An efficient transient gene expression system for protein subcellular localization assay and genome editing in citrus protoplasts

Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression system is a powerful tool for gene functional characterization and CRISPR/Cas9 genome editing in higher plants, but it has not been widely used in citrus. In this study, the polyethylene glycol(PEG)-mediated method was optimized for citrus callus protoplast transfection, with an improved transfection efficiency of 68.4%. Consequently, the efficiency of protein subcellular localization assay was increased to 65.8%, through transient expression of the target gene in protoplasts that stably express the fluorescent organelle marker protein. The gene editing frequencies in citrus callus protoplasts reached 14.2% after transient expression of CRISPR/Cas9 constructs. We demonstrated that the intronic polycistronic tRNAgRNA(inPTG) genome editing construct was functional in both the protoplast transient expression system and epicotyl stable transformation system in citrus. With this optimized protoplast transient expression system, we improved the efficiency of protein subcellular localization assay and developed the genome editing system in callus protoplasts, which provides an approach for prompt test of CRISPR vectors.

Extract from Phyllanthus urinaria L.Inhibits Hepatitis B Virus Replication and Expression in Hepatitis B Virus Transfection Model in Vitro

Objective： To explore the effects of the extract from Phyllanthus urinaria L. on hepatitis B virus （HBV） replication and expression in HBV transient transfection model in vitro. Methods： The eukaryotic expression plasmid pHBVI.1, which contains l.l-fold-overlength genome of HBV, was transfected into the human hepatoma cell line, HepG2, to establish and assess the HBV transient transfection model. The extract from Phyllanthus urinaria L. was prepared in different concentrations and methyl thiazolyl tetrazolium was used to detect the maximum nontoxic concentration of the drug. The extract from Phyllanthus urinaria L. were added into the transfected cell, at the concentrations of 0.8, 0.2 and 0.05 g/L, respectively. Four days after drug application, enzyme-linked immuno sorbent assay was used to detect the concentration of HBsAg in the supernatants, Southern blot was applied to analyze HBV DNA level, and Western blot was used to detect the expression of HBcAg in cells. Results： After the transfection of plasmid pHBVI.1 into HepG2 cells, the concentration of HBsAg in supernatants was increased obviously as compared with that of the normal cells （P〈0.05）, and all expected HBV replicative intermediates were confirmed by Southern blot analysis, which ensured the successful establishment of the HBV transient transfection model. After the application of drugs at the concentrations of 0.8 and 0.2 g/L, the level of HBsAg was obviously decreased in the supernatants, as compared with that of the virus group （P〈0.05）; Southern blot showed that the level of HBV rc DNA, ds DNA, ss DNA was obviously reduced compared with that of the virus group （P〈0.01）; Western blot revealed that the expression of HBcAg in the drug group was obviously inhibited, as compared with that of the virus group （P〈0.01）. Conclusions： The extract from Phyllanthus urinaria L. obviously inhibited replication and expression of HBV in HBV transfected cell lines in vitro, thus exerting distinctive anti-HBV effects.