ABSTRACT
Post-transcriptional gene silencing (PTGS)-mediated gene silencing exploits the cellular mechanism whereintranscripts having sequence similarity to the double-stranded RNA (dsRNA) molecules present in the cell will besubjected to degradation. PTGS is closely related to natural processes such as RNA-mediated virus resistance andcross-protection in plants. Gene silencing and the cellular machinery for affecting this phenomenon might haveevolved as a natural protective measure against viral infection in plants. In PTGS, small interfering RNA (siRNA)molecules of 21–23 nucleotides length act as homology guides for triggering the systemic degradation of transcriptshomologous to the siRNA molecules. PTGS phenomenon, first discovered in transgenic petunia plants harbouringchalcone synthase gene and termed co-suppression, has been subsequently exploited to target specific gene transcripts for degradation leading to manifestation of desirable traits in crop plants. Targeted gene silencing has beenachieved either through the introduction of DNA constructs encoding dsRNA or antisense RNA or by deploying cosuppression constructs producing siRNAs against the transcript of interest. Understanding the mechanism of genesilencing has led to the development of several alternative strategies for inducing gene silencing in a precise andcontrolled way. This has paved the way for using PTGS as one ofthe chief functional genomicstools in plants and hashelped in unraveling the mechanism of many cellular processes and identifying the focal points in pathways, besides,opening new vistas in genetic engineering of plants for human benefits. PTGS has shown great potential in silencingthe deleterious genes efficiently so that value-added plant products could be obtained. Thus, PTGS has ushered in anew era in the genetic manipulation of plants for both applied and basic studies. In this review, we have outlined thebasics of RNAi-mediated gene silencing and summarized the work carried out at our institute using this approach, ascase studies. In particular, adopting RNAi-mediated gene silencing (a) as a method to restore fertility in transgenicmale sterile lines developed based on orfH522 gene from sunflower PET1-CMS source, (b) as a tool to suppress theproduction of toxic proteins, ricin and RCA, in castor, and (c) as an approach to induce bud necrosis virus resistancein sunflower has been discussed. Examples from other plant systems also have been mentioned to exemplify theconcept and utility of gene silencing in crop plants.
ABSTRACT
Flower development is a complex procedure regulated by combinatorial factors, such as transcription factors,peptides, hormones, and small RNAs. One of the important gene determining the floral structure and floral meristem isAPETALA2 (AP2) which belongs to a large family of transcription factors. AP2 contributes stochastically in signalingpathway in flower development and in various bioactive components synthesis. The presence of GbAP2 transcriptsin live fossil Ginkgo biloba leaves and female strobili tissue showed that GbAP2 might be involved directly in leafand female strobili development, whereas it may possible that GbAP2 indirectly involved in synthesis of bioactivecompounds such as flavonoids, terpenoids, ginkgolides, and organic acids. Gingko or Ginkgo biloba is among themost popular plant used in United States. Bioactive compounds isolated from the ginkgo plant are thought to exhibitas antioxidant and antiplatelet activity. Due to the pleotropic nature of AP2, it is involved in various tissues such asregulating in floral pattern, stem cell maintenance, floral organ identity, floral meristem, leaves, development of stems,and seed development. AP2 also regulate number of downstream genes but its own expression is negatively regulatedat translational or post-translational levels by miRNA172 which is a small RNA (22 bp) and binds to complementaryregion of AP2 transcript. Mutation in AP2 showed increases in seed size and seed mass, this property of AP2 could beused in medicinal plant to enhance the valuable product. Since AP2 is engaged in various pathways it is essential tocompile the functioning in the form of presented manuscript, which discusses the structure and functioning of AP2.We likewise explain how AP2 involved in various expressions and its regulatory mechanism, especially in the plant.
ABSTRACT
With the understanding of the function mechanism of RNAi,it has been widely applied in various fields.RNAi is a powerful tool to study gene functions and provides a new technical method for the specific gene treatment as well.The further development of RNAi technique opens up a new way for tumor gene treatment.
ABSTRACT
Los microARNs son ARN pequeños de aproximadamente 22 nucleótidos de longitud que participan en la regulación de muchos procesos celulares y, su alteración está asociada con el desarrollo de diferentes patologías, especialmente cáncer. Gracias al uso de las herramientas de bioinformática es posible determinar su distribución en el genoma y sus funciones en diferentes tejidos. La mayoría de microARNs son producidos en una vía canónica a partir de un transcripto primario largo, en un proceso secuencial de dos reacciones guiadas principalmente por dos enzimas, Drosha en el núcleo y Dicer en el citoplasma. Sin embargo, actualmente han sido descritas algunas vías no canónicas de generación de microARNs. El objetivo de esta revisión es describir el proceso de generación de microARNs y la maquinaria involucrada en su generación con el propósito de lograr alcanzar un mejor entendimiento de los diferentes procesos en los cuales están involucrados. Salud UIS 2011; 43 (3): 289-297.
MicroRNAs are small RNAs of approximately 22 nucleotides in length that participate in the regulation of many cellular processes and it alteration is associated with the development of different pathologies in particular, cancer. Using bioinformatics tools is possible determine their wide distribution in the genome and their functions in different tissues. Mostly of microRNAs are produced in a canonical way from a long primary transcript, in a sequential process of two reactions guided mostly by two enzymes: Drosha in the nucleus and Dicer in the cytoplasm. However, it has been described some pathways non-canonical for the generation of microRNAs. The aim of this review is to describe the generation process of microRNAs and the machinery involved in their generation for the purpose of achieving a better understanding of the different processes they are involved. Salud UIS 2011; 43 (3): 289-297.
Subject(s)
Transcription, Genetic , MicroRNAs , RNA, Double-Stranded , RNA Processing, Post-Transcriptional , RNA-Induced Silencing ComplexABSTRACT
MicroRNAs (miRNAs) are a class of small RNAs of 19-23 nucleotides that regulate gene expression through target mRNA degradation or translational gene silencing. The miRNAs are reported to be involved in many biological processes, and the discovery of miRNAs has been provided great impacts on computational biology as well as traditional biology. Most miRNA-associated computational methods comprise the prediction of miRNA genes and their targets, and increasing numbers of computational algorithms and web-based resources are being developed to fulfill the need of scientists performing miRNA research. Here we summarize the rules to predict miRNA targets and introduce some computational algorithms that have been developed for miRNA target prediction and the application of the methods. In addition, the issue of target gene validation in an experimental way will be discussed.
ABSTRACT
Objective:To generate rabbit polyclonal antibody against human Argonaute2 (Ago2) protein and to identify its functional characterization for determination of differential expression and cellular localization of Ago2 protein in various cell lines.Methods:DNAstar software was applied for searching the high antigenicity region of Ago2 gene sequence termed k-Ago2.Prokaryotic expressing plasmid was constructed and transformed to E.coli BL21 (DE3) to induce expression by IPTG.The fusion protein was injected into rabbits subcutaneously to produce polyclonal antibodies after purification by gel regaining.ELISA was operated to detect antibody titer.Western blot was used to identify the specificity and sensitivity of the antibodies and detect the differential expression of Ago2 protein in various cell lines.Meanwhile,immunofluorescence experiments were arranged to show cellular localization of Ago2 protein.Results:The prokaryotic expressing plasmid was constructed correctly.K-Ago2 protein was expressed and purified,and then rabbit polyclonal antibodies against Ago2 were generated after immunization with k-Ago2 protein.The titer detected by ELISA was 1∶19 000.Western blot results demonstrated the high specificity of the antibodies.Finally,we successfully observed the differential expression and cellular localization of Ago2 protein in various cell lines.Conclusion:The polyclonal antibody against Ago2 protein has been achieved successfully.It will be propitious for the intensive study of the RNAi mechanism and even profound clinical application.