (The m-miR matches in 5'utr and cds should be by far fewer, but could follow similar patterns). To ascertain if the sense and antisense m-miR fragments in 3'utr occur at similar or different levels. Frequency of sense and antisense m-miR matches in 3'utr was examined in the range of 7-22 nucleotides. The fragmentation occurs at gene level by mutation within one of the paired m-miRs, which upon transcription results in increased interactive capability for both former pre-micro (premir) RNA stem partners. The non-mutated stem partner can persist in 3'utr sequences, as is apparent from significant presence of miR-619-5p and miR-5096 and some conservation of 20 other simian- specific m-miR sequences. However, most of m-mir sequences in 3'utr are extensively fragmented, with low preservation of long matches. In flanks of individual m-miR embeds the mutated pre-mir positions are to a degree defined specifically. The m-mir matches of various sizes in 3'utr apparently reflect accumulation, on a phylogenetic time scale, of in-sequence point mutations. Across human 3'utr this fragmentation is significantly less for evolutionarily recent human m-miRs that originate in simians compared to human m-miRs first appearing in lower primates, and especially to human m-miRs introduced in nonprimates.

History and main directions of research on plant protection in the Nikitsky Botanical Gardens

The article provides information on the history of the creation and development of the Department of plant protection in the Nikitsky Botanical Gardens and the results of modern theoretical and applied research on the protection of native and introduced plants for 2000-2020. The main concepts, directions and methods in the development of integrated plant protection systems for fruit and park plantations are considered. There are five stages in solving the main task of plant protection-the rational management of the phytosanitary state of agrobiocenoses: 1. use of agrotechnical methods of control, including mechanical destruction of phytophages; 2. chemical method of plant protection using polytoxic broad-spectrum pesticides; 3. integrated control in the form of the concept of a reduced treatment scheme, taking into account the economic thresholds of abundance and harmfulness and combining all existing methods of control; 4. managing the growth of populations of harmful organisms using biologically active substances of the information type according to integrated indicators; 5. development of a post-genomic approach to limiting the number of phytophages, based on the use of fragments of natural polymers of nucleic acids, development of contact DNA insecticides based on short antisense fragments of genes, as well as preparations based on double-stranded RNA fragments. Modern research has a high theoretical and applied significance for the development of integrated protection systems for fruit, ornamental, and forest crops in the Russian Federation and Crimea. It allows us to objectively assess the phytosanitary state of the garden and park agrobiocenosis, identify the dominant types of pathogens and pests, predict their development trends, and select environmentally safe, cost-effective methods for controlling their numbers.

Adaption of an Episomal Antisense Silencing Approach for Investigation of the Phenotype Switch of Staphylococcus aureus Small-Colony Variants.

Staphylococcus aureus small-colony variants (SCVs) are associated with chronic, persistent, and relapsing courses of infection and are characterized by slow growth combined with other phenotypic and molecular traits. Although certain mechanisms have been described, the genetic basis of clinical SCVs remains often unknown. Hence, we adapted an episomal tool for rapid identification and investigation of putative SCV phenotype-associated genes via antisense gene silencing based on previously described Tnl0-encoded tet-regulatory elements. Targeting the SCV phenotype-inducing enoyl-acyl-carrier-protein reductase gene (fabI), plasmid pSN1-AS‘fabI’ was generated leading to antisense silencing, which was proven by pronounced growth retardation in liquid cultures, phenotype switch on solid medium, and 200-fold increase of antisense ‘fabI’ expression. A crucial role of TetR repression in effective regulation of the system was demonstrated. Based on the use of anhydrotetracycline as effector, an easy-to-handle one-plasmid setup was set that may be applicable to different S. aureus backgrounds and cell culture studies. However, selection of the appropriate antisense fragment of the target gene remains a critical factor for effectiveness of silencing. This inducible gene expression system may help to identify SCV phenotype-inducing genes, which is prerequisite for the development of new antistaphylococcal agents and future alternative strategies to improve treatment of therapy-refractory SCV-related infections by iatrogenically induced phenotypic switch. Moreover, it can be used as controllable phenotype switcher to examine important aspects of SCV biology in cell culture as well as in vivo.

Functional characterization of poplar NAC13 gene in salt tolerance

Transcription factor (TF) genes play a critical role in plant abiotic and biotic stress responses. In this study, we cloned a poplar TF NAC13 gene (Potri.001G404100.1), which is significantly up-regulated to salt stress. Then we developed gene overexpression and antisense suppression constructions driven by CaMV35S, and successfully transferred them to a poplar variety 84 K (Populus alba × P. glandulosa), respectively. Evidence from molecular assay indicated that NAC13 overexpression and antisense suppression fragments have been integrated into the poplar genome. The morphological and physiological characterization and salt treatment results indicated the NAC13-overexpressing transgenic plants enhance salt tolerance significantly, compared to wide type. In contrast, the NAC13-suppressing transgenic plants are significantly sensitive to salt stress, compared to wide type. Evidence from transgenic Arabidopsis expressing GUS gene indicated that the gene driven by NAC13 promoter is mainly expressed in the roots and leaves of young plants. These studies indicate that the NAC13 gene plays a vital role in salt stress response.

RNA-mediated silencing of PKS1 gene in Colletotrichum falcatum causing red rot in sugarcane

Red rot is one of the major threatening diseases of sugarcane. Due to frequent breakdown of resistance by simultaneous evolution of pathogen variants with the introduction of newer varieties red rot remains as a challenge for successful breeding programme in India. Earlier studies established that the pathogen has limited genotypic variation with enormous phenotypic variation, which could be mainly due to adaptability of pathogen to the newly released varieties and expression of pathogenicity factors including secondary metabolites viz., toxins, enzymes and pigments like melanin. It is also proved that a positive correlation remains between the production of melanin and disease expression and hence the melanin biosynthesizing genes viz., Polyketide synthase1 (PKS1), Scytalone dehydratase1 (SCD1) and Trihydroxynapthalene reductase1 (THR1) were characterized. In this study, we have utilized RNA interference (RNAi) approach using pSilent-1 vector which facilitates the generation of hair pin constructs that suppress the expression of target gene through Agrobacterium mediated transformation (ATMT) to functionally analyze the major gene PKS1 involved in the production of dihydroxy naphthalene (DHN) melanin to determine its role in virulence in C. falcatum. Functionally active sites of the PKS1 gene i.e., acyl transferase (AT) domain and the region responsible for conidial pigment (CP) polyketide synthase were chosen for knockdown approach. The above said regions were amplified in the virulent C. falcatum isolate (Cf671) and both the sense and antisense fragments were separately cloned into pSilent-1 vector. The expression cassette was then cloned into the binary vector and introduced into Agrobacterium tumefaciens LBA4404 which was later co-cultivated with the fungal spore suspension. Results indicated that the knockdown AT mutant failed to produce spores whereas the CP mutants produced spores as that of the parental strains. Further pathogenicity testing revealed that the disease symptoms of both AT and CP mutants were restricted within the inoculated internode until 15th and 5th dpi respectively while the symptoms of wild type were prominent and crossed beyond 3rd internode at 15 dpi during the host-pathogen interaction. Molecular confirmation of the knockdown mutants with the expression of hygromycin gene and absence of functional domains under RT-PCR clearly indicated that the PKS1 gene has a possible role in C. falcatum pathogenesis. However expression analysis by qRT-PCR indicated regaining of virulence in mutants during later part of host pathogen interaction. The results confirm the applicability of RNAi mediated silencing for functional analysis of pathogenicity gene homologs in C. falcatum.

LncRNA ZFAS1 as a SERCA2a Inhibitor to Cause Intracellular Ca2+ Overload and Contractile Dysfunction in a Mouse Model of Myocardial Infarction.

Ca2+ homeostasis-a critical determinant of cardiac contractile function-is critically regulated by SERCA2a (sarcoplasmic reticulum Ca2+-ATPase 2a). Our previous study has identified ZFAS1 as a new lncRNA biomarker of acute myocardial infarction (MI). To evaluate the effects of ZFAS1 on SERCA2a and the associated Ca2+ homeostasis and cardiac contractile function in the setting of MI. ZFAS1 expression was robustly increased in cytoplasm and sarcoplasmic reticulum in a mouse model of MI and a cellular model of hypoxia. Knockdown of endogenous ZFAS1 by virus-mediated silencing shRNA partially abrogated the ischemia-induced contractile dysfunction. Overexpression of ZFAS1 in otherwise normal mice created similar impairment of cardiac function as that observed in MI mice. Moreover, at the cellular level, ZFAS1 overexpression weakened the contractility of cardiac muscles. At the subcellular level, ZFAS1 deleteriously altered the Ca2+ transient leading to intracellular Ca2+ overload in cardiomyocytes. At the molecular level, ZFAS1 was found to directly bind SERCA2a protein and to limit its activity, as well as to repress its expression. The effects of ZFAS1 were readily reversible on knockdown of this lncRNA. Notably, a sequence domain of ZFAS1 gene that is conserved across species mimicked the effects of the full-length ZFAS1. Mutation of this domain or application of an antisense fragment to this conserved region efficiently canceled out the deleterious actions of ZFAS1. ZFAS1 had no significant effects on other Ca2+-handling regulatory proteins. ZFAS1 is an endogenous SERCA2a inhibitor, acting by binding to SERCA2a protein to limit its intracellular level and inhibit its activity, and a contributor to the impairment of cardiac contractile function in MI. Therefore, anti-ZFAS1 might be considered as a new therapeutic strategy for preserving SERCA2a activity and cardiac function under pathological conditions of the heart.

A small molecule for a big transformation: Topical application of a 20-nucleotide-long antisense fragment of the DIAP-2 gene inhibits the development of Drosophila melanogaster female imagos

Several genes have been identified to play important roles associated with sex selection in Drosophila melanogaster . An essential part is attributed to the sex-lethal gene that depends on the expression of the X:A (number of chromosomes to autosomes) ratio signal controlling both sex selection and dosage compensation processes in D. melanogaster . Interestingly, for sex selection in D. melanogaster there are no documented data addressing the role of the inhibitor of apoptosis (IAP) genes and their signaling influence on this biological process. In this study, we found that topical application of a 20-nucleotide-long antisense DNA fragment (oligoDIAP-2) from the death-associated inhibitor of apoptosis (DIAP)-2 gene interferes with D. melanogaster development and significantly decreases the number of female imagos and their biomass. We show that the applied antisense oligoDIAP-2 fragment downregulates the target DIAP-2 gene whose normal concentration is necessary for the development of female D. melanogaster . These data correspond to the results on downregulation of the target host IAP-Z gene of Lymantria dispar L. female imagos after topical treatment with an 18-nucleotide-long antisense DNA fragment from the Lymantria dispar multicapsid nuclear polyhedrosis virus IAP-3 gene at the larval stage. The observed novel phenomenon linking the downregulation of insect IAP genes and the low rate of female imago development could have practical application, especially in insect pest control and molecular pathology. https://doi.org/10.2298/ABS170302023N Received: March 2, 2017; Revised: April 29, 2017; Accepted: May 17, 2017; Published online: July 19, 2017 How to cite this article: Nyadar PM, Oberemok VV, Zubarev IV. A small molecule for a big transformation: Topical application of a 20-nucleotide-long antisense fragment of the DIAP-2 gene inhibits the development of Drosophila melanogaster female imagos. Arch Biol Sci. 2018;70(1):33-9.

Search for oligonucleotides selectively binding oncogenic miR-21

In the pathogenesis of malignancies, an active regulatory role belongs to small noncoding RNAs, miRNA (miR). miRNA expression profiles are often associated with the prognosis and therapeutic outcome of different oncological diseases. It is well known that in comparison with normal tissues cancer cells are characterized by hyperexpression of oncogenic miRNAs which leads to oncogenic transformation, carcinogenesis and metastasis progression. From this point of view, selective down-regulation of miRNA expression by specific agents, such as antisense oligonucleotides that recognize particular sequences, therefore, can be an effective tool to regulate the amount of miRNA in cancer cells and decrease tumor malignancy. In this paper, we have designed a series of antisense oligonucleotides addressed to the oncogenic miR-21 with a view to its selective binding and studied patterns of interaction of miR-21 with these oligonucleotides in vitro. The series included linear and hairpin oligonucleotides with the length of antisense fragment of 10–16 nucleotides (nt) complementary to the 5'- or the 3'-end of miRNA target. Hairpin oligonucleotides consist of a sequence complementary to miR-21 and a hairpin containing a four-nucleotide loop and stem of 6–9 bp necessary for stabilizing the complex with miR-21. It has been shown that inclusion of the hairpin with the stem of 6 bp to the oligonucleotide structure leads to a 1.6-fold increase in binding efficiency with miR-21 in comparison with a linear oligonucleotide and elongation of the stem from six to nine bp does not increase binding efficiency. Hairpin oligonucleotides with an antisense sequence of 14 nt effectively hybridize with miR-21 and are not inferior to 16-mer linear and hairpin oligonucleotides in the efficiency of complex formation. Thus, we have shown that hairpin oligonucleotides with antisense fragment of 14 nt and a hairpin, including the stem of 6 bp, are optimal for selective and effective sequestering of mature miR-21.

Original Article. Topical treatment of LdMNPV-infected gypsy moth caterpillars with 18 nucleotides long antisense fragment from LdMNPV IAP3 gene triggers higher levels of apoptosis in infected cells and mortality of the pest

Abstract The high efficiency of baculovirus infection is partially explained by the ability of the virus to suppress host defense machinery connected with the apoptosis pathway. Members of the baculovirus gene family, inhibitors of apoptosis (vIAPs), have been shown to inhibit apoptosis in baculovirus-infected cells. Here we showed that treatment of the LdMNPV-infected 1st instar gypsy moth (Lymantria dispar) caterpillars with sense (oligoBIR) and antisense (oligoRING) DNA oligonucleotides from the LdMNPV IAP3 gene induced elevated mortality of the insects. Apoptotic DNA ladder assay showed that the leading role in this phenomenon is played by the antisense oligoRING fragment of the vIAP3 gene. These results imply that the application of both antisense DNA oligonucleotides from vIAP genes and baculovirus preparations (one following the other) may be a potential method for plant protection against insect pests.

The RING for gypsy moth control: Topical application of fragment of its nuclear polyhedrosis virus anti-apoptosis gene as insecticide

Numerous studies suggest a cellular origin for the Lymantria dispar multicapsid nuclear polyhedrosis virus (LdMNPV) anti-apoptosis genes IAPs, thus opening a possibility to use the fragments of these genes for modulation of host metabolism. We report here the strong insecticidal and metabolic effect of single-stranded antisense DNA fragment from RING (really interesting new gene) domain of gypsy moth LdMNPV IAP-3 gene: specifically, on reduction of biomass (by 35%) and survival of L. dispar caterpillars. The treatment with this DNA fragment leads to a significantly higher mortality rates of female insects (1.7 fold) accompanied with the signs of apoptosis. Additionally, we show increased expression of host IAP-1, caspase-4 and gelsolin genes in eggs laid by survived females treated with RING DNA fragment accompanied with calcium and magnesium imbalance, indicating that the strong stress reactions and metabolic effects are not confined to treated insects but likely led to apoptosis in eggs too. The proposed new approach for insect pest management, which can be considered as advancement of “microbial pesticides”, is based on the application of the specific virus DNA, exploiting the knowledge about virus–pest interactions and putting it to the benefit of mankind.

Effect of overexpression of HOX genes on its invasive tendency in cerebral glioma.

Transcription factors encoded by HOX genes are vital in the determination of cell fate and identity during embryonic development. In certain malignancies, HOX genes also behave as oncogenes. The present study demonstrated suppression of the invasive tendency of glioblastoma multiforme U-118 and U-138 cells by the introduction of the antisense fragments of HOXA6 and B13 genes using electroporation. The invasion index indicated 79 and 72% reductions in the invasive ability of antisense HOXA6 and B13, respectively. No significant differences in the invasive index of the parental and mock cells of each HOX gene were observed (invasive index, 0.75-0.91; P=0.05). A reduction in invasion tendency was also observed following betulinic acid (BA) treatment: The results from the matrigel assay analysis clearly demonstrated a significant inhibition in the invasive behaviour of U-118 and U-138 cell lines from day 15 following BA treatment, with a maximum effect on day 30. The invasion index demonstrated 62 and 65% reductions in invasion ability in the U-118 and U-138 cell lines, respectively. The suppression of HOXC6 and B13 expression by the introduction of the corresponding antisense fragments in addition to BA reduced invasion tendency in U-118 and U-138 cell lines. The mechanism underlying the association between the HOX gene and invasive behavior in glioma cells is yet to be understood. However, the anti-invasive behavior of BA may aid understanding of the mechanism in future studies.

Untranslatable tospoviral NSs fragment coupled with L conserved region enhances transgenic resistance against the homologous virus and a serologically unrelated tospovirus.

Tospoviruses cause severe damages to important crops worldwide. In this study, Nicotiana benthamiana transgenic lines carrying individual untranslatable constructs comprised of the conserved region of the L gene (denoted as L), the 5' half of NSs coding sequence (NSs) or the antisense fragment of whole N coding sequence (N) of Watermelon silver mottle virus (WSMoV), individually or in combination, were generated. A total of 15-17 transgenic N. benthamiana lines carrying individual transgenes were evaluated against WSMoV and the serologically unrelated Tomato spotted wilt virus (TSWV). Among lines carrying single or chimeric transgenes, the level of resistance ranged from susceptible to completely resistant against WSMoV. From the lines carrying individual transgenes and highly resistant to WSMoV (56-63% of lines assayed), 30% of the L lines (3/10 lines assayed) and 11% of NSs lines (1/9 lines assayed) were highly resistant against TSWV. The chimeric transgenes provided higher degrees of resistance against WSMoV (80-88%), and the NSs fragment showed an additive effect to enhance the resistance to TSWV. Particularly, the chimeric transgenes with the triple combination of fragments, namely L/NSs/N or HpL/NSs/N (a hairpin construct), provided a higher degree of resistance (both 50%, with 7/14 lines assayed) against TSWV. Our results indicate that the untranslatable NSs fragment is able to enhance the transgenic resistance conferred by the L conserved region. The better performance of L/NSs/N and HpL/NSs/N in transgenic N. benthamiana lines suggests their potential usefulness in generating high levels of enhanced transgenic resistance against serologically unrelated tospoviruses in agronomic crops.

Antiviral effect of hepatitis B virus S/C gene loci antisense locked nucleic acid on transgenic mice in vivo.

We investigated the effects of hepatitis B virus (HBV) S/C double gene loci antisense locked nucleic acid on replication and expression of HBV in hepatitis transgenic mice. HBV mice (N = 30) were randomly divided into five groups of six mice: 5% glucose solution control, empty liposome control, single-target S, single-target C, and dual-target SC groups. An antisense locked nucleic acid fragment was injected into the mice. Serum HBsAg, serum HBV DNA, HBV C-mRNA expression in liver tissue, HbsAg and HbcAg expression in hepatocytes, serum albumin, alanine transaminase (ALT), urea nitrogen, and creatinine were detected. Liver and kidney sections were examined for the effects of antisense locked nucleic acid. The expression of HBsAg was markedly inhibited; the inhibition rates of the S, C, and SC target groups were 36.63, 31.50, and 54.87%, respectively; the replication of HBV DNA was also inhibited: 23.97, 21.13, and 35.83%, respectively. After injection at 1, 3, and 5 days, the corresponding rates for HBsAg inhibition were 14.40, 25.61, and 31.33%, and for HBV DNA inhibition they were 11.04, 19.24, and 24.13%. Compared with the control group, the differences in serum albumin, ALT, urea nitrogen, and creatinine in each group were not statistically significant, and the number of HbsAg- and HBcAg-positive cells in the mouse liver was significantly reduced. The liver and kidney tissues were normal. The gene therapy had significant inhibitory effects on the replication and expression of HBV in transgenic mice, and double-gene targeting was better than single-gene targeting.

RNA silencing of the anionic peroxidase gene impairs potato plant resistance to Phytophthora infestans (Mont.) de Bary

Potato Solanum tuberosum L. plants expressing an antisense M21334 fragment were obtained by agrobacterial transformation. A manifold decrease in activity of anionic isoperoxidase with pI ∼ 3.5 in the transformed plants demonstrated that the enzyme is encoded by M21334. The transformed plants showed a decrease in lignin accumulation and a dramatically lower resistance to the late blight agent Phytophthora infestans, implicating the enzyme in the response to P. infestans infection.

Downregulation of Glucan Synthase-Like (TaGSL) genes in wheat leads to inhibition of transgenic plant regeneration

RNA interference (RNAi) cassettes for gene silencing in plants consist of an inverted repeat sequence of the targeted gene and a spacer region separating sense and antisense fragments. The sequences are generally placed under the control of a strong promoter and terminator and produce hairpin RNA structures that generate small interfering RNAs (siRNAs). These siRNAs destroy the mRNA of the matching gene in the cytoplasm. The number and efficiency of siRNAs depend upon the sequence length of inverted repeats and the region in the gene. In the present study, our objective was to downregulate three members of the Glucan Synthase-Like family in wheat. Initially, a 230-nucleotide fragment was cloned under the maize ubiquitin-1 promoter and nos terminator in both sense and antisense directions, and separated by a spacer including the Escherichia coli uidA (GUS) gene. RNAi constructs were prepared for TaGSL8 and TaGSL10. Biolistic delivery of RNAi constructs for TaGSL8 and TaGSL10 into immature zygotic embryos (IZEs) of wheat did not yield any transgenic plants. The experiment was repeated and comparisons were made with a control bar gene construct. IZEs bombarded with a bar selection cassette alone generated healthy calli on selection medium and transgenic plants were recovered. IZEs co-bombarded with the combination of RNAi and bar gene constructs produced only unhealthy calli when cultured on selection medium and no transgenic plants were recovered. This indicated that the RNAi constructs with 230-bp long inverted repeats of TaGSL8 or TaGSL10 were inhibiting regeneration. In the second experiment, the length of the inverted repeats was reduced to 150 and 122 nucleotides for TaGSL3 and TaGSL8, respectively. Two independent transgenic plants were recovered for each of the TaGSL3 and TaGSL8 target genes. These transgenic plants showed transcription of the introduced RNAi constructs, and the transcript levels of the corresponding endogenous genes were reduced. RNAi transgenic lines of TaGSL3 and TaGSL8 showed slightly reduced resistance against Fusarium graminearum compared to nontransgenic control plants.