ABSTRACT
Background: Cucumber target leaf spot (TLS), caused by Corynespora cassiicola (C. Cassiicola), is a serious disease in cucumber (Cucumis sativus) production worldwide. Therefore, cultivating new varieties of TLS resistance of C. sativus is an important goal of cucumber breeding. Previous studies have shown that subtilisin-like protease (SUBP) plays an important role in response to C. Cassiicola infection in resistant plants. Objective: In this study, the full-length cDNA of the CsSUBP gene was cloned, and the prokaryotic expression vector was successfully constructed in order to study the effects of subtilisin. Futhermore, vital clues regarding CsSUBP gene involved in TLS resistance of C. sativus are gained from the bioinformatics assay. Method: The CsSUBP gene was identified by sequencing with the intermediate vector pMD18 by designing specific primers and PCR amplification techniques. The prokaryotic expression vector pET30a-CsSUBP was further constructed and identified by colony PCR and EcoR V and SalⅠ double digestion. Result: The primary structure of CsSUBP was predicted and analyzed by bioinformatics analysis. The results showed that CsSUBP was weakly acidic protein, N-terminal signal peptide region, including a Inhibitor_I9 domain domain. Conclusion: The pET30a-CsSUBP prokaryotic expression vector was constructed successfully. This study is convenient for the study of prokaryotic expression and its kinase activity.
ABSTRACT
BACKGROUND: The occurrence and prevalence of integrons in clinical microorganisms and their role played in antimicrobial resistance have been well studied recently. As screening and detection of integrons are concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for integrons detection. RESULTS: In this study, three loop-mediated isothermal amplification (LAMP) assays targeting on class 1, 2 and 3 integrons were implemented and evaluated. Optimization of these detection assays were performed, including studing on the reaction temperature, volume, time, sensitivity and specificity (both primers and targets). Application of the established LAMP assays were further verified on a total of 1082 isolates (previously identified to be 397 integron-positive and 685 integron-negative strains). According to the results, the indispensability of each primer had been confirmed and the optimal reaction temperature, volume and time were found to be 65°C, 45 min and 25 µL, respectively. As application was concerned, 361, 28 and 8 isolates carrying intI1, intI2 and intI3 yielded positive amplicons, respectively. Other 685 integron-negative bacteria were negative for the integron-screening LAMP assays, totaling the detection rate and specificity to be 100%. CONCLUSIONS: The intI1-, intI2- and intI3-LAMP assays established in this study were demonstrated to be the valid and rapid detection methodologies for the screening of bacterial integrons.
Subject(s)
DNA, Bacterial/isolation & purification , Nucleic Acid Amplification Techniques/methods , Integrons , Organic Chemicals , Salmonella/genetics , Serratia marcescens/genetics , Staphylococcus/genetics , Vibrio cholerae/genetics , Colony Count, Microbial , Microbial Sensitivity Tests , Polymerase Chain Reaction/methods , Sensitivity and Specificity , DNA, Complementary , DNA Primers , Integrases/genetics , Drug Resistance, Bacterial/genetics , Electrophoresis, Agar Gel , Escherichia coli/genetics , Fluorescent Dyes , Hot TemperatureABSTRACT
Aims: Find a suitable method for the protein extraction from flower buds of Solanum lycopersicum. Study Design: Compare some kinds of protein extraction methods and find the best one among them suitable to tomato flower buds. Place and Duration of Study: Biological Science and Technology College, between June 2010 and July 2011. Methodology: The proteins for electrophoresis were extracted using different methods, such as trichloroacetic acid /acetone (TCA/acetone), Sodium dodecyl sulfate (SDS), Trissaturated phenol (Tris-Phen), Phenol/SDS and Direct lysis method. After silver staining, different patterns of protein spots were observed in the gels. Results: Few spots were found by SDS and Phenol/SDS extractions, more spots by immediate dissolution but the most impurities, less protein productivity though more spots by Tris-Phen extractions, and more protein productivity and better apart effect by TCA/acetone. The 2-DE image background was the clear and the protein spots were the most by TCA/acetone method. Conclusion: TCA/acetone method is much more suitable as extraction method for protein two-dimensional electrophoresis of tomato flower buds.