Overexpression of cucumber CYP82D47 enhances resistance to powdery mildew and Fusarium oxysporum f. sp. cucumerinum.

Cytochrome P450s are a large family of protein-encoding genes in plant genomes, many of which have not yet been comprehensively characterized. Here, a novel P450 gene, CYP82D47, was isolated and functionally characterized from cucumber (Cucumis sativus L.). Quantitative real-time reverse-transcription polymerase chain reaction analysis revealed that CYP82D47 expression was triggered by salicylic acid (SA) and ethephon (ETH). Expression analysis revealed a correlation between CYP82D47 transcript levels and plant defense responses against powdery mildew (PM) and Fusarium oxysporum f. sp. cucumerinum (Foc). Although no significant differences were observed in disease resistance between CYP82D47-RNAi and wild-type cucumber, overexpression (OE) of CYP82D47 enhanced PM and Foc resistance in cucumber. Furthermore, the expression levels of SA-related genes (PR1, PR2, PR4, and PR5) increased in CYP82D47-overexpressing plants 7 days post fungal inoculation. The levels of ETH-related genes (EIN3 and EBF2) were similarly upregulated. The observed enhanced resistance was associated with the upregulation of SA/ETH-signaling-dependent defense genes. These findings indicate the crucial role of CYP82D47 in pathogen defense in cucumber. CYP82D47-overexpressing cucumber plants exhibited heightened susceptibility to both diseases. The study results offer important insights that could aid in the development of disease-resistant cucumber cultivars and elucidate the molecular mechanisms associated with the functions of CYP82D47.

Gene cloning and expression of MAP30 in Pichia pastoris.

MAP30, a single-stranded type-I ribosome inactivating protein found in Momordica charantia, shows anti-HIV and anti-tumour activity. It could significantly inhibit the HIV-1 and herpes simplex virus infection. In this study, we tried a safe and convenient expression system supplying MAP30 protein for medical practice. The gene encoding MAP30 was cloned into pMD18-T vector. The pMD18-MAP30 plasmid was transformed into competent Escherichia coli JM109 by a chemical method. The MAP30 gene was obtained from the pMD18-MAP30 plasmid digested with NotI and SnaBI and the MAP30 gene was ligated into pGAPHα. Then, pGAPHα-MAP30 was transformed into Pichia pastoris GS115 by electroporation. GS115 transformants were analysed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and Western blot. SDS-PAGE revealed an extra band of approximately 32 kDa in the supernatant protein of the GS115 transformants and in their intracellular protein fraction. The result of Western-blot analysis showed that the supernatant and the cell pellet from GS115 with pGAPHα-MAP30 could specially bind to monoclonal antibodies against His in the 32 kDa site. These results demonstrated that the expression of MAP30 in P. pastoris was successful; the process of the expression did not need methanol induction or introduction of an antibiotic-resistance gene. The study may provide a new way for MAP30 synthesis. Owing to its safety, this new approach is expected to be widely used in the medical field.

Changes of DNA methylation of Isoetes sinensis under Pb and Cd stress.

To investigate the molecular response of ancient plants to heavy metal stress and to explore the feature of DNA methylation in endangered plants after exposure to heavy metals, the Isoetes sinensis, an endangered plant, was stressed with three different concentrations of two heavy metals lead (Pb) and cadmium (Cd), respectively. Then the degrees and the patterns of DNA methylation in the leaves were measured on the 14th day using Methylation Sensitive Amplified Polymorphism (MSAP) technique. The results showed that the DNA methylated profile of I. sinensis was affected by Pb and Cd stress. There was no significant difference in the amount of DNA methylation among control check (CK), Pb stress group, and Cd stress group (CK 46.96%, Pb 48.23%, and Cd 48.1%). However, full-methylation level of Pb stress group (28.34%) and Cd stress group (20.25%) was lower than control (33.91%), in contrast, hemi-methylation level Pb stress group (19.89%) and Cd stress group (27.85%) were higher than control (13.04%). The change of patterns from no methylation or hemi-methylation of internal and external cytosines into full-methylation of internal and external cytosines accounted for a large proportion in enhanced methylation aspects. The full-methylation into no methylation or hemi- or full-methylation of internal and external cytosines occupied most of demethylation. The proportion of DNA methylation (including hypermethylation) by both Pb and Cd stresses is nearly equal (39.04% and 39.71%), but the proportion of DNA demethylation by Cd is higher than that by Pb (46.86% than 33.92%).