Two candidate small interfering RNAs (siRNAs) corresponding to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike gene were designed and in vitro transcribed to explore the possibility of silencing SARS-CoV S gene. The plasmid pEGFP-optS, which contains the codon-optimized SARS-CoV S gene and expresses spike-EGFP fusion protein (S-EGFP) as silencing target and expressing reporter, was transfected with siRNAs into HEK 293T cells. At various time points of posttransfection, the levels of S-EGFP expression and amounts of spike mRNA transcript were detected by fluorescence microscopy, flow cytometry, Western blot, and real-time quantitative PCR, respectively. The results showed that the cells transfected with pEGFP-optS expressed S-EGFP fusion protein at a higher level compared with those transfected with pEGFP-S, which contains wildtype SARS-CoV spike gene sequence. The green fluorescence, mean fluorescence intensity, and SARS-CoV S RNA transcripts were found significantly reduced, and the expression of SARS-CoV S glycoprotein was strongly inhibited in those cells co-transfected with either EGFP- or S-specific siRNAs. Our findings demonstrated that the S-specific siRNAs used in this study were able to specifically and effectively inhibit SARS-CoV S glycoprotein expression in cultured cells through blocking the accumulation of S mRNA, which may provide an approach for studies on the functions of SARS-CoV S gene and development of novel prophylactic or therapeutic agents for SARS-CoV.

BackgroundCucumber downy mildew is among the most important diseases that can disrupt cucumber production. Propamocarb, also known as propyl-[3-(dimethylamino)propyl]carbamate (PM), is a systemic carbamate fungicide pesticide that is widely applied in agricultural production because of its high efficiency of pathogens control, especially cucumber downy mildew. However, residual PM can remain in cucumbers after the disease has been controlled. To explore the molecular mechanisms of PM retention, cucumber cultivars ‘D9320’ (with the highest residual PM content) and ‘D0351’ (lowest residual PM content) were studied. High-throughput tag-sequencing (Tag-Seq) results showed that the CsDIR16 gene was related to PM residue, which was verified using transgenic technology.ResultsWe investigated the activity of a dirigent cucumber protein encoded by the CsDIR16 in gene response to stress induced by PM treatment. Gene-expression levels of CsDIR16 were up-regulated in the fruits, leaves, and stems of ‘D0351’ plants in response to PM treatment. However, in cultivar ‘D9320’, CsDIR16 levels were down-regulated in the leaves and stems after PM treatment, with no statistically significant differences observed in the fruits. Induction by jasmonic acid, abscisic acid, polyethylene glycol 4000, NaCl, and Corynespora cassiicola Wei (Cor) resulted in CsDIR16 up-regulation in ‘D0351’ and ‘D9320’. Expression after salicylic acid treatment was up-regulated in ‘D0351’, but was down-regulated in ‘D9320’. CsDIR16 overexpression lowered PM residues, and these were more rapidly reduced in CsDIR16(+) transgenic ‘D9320’ plants than in wild-type ‘D9320’ and CsDIR16(−) transgenic plants.ConclusionsAnalyses of the CsDIR16-expression patterns in the cucumber cultivars with the highest and lowest levels of PM residue, and transgenic validation indicated that CsDIR16 plays a positive role in reducing PM residues. The findings of this study help understand the regulatory mechanisms occurring in response to PM stress in cucumbers and in establishing the genetic basis for developing low-pesticide residue cucumber cultivars.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1236-2) contains supplementary material, which is available to authorized users.

Riboflavin prolonged the lifespan and increased the reproduction of fruit flies through anti-oxidative stress pathway involving enhancing the activity of SOD1 and CAT and inhibiting LF accumulation. Riboflavin deserves more attention for slowing human aging.

Objectives: Hepatitis C virus (HCV) F protein is a newly identified protein encoded by an alternative open reading frame that +1 overlaps core-encoding gene. It has been found that regulation of c-myc and p53 genes by HCV core protein is involved in liver cancer genesis. We wondered whether HCV F protein exerts similar or adverse regulatory effects on the transcription of c-myc and p53 genes. Methods: HCV F gene-containing, plasmid pcDNA3.1-F and HCV core gene-containing pcDNA3.1-C were constructed and transiently transfected into HepG₂ cells. Real-time quantitative PCR or Western blotting was used to determine the changes at transcription or translation levels of c-myc and p53 genes. Results: The transcription level of c-myc was much higher in pcDNA3.1-F transfected cells than those without plasmid transfected. Whereas the level of p53 transcription in pcDNA3.1-F transfected cells was lower than those in the parental cells. Moreover, levels of c-myc expression were up-regulated and those of p53 expression were down-regulated by HCV F protein. Conclusions: HCV F protein is of regulatory properties in cellular oncogene c-myc and anti-oncogene p53, which may be implicated in the formation of hepatocellular carcinoma.

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