A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids

BACKGROUND: Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule. RESULTS: We describe a real-time PCR technique employing attached universal duplex probes (AUDP), which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP) and a complementary quenching probe (QP) lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT) and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO) quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods. CONCLUSION: The results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost.Litao Yang, Wanqi Liang, Lingxi Jiang, Wenquan Li, Wei Cao, Zoe A Wilson, and Dabing Zhan

DWT1/DWL2 act together with OsPIP5K1 to regulate plant uniform growth in rice

•Uniform growth of the main shoot and tillers significantly influences rice plant architecture and grain yield. The WUSCHEL‐related homeobox transcription factor DWT1 is a key regulator of this important agronomic trait, disruption of which causes enhanced main shoot dominance and tiller dwarfism by an unknown mechanism. •Here, we have used yeast‐two‐hybrid screening to identify OsPIP5K1, a member of the rice phosphatidylinositol‐4‐phosphate 5‐kinase family, as a protein that interacts with DWT1. Cytological analyses confirmed that DWT1 induces accumulation of OsPIP5K1 and its product PI(4,5)P2, a phosphoinositide secondary messenger, in nuclear bodies. •Mutation of OsPIP5K1 compounds the dwarf dwt1 phenotype but abolishes the main shoot dominance. Conversely, overexpression of OsPIP5K1 partially rescues dwt1 developmental defects. Furthermore, we showed that DWL2, the homologue of DWT1, is also able to interact with OsPIP5K1 and shares partial functional redundancy with DWT1 in controlling rice uniformity. •Overall, our data suggest that nuclear localised OsPIP5K1 acts with DWT1 and/or DWL2 to coordinate the uniform growth of rice shoots, likely to be through nuclear phosphoinositide signals, and provides insights into the regulation of rice uniformity via a largely unexplored plant nuclear signalling pathway

GMDD: a database of GMO detection methods

<p>Abstract</p> <p>Background</p> <p>Since more than one hundred events of genetically modified organisms (GMOs) have been developed and approved for commercialization in global area, the GMO analysis methods are essential for the enforcement of GMO labelling regulations. Protein and nucleic acid-based detection techniques have been developed and utilized for GMOs identification and quantification. However, the information for harmonization and standardization of GMO analysis methods at global level is needed.</p> <p>Results</p> <p>GMO Detection method Database (GMDD) has collected almost all the previous developed and reported GMOs detection methods, which have been grouped by different strategies (screen-, gene-, construct-, and event-specific), and also provide a user-friendly search service of the detection methods by GMO event name, exogenous gene, or protein information, etc. In this database, users can obtain the sequences of exogenous integration, which will facilitate PCR primers and probes design. Also the information on endogenous genes, certified reference materials, reference molecules, and the validation status of developed methods is included in this database. Furthermore, registered users can also submit new detection methods and sequences to this database, and the newly submitted information will be released soon after being checked.</p> <p>Conclusion</p> <p>GMDD contains comprehensive information of GMO detection methods. The database will make the GMOs analysis much easier.</p

Aborted microspores acts as a master regulator of pollen wall formation in Arabidopsis

Mature pollen is covered by durable cell walls, principally composed of sporopollenin, an evolutionary conserved, highly resilient, but not fully characterized, biopolymer of aliphatic and aromatic components. Here, we report that ABORTED MICROSPORES (AMS) acts as a master regulator coordinating pollen wall development and sporopollenin biosynthesis in Arabidopsis thaliana. Genome-wide coexpression analysis revealed 98 candidate genes with specific expression in the anther and 70 that showed reduced expression in ams. Among these 70 members, we showed that AMS can directly regulate 23 genes implicated in callose dissociation, fatty acids elongation, formation of phenolic compounds, and lipidic transport putatively involved in sporopollenin precursor synthesis. Consistently, ams mutants showed defective microspore release, a lack of sporopollenin deposition, and a dramatic reduction in total phenolic compounds and cutin monomers. The functional importance of the AMS pathway was further demonstrated by the observation of impaired pollen wall architecture in plant lines with reduced expression of several AMS targets: the abundant pollen coat protein extracellular lipases (EXL5 and EXL6), and CYP98A8 and CYP98A9, which are enzymes required for the production of phenolic precursors. These findings demonstrate the central role of AMS in coordinating sporopollenin biosynthesis and the secretion of materials for pollen wall patterning

MADS1 maintains barley spike morphology at high ambient temperatures

Temperature stresses affect plant phenotypic diversity. The developmental stability of the inflorescence, required for reproductive success, is tightly regulated by the interplay of genetic and environmental factors. However, the mechanisms underpinning how plant inflorescence architecture responds to temperature are largely unknown. We demonstrate that the barley SEPALLATA MADS-box protein HvMADS1 is responsible for maintaining an unbranched spike architecture at high temperatures, while the loss-of-function mutant forms a branched inflorescence-like structure. HvMADS1 exhibits increased binding to target promoters via A-tract CArG-box motifs, which change conformation with temperature. Target genes for high-temperature-dependent HvMADS1 activation are predominantly associated with inflorescence differentiation and phytohormone signalling. HvMADS1 directly regulates the cytokinin-degrading enzyme HvCKX3 to integrate temperature response and cytokinin homeostasis, which is required to repress meristem cell cycle/division. Our findings reveal a mechanism by which genetic factors direct plant thermomorphogenesis, extending the recognized role of plant MADS-box proteins in floral development.Gang Li, Hendrik N. J. Kuijer, Xiujuan Yang, Huiran Liu, Chaoqun Shen, Jin Shi ... et al