Functional Analysis of a Viral Promoter From the Strawberry Vein Banding Virus (SVBV) Chinese Isolate

BackgroundPromoter is an important factor during gene expression in cells. In this study, we cloned a full-length promoter from the strawberry vein banding virus (SVBV) Chinese isolate and produced several its deletion mutants.MethodsThe full-length promoter of SVBV (SP1) and its three deletion mutants (SP2, SP3, and SP4) were amplified using polymerase chain reaction (PCR). The expression activities controlled by the SVBV SP1, SP2, SP3, and SP4 were evaluated using β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes.ResultsOur transient expression assays showed that the SVBV SP1 promoter as well as its three deletion mutants all expressed the reporter genes, but to very different levels. Interestingly, the expression activity driven by the SP1 promoter was much higher than that shown by the CaMV 35S promoter. After stable transformation of a GUS gene into Nicotiana tabacum plants, the transgene expression level driven by the SVBV SP1 promoter was about 2.6-fold greater than that driven by the CaMV 35S promoter. In addition, the GUS gene expression levels could be enhanced by co-infiltrating the plants with the SP1 promoter-driven vector carrying the GUS gene and the vector expressing the SVBV ORF V or ORF VI.ConclusionsThe SVBV Chinese isolate promoter SP1 is a stronger promoter than the CaMV 35S and FLt-US promoter, may be more useful for production of stable transgenic plants.

Targeted Transgene Expression in Rice Using a Callus Strong Promoter for Selectable Marker Gene Control

Precise expression of a transgene in the desired manner is important for plant genetic engineering and gene function deciphering, but it is a challenge to obtain specific transgene expression free from the interference of the constitutive promoters used to express the selectable marker gene, such as the Cauliflower mosaic virus (CaMV) 35S promoter. So, the solutions to avoid these inappropriate regulations are largely demanded. In this study, we report the characterization of a callus strong promoter (CSP1) in rice and its application for accurate transgene expression. Our results indicate that the high expression of the CSP1 promoter in the callus enables efficient selection of hygromycin equivalent to that provided by the CaMV 35S promoter, whereas its expression in other tissues is low. To evaluate possible leaky effects, the expression of a β-glucuronidase reporter driven by six specific promoters involving hormone signaling, pathogen response, cell fate determination, and proliferation was observed in transgenic rice plants generated by CSP1-mediated selection. Distinct β-glucuronidase expression was found consistently in most of the transgenic lines obtained for each promoter. In addition, we applied these specific marker lines to investigate the root cellular responses to exogenous cytokinin and auxin treatment. The results reveal that the root growth inhibition by cytokinin was differently regulated at high and low concentrations. In summary, we have established the feasibility of using callus-specific promoter-dependent selection to mitigate the transgene misexpression in rice. By enabling efficient transformation, rice plants with reliable transgene expression will be easily acquired for broad applications.

Evaluation of Plant-Derived Promoters for Constitutive and Tissue-Specific Gene Expression in Potato

Various plant-derived promoters can be used to regulate ectopic gene expression in potato. In the present study, four promoters derived from the potato genome have been characterized by the expression of identical cassettes carrying the fusion with the reporter β-glucuronidase (gusA) gene. The strengths of StUbi, StGBSS, StPat, and StLhca3 promoters were compared with the conventional constitutive CaMV 35S promoter in various organs (leaves, stems, roots, and tubers) of greenhouse-grown plants. The final amount of gene product was determined at the post-transcriptional level using histochemical analysis, fluorometric measurements, and Western blot analysis. The promoter strength comparison demonstrated that the StUbi promoter generally provided a higher level of constitutive β-glucuronidase accumulation than the viral CaMV 35S promoter. Although the StLhca3 promoter was predominantly expressed in a green tissue-specific manner (leaves and stems) while StGBSS and StPat mainly provided tuber-specific activity, a “promoter leakage” was also found. However, the degree of unspecific activity depended on the particular transgenic line and tissue. According to fluorometric data, the functional activity of promoters in leaves could be arranged as follows: StLhca3 > StUbi > CaMV 35S > StPat > StGBSS (from highest to lowest). In tubers, the higher expression was detected in transgenic plants expressing StPat-gusA fusion construct, and the strength order was as follows: StPat > StGBSS > StUbi > CaMV 35S > StLhca3. The observed differences between expression patterns are discussed considering the benefits and limitations for the usage of each promoter to regulate the expression of genes in a particular potato tissue.

A simple and accurate PCR method for detection of genetically modified rice

Background Legislation regulating for labeling and use of genetically modified (GM) crops are increased considerably worldwide in order to health and safety assurance of consumers. For this purpose, a polymerase chain reaction (PCR) method has been developed for detection of GM rice in people’s food diet. Methods In this study, eighty-one non-labeled rice samples were collected randomly from different market sites of Tehran, Iran. In order to analysis, rice genomic DNA was extracted using MBST DNA extraction kit and subsequently, sucrose phosphate synthase (SPS) gene was used to confirm the quality of extracted DNA. Then, cauliflower mosaic virus (CaMV) 35S promoter and Agrobacterium nopaline synthase (NOS) terminator were selected as screening targets for detection of GM rice sequences by PCR. Results According to our results, 2 out of 81 (2.4%) samples tested were positive for CaMV 35S promoter while no positive result was detected for NOS terminator. Conclusion The obtained data indicated that this method is capable to identify the GM rice varieties. Furthermore, it can demonstrate the possibility of the presence of GM rice in Tehran’s market, thus putting emphasis on the requirement for developing a precise approach to evaluate this product.

A short history of the CaMV 35S promoter

In an organism, be it plant, animal or human, almost every gene has its own promoter sequence, which is typified as a DNA stretch that controls how a gene is expressed in a cell. Hence, the activity of a promoter controls in which cell type, during which developmental stage or during what environmental condition a certain gene is expressed. However, the most widely used promoter in plant biotechnology is actually not derived from a plant, but a pathogenic virus. How and why did that happen? Here's a short history of the CaMV 35S promoter.

A short history of the CaMV 35S promoter

In an organism, be it plant, animal or human, almost every gene has its own promoter sequence, which is typified as a DNA stretch that controls how a gene is expressed in a cell. Hence, the activity of a promoter controls in which cell type, during which developmental stage or during what environmental condition a certain gene is expressed. However, the most widely used promoter in plant biotechnology is actually not derived from a plant, but a pathogenic virus. How and why did that happen? Here's a short history of the CaMV 35S promoter.

Construction and Transient Expression of Chimeric Cassettes Containing CaMV 35S or OsAER1 Promoter and GUS Gene Fusion in Tobacco

<p>Reporter gene assays are commonly used to study the expression pattern of a gene and the promoter activity. The purpose of this study was to assemble the chimeric gene constructs consisting of CaMV 35S promoter orOsAER1 gene promoter connected to the β-glucuronidase (GUS) reporter gene encoding the β-glucuronidase enzyme and to obtain an efficient method for Agrobacterium tumefaciens-mediated transient transformation of tobacco sprouts. The CaMV 35S promoter fragment reamplified from pCAMBIA1301 binary vector and the OsAER1 gene promoter fragment amplified from rice cv. Awan Kuning were ligated into pCAMBIA1300int::gus::tNOS to produce binary vectors pCAMBIA1300int::p35S::gus::tNOS and pCAMBIA1300int::prOsAER1::gus::tNOS. The vectors were used for transient transformation of 5–day old tobacco seedlings. The transformation was carried out using two bacterial cultures with densities of OD600 = 0.5 or OD600 = 1.0 combined with a vacuum for 15 or 30 minutes. Tobacco seedlings transformed with pCAMBIA1300int::p35S::gus::tNOS showed higher transformation efficiency as compared tothe ones transformed with pCAMBIA1300int::prOsAER1::gus::tNOS. A higher efficiency was obtained from transformation using bacterial culture with density of OD600 = 0.5 in combination with a vacuum for 30 minutes. Expression of GUS gene in the tobacco sprouts transformed with CaMV 35S promoter construct was observed through out the sprouts area (root, hypocotyl, cotyledon, and leaf), where as expression of GUS gene was observed in root, hypocotyl, and cotyledon, but not in leaf on tobacco sprouts transformed with OsAER1 promoter construct. These results indicate that the transient transformation is a quick and simple method for testing a chimeric gene construct.</p>