Jinyang Zhang

Structural analysis and tissue-specific expression patterns of a novel salt-inducible NAC transcription factor gene from Nicotiana tabacum cv. Xanthi

Summary The NAC (acronym derived from the names of the first three cloned genes NAM, ATAF1/ATAF2, and CUC) transcription factor family is important for plant development and environmental stress responses. We isolated NtNAC2, a new salt-inducible gene in the NAC gene family from tobacco (Nicotiana tabacum var. Xanthi). NtNAC2 encoded 311 amino acids and the deduced protein sequence contained five typical NAC transcription factor domains. Most of the secondary structure of the predicted NtNAC2 protein was made up of αhelices and βsheets. We analysed tissue-specific expression profiles and found that the NtNAC2 gene was expressed mainly in roots, stems, and flowers. We were able to induce NtNAC2 gene expression by exposing tobacco plants to high levels of salt. A phylogenetic analysis of putative NAC-family proteins from the tobacco genome indicated a wide diversity of family members. Our results suggest that the NtNAC2 gene may play an important role in the tolerance of tobacco to salinity.

Analysis of Subcellular Prefoldin 1 Redistribution During Rabies Virus Infection

Background: Rabies virus (RABV) is one of the old deadly zoonotic viruses. It attacks the central nervous system and causes acute encephalitis in humans and animals. Host factors are known to be essential for virus infection and replication in cells. The identification of the key host factors required for RABV infection may provide important information on RABV replication and may provide new potential targets for RABV drug discovery. Objectives: This study aimed to investigate the change in the subcellular distribution and expression of the host protein Prefoldin subunit 1 (PFDN1) in RABV-infected cells and the viral expression of plasmids in the transfected cells. Materials and Methods: Mouse Neuro-2a (N2a) cells were infected by RABV or transfected with the plasmids of the nucleoprotein (N) and/or phosphoprotein (P) gene of RABV. The subcellular distribution of PFDN1 was analyzed by confocal microscopy, and the transcription levels of PFDN1 in the N and/or P gene of the RABV-transfected or RABV-infected N2a cells were assessed via real-time quantitative polymerase chain reaction. Results: Confocal microscopy showed that PFDN1 was colocalized with the N protein of RABV in the infected N2a cells and was mainly recruited to the characteristic Negri-Body-Like (NBL) structures in the cytoplasm, as well as the cotransfection of the N and P genes of RABV. The transcription of PFDN1 in the RABV-infected N2a cells was upregulated, whereas the transfection of the N and/or P genes did not result in the upregulation of PFDN1. Conclusions: The results of this work demonstrated that the subcellular distribution of PFDN1 was altered in the RABV-infected N2a cells and colocalized with the N protein of RABV in the NBL structures.

Studies on the role of the SlNAC3 gene in regulating seed development in tomato (Solanum lycopersicum)

Summary Plant seeds contain the sporophytic embryo and nutrients for embryo development. To ensure correct seed formation, co-ordination between the embryo, the endosperm, and the maternal seed components must be achieved. However, this regulatory mechanism is poorly understood. Here, we demonstrate that an NAC gene (SlNAC3) was essential for young embryo and endosperm development in tomato (Solanum lycopersicum). Transgenic tomato seeds expressing SlNAC3 RNA interference (RNAi) exhibited defects in pollen viability and a reduced seed size. Ultrastructural analysis revealed the absence of an embryo sac, while the endosperm collapsed in the seed of transgenic tomato plants expressing lower levels of SlNAC3 transcripts due to RNAi. Real-time quantitative RT-PCR analysis revealed the down-regulation (to < 10% of wild-type levels) of four genes involved in jasmonic acid (JA) biosynthesis in the flowers of transgenic plants. These results demonstrate that the SlNAC3 gene plays a critical role in the development of the embryo in tomato seed.

Prokaryotic Expression, Purification, and Polyclonal Antibody Production of a Truncated Recombinant Rabies Virus L Protein

BACKGROUND Rabies virus (RABV) is a deadly neurotropic virus that causes the disease of rabies in humans and animals. L protein is one of the large structural protein of rabies virus, which displays multiple enzymatic activities, and is required for viral transcription and replication. OBJECTIVES A truncated L protein of Rabies virus is being cloned, expressed and purified to produce relevant polyclonal antibody. MATERIALS AND METHODS The gene fragment of L protein of RABV was subcloned into prokaryotic expression vector pET- 28a and transformed into E. coli Rosetta DE3 host strain. The recombinant L protein of RABV was expressed and characterized by SDS-PAGE and western blot analysis using anti-his tag antibody. Mice were immunized with the purified recombinant L protein, the reaction of the anti-serum was checked by immunofluorescence and dot-blot, respectively. RESULTS The results of PCR and sequencing confirmed that the fragment of L gene of RABV was successfully cloned into the expression vector. The expression of recombinant L protein fragment induced by IPTG was confirmed by the band of 43 kDa in SDS-PAGE and western blot. The antiserum of purified L protein immunized mice was reacted with RABV infected N2a cells and suckling mouse brain tissue lysates. CONCLUSIONS Our data showed that the recombinant L protein produced by pET-28a vector was very successful, and the purified L protein could efficiently induce the antibody response in mice. The antiserum could recognize the virus in RABV infected cells and tissue very well.

Detection of Shigella in Milk and Clinical Samples by Magnetic Immunocaptured-Loop-Mediated Isothermal Amplification Assay

Shigella is an important human food-borne zoonosis bacterial pathogen, and can cause clinically severe diarrhea. There is an urgent need to develop a specific, sensitive, and rapid methodology for detection of this pathogen. In this study, loop-mediated isothermal amplification (LAMP) combined with magnetic immunocapture assay (IC-LAMP) was first developed for the detection of Shigella in pure culture, artificial milk, and clinical stool samples. This method exhibited a detection limit of 8.7 CFU/mL. Compared with polymerase chain reaction, IC-LAMP is sensitive, specific, and reliable for monitoring Shigella. Additionally, IC-LAMP is more convenient, efficient, and rapid than ordinary LAMP, as it is more efficiently enriches pathogen cells without extraction of genomic DNA. Under isothermal conditions, the amplification curves and the green fluorescence were detected within 30 min in the presence of genomic DNA template. The overall analysis time was approximately 1 h, including the enrichment and lysis of the bacterial cells, a significantly short detection time. Therefore, the IC-LAMP methodology described here is potentially useful for the efficient detection of Shigella in various samples.

Optimized Expression, Purification of Herpes B Virus gD Protein in Escherichia coli , and Production of Its Monoclonal Antibodies

Background Herpes B virus (BV) is a zoonotic disease caused by double-stranded enveloped DNA virus with cercopithecidae as its natural host. The mortality rate of infected people could be up to 70% with fatal encephalitis and encephalomyelitis. Up to now, there are no effective treatments for BV infection. Among the various proteins encoded by monkey B virus, gD, a conserved structural protein, harbors important application value for serological diagnosis of frequent variations of the monkey B virus. Objectives This study aimed to expressed the gD protein of BV in Escherichia coli by a recombinant vector, and prepare specific monoclonal antibodies against gD of BV to pave the way for effective and quick diagnosis reagent research. Materials and Methods The gD gene of BV was optimized by OptimWiz to improve codon usage bias and synthesis, and the recombinant plasmid, pET32a/gD, was constructed and expressed in E. coli Rosetta (DE3). The expressed fusion protein, His-gD, was purified and the BALB/c mice were immunized by this protein. Spleen cells from the immunized mice and SP2/0 myeloma cells were fused together, and the monoclonal cell strains were obtained by indirect enzyme-linked immunosorbent assay (ELISA) screening, followed by preparation of monoclonal antibody ascetic fluid. Results The optimized gD protein was highly expressed in E. coli and successfully purified. Five monoclonal antibodies (mAbs) against BV were obtained and named as 4E3, 3F8, 3E7, 1H3 and 4B6, and with ascetic fluid titers of 2 × 106, 2 × 105, 2 × 105, 2 × 103 and 2 × 102, respectively. The 1H3 and 4E3 belonged to the IgG2b subclass, while 3E7, 3F8 and 4B6 belonged to the IgG1 subclass. Conclusions The cell lines obtained in this work secreted potent, stable and specific anti-BV mAbs, which were suitable for the development of herpes B virus diagnosis reagents.