[Synergistic effect on biosynthesis of Panax notoginseng saponins by overexpressing a transcription factor PnbHLH and RNA interference of cycloartenol synthase gene].

This study cloned the transcription factor gene PnbHLH which held an open reading frame of 966 bp encoding 321 amino acids. This study constructed the overexpression vector of transcription factor PnbHLH of Panax notoginseng. The combination of PnbHLH overexpression and RNAi of the key enzyme gene PnCAS involved in the phytosterol biosynthesis was achieved in P. notoginseng cells, thus exploring the biosynthetic regulation of P. notoginseng saponins(PNS) by the synergistic effect of PnbHLH overexpression and PnCAS RNAi. The results showed that the PnbHLH transcription factor interacted with the promoters of key enzyme genes PnDS, PnSS and PnSE in the biosynthetic pathway of PNS, and then regulated the expression levels of key enzyme genes and affected the biosynthesis of saponins indirectly. Further study indicated that the synergistic effect of PnbHLH overexpression and PnCAS RNAi was a more effective approach to regulate the biosynthesis of saponins. Compared with the wild type and PnCAS RNAi cells of P. notoginseng, the contents of total saponins and monomeric saponins(Rd, Rb_1, Re, Rg_1 and R_1) were increased to some extent in the cell lines of PnbHLH overexpression and PnCAS RNAi. This indicated that the two ways of forward regulation and reverse regulation of saponin biosynthesis showed superposition effect. This study explored a more rational and efficient regulation strategy of PNS biosynthesis based on the advantages of multi-point regulation of transcription factors as well as the down-regulation of by-product synthesis of saponins.

[Functional analysis of a chitinase gene PnCHI1 from Panax notoginseng].

Chitinases, a glycosidase enzyme that hydrolyzes chitin to N-acetylglucosamine, are widely found in plant cells, and they are an important part of plant antifungal defense system. The function of a Panax notoginseng chitinase gene PnCHI1 was characterized in this paper. Expression vector of PnCHI1 was constructed and transiently expressed in onion epidermal cells, and laser scanning confocal microscopy demonstrated that PnCHI1 was localized in the cell wall. Prokaryotic expression vector of PnCHI1 was also constructed, and recombinant protein of PnCHI1 was induced and purified. In vitro antibacterial assay showed that recombinant PnCHI1 protein had strong inhibitory activity on the mycelium growth of Fusarium solani, F. oxysporum and F. verticillioide. The function of PnCHI1 was further verified by reverse genetics. PnCHI1 expression vector was transferred into tobacco by Agrobacterium tumefaciens and expression of PnCHI1 was confirmed by qRT-PCR. It was found by leaf inoculation experiment that resistance of transgenic tobacco to F. solani was significantly increased. It is conclnded that: PnCHI1 is a chitinase localized in the cell wall, which inhibits several fungi which cause the root rot disease of P. notoginseng. Overexpression of this chitinase gene in tobacco greatly increased resistance to F. solani. PnCHI1 may be an important resistance gene in P. notoginseng that participates in the defense against root rot disease.

[Cloning and expression analysis of a chitinase gene PnCHI1 from Panax notoginseng].

Chitinases(EC3.2.1.14), which are present in various organisms, catalyze the hydrolytic cleavage of chitin and play a vital role in plant defense mechanisms against fungal pathogens.In addition, the chitinases are well known to regulate plant growth and development and are involved in programmed cell death(PCD).A chitinase expressed sequence tag(EST) was isolated from Panax notoginseng, and the full-length cDNA of this EST was cloned with the method of rapid amplification of cDNA ends and named as PnCHI1. PnCHI1 was 1 022 bp in length and contained an intact open reading frame(ORF) of 822 bp, a 26 bp 5'-untranslated region(UTR), and a 174 bp 3'-UTR.The predicted protein of PnCHI1 with 273 amino acid residues belongs to glycoside hydrolase family 19 and fell into the class IV of chitinases through phylogenetic analysis.QRT-PCR analysis showed that the expression of PnCHI1 was induced by methyl jasmonate, ethylene, H2O2, and salicylic acid.PnCHI1 was quickly induced after inoculation with Alternaria panax.Moreover, the expression level of PnCHI1 was increased after pretreatment with methyl jasmonate, and then the transcription level of PnCHI1was sharp increased after inoculation with Fusarium solani,and the highest transcription level was achieved at 4 h post inoculation.But the expression level of PnCHI1 in the sterile water pretreated P.notoginseng was increased gradually after inoculation with F.solani, and the highest expression level was achieved at 48 h post inoculation.All the results of present study indicated that PnCHI1 was involved in defense response of P.notoginseng against the F.solani and A.panax.

[The construction of over-expression vector for Panax notoginseng SS gene and its transformation].

PNS (Panax notoginseng saponins) is the main medical bioactive component in Panax notoginseng. The medical value of PNS cannot be extended because of its low production. With the deep study of saponins biosynthetic pathway, the control of PNS biosynthesis through metabolic engineering has gradually become possible. In this study, the Squalene synthase (SS) over-expression vector was established. By the way of agrobacterium-mediated method, the vector was transfered and integrated into the Panax notoginseng genome. The result of the PCR detection and the saponin content detection shows that over-expression SS is able to produce high level of Panax notoginseng saponins, and confirms the regulatory function of SS in the biosynthesis of ginsenosides in Panax notoginseng. It provides a theoretical basis and technical basis for the construction of PNS homologous or heterologous efficient expression system in the future.

High-level mucosal and systemic immune responses induced by oral administration with Lactobacillus-expressed porcine epidemic diarrhea virus (PEDV) S1 region combined with Lactobacillus-expressed N protein.

To develop effective mucosal vaccine formulation against porcine epidemic diarrhea virus (PEDV) infection, the DNA fragments encoding spike protein immunodominant region S1 and nucleocapsid N of PEDV were inserted into pPG1 (surface-displayed) or pPG2 (secretory) plasmids followed by electrotransformation into Lactobacillus casei (Lc) to yield four recombinant strains: PG1-S1, PG2-S1, PG1-N, and PG2-N. After intragastric administration, it was observed that live Lc-expressing S1 protein combined with Lc-expressing N protein could elicit much more potent mucosal and systemic immune responses than the former alone (P < 0.001), however slightly inferior to the latter alone (P > 0.05). Furthermore, the surface-displayed mixture (PG1-S1+ PG1-N) revealed stronger immunogenicity than the secretory mixture (PG2-S1+ PG2-N) as well as PEDV-neutralizing potency in vitro (P < 0.001). On 49th day after the last immunization, splenocytes were prepared from mice immunized with surface-displayed mixture, secretory mixture and negative control to be stimulated by purified N and S protein, respectively. The results of ELISA analysis showed that N protein was capable of inducing a higher level of IL-4 (P < 0.001) and IFN-γ (P < 0.001) than S1 protein in the immunized mice. Taken together, Lc-expressed N protein as molecular adjuvant or immunoenhancer was able to effectively facilitate the induction of mucosal and systemic immune responses by Lc-expressing S1 region.

Construction of recombinant lactobacilli expressing the core neutralizing epitope (COE) of porcine epidemic diarrhea virus and a fusion protein consisting of COE and Escherichia coli heat-labile enterotoxin B, and comparison of the immune responses by orogastric immunization.

The core neutralizing epitope (COE) region of porcine epidemic diarrhea virus (PEDV) plays an important role in the development of the subunit vaccine against PEDV infection. To enhance the vaccine's immunogenicity, Escherichia coli heat-labile enterotoxin B (LTB) has usually been adopted as a molecular adjuvant. In this study, the COE and LTB-COE genes were engineered into the Lactobacillus -Escherichia coli shuttle vectors pSAPG1 (surface-displaying) and pSAPG2 (secreting) followed by electrotransformation into Lactobacillus casei (Lc) to yield the following recombinant strains: Lc:PG1-LTB-COE, Lc:PG2-LTB-COE, Lc:PG1-COE, and Lc:PG2-COE. Our results showed that mice immunized orogastrically with L. casei expressing COE or LTB-COE produced secretory immunoglobulin A and immunoglobulin G with the ability to neutralize PEDV in sera and mucus. Moreover, higher levels of interleukin-4 and gamma interferon were also exhibited compared with negative control. These data displayed the tendency of Lc:PG2-LTB-COE > Lc:PG1-LTB-COE > Lc:PG2-COE > Lc:PG1-COE at the same time point. Taken together, LTB-COE is more suitable for Lactobacillus expressing system to engineer mucosal vaccine against PEDV infection.

Genome-wide search for the genes accountable for the induced resistance to HIV-1 infection in activated CD4+ T cells: apparent transcriptional signatures, co-expression networks and possible cellular processes.

BACKGROUND: Upon co-stimulation with CD3/CD28 antibodies, activated CD4 + T cells were found to lose their susceptibility to HIV-1 infection, exhibiting an induced resistant phenotype. This rather unexpected phenomenon has been repeatedly confirmed but the underlying cell and molecular mechanisms are still unknown. METHODS: We first replicated the reported system using the specified Dynal beads with PHA/IL-2-stimulated and un-stimulated cells as controls. Genome-wide expression and analysis were then performed by using Agilent whole genome microarrays and established bioinformatics tools. RESULTS: We showed that following CD3/CD28 co-stimulation, a homogeneous population emerged with uniform expression of activation markers CD25 and CD69 as well as a memory marker CD45RO at high levels. These cells differentially expressed 7,824 genes when compared with the controls on microarrays. Series-Cluster analysis identified 6 distinct expression profiles containing 1,345 genes as the representative signatures in the permissive and resistant cells. Of them, 245 (101 potentially permissive and 144 potentially resistant) were significant in gene ontology categories related to immune response, cell adhesion and metabolism. Co-expression networks analysis identified 137 "key regulatory" genes (84 potentially permissive and 53 potentially resistant), holding hub positions in the gene interactions. By mapping these genes on KEGG pathways, the predominance of actin cytoskeleton functions, proteasomes, and cell cycle arrest in induced resistance emerged. We also revealed an entire set of previously unreported novel genes for further mining and functional validation. CONCLUSIONS: This initial microarray study will stimulate renewed interest in exploring this system and open new avenues for research into HIV-1 susceptibility and its reversal in target cells, serving as a foundation for the development of novel therapeutic and clinical treatments.

Expression of infectious pancreatic necrosis virus (IPNV) VP2-VP3 fusion protein in Lactobacillus casei and immunogenicity in rainbow trouts.

Infectious pancreatic necrosis virus (IPNV) infects wild and cultured salmonids, causing high mortality in juvenile trouts and salmons. IPNV VP2-VP3 fusion gene was constructed by splicing overlap extension (SOE) PCR and inserted into Lactobacillus/Escherichia coli shuttle vectors (pPG1and pPG2) followed by transformation of Lactobacillus casei competent cell to yield two recombinant strains: Lc:PG1-VP2-VP3 (surface-displayed) and Lc:PG2-VP2-VP3 (secretory). Subsequently, juvenile rainbow trouts were inoculated with the recombinant strains via orogastric route. Our results demonstrated that Lactobacillus-derived VP2-VP3 fusion protein could induce production of serum IgM specific for IPNV with neutralizing activity in rainbow trouts. Statistical analyses of IgM levels showed that immunogenicity of Lc:PG1-VP2-VP3 was more powerful than that of Lc:PG2-VP2-VP3 (P<0.001) in rainbow trouts. This result has been confirmed by viral loads reduction analyzed by real-time RT-PCR in orogastrically immunized rainbow trouts after virus challenging. Comparing to trouts received Lactobacillus (control), rainbow trouts orogastrically dosed with Lc:PG1-VP2-VP3 resulted in ∼10-fold reduction in viral loads on day 10 post-virus challenging, and ∼4-fold did by Lc:PG2-VP2-VP3. Taken together, Lc:PG1-VP2-VP3 functions as novel mucosal vaccine against IPNV infection in rainbow trouts, which most likely come true.

Intracellular overexpression of HIV-1 Nef impairs differentiation and maturation of monocytic precursors towards dendritic cells.

Nef functions as an immunosuppressive factor critical for HIV-1 replication, survival and development of AIDS following HIV-1 infection. What effects Nef exerts on differentiation and maturation of monocytes towards dendritic cells (DCs) remains greatly controversial. In this study, we used THP-1 (human monocytic leukemia cell line) as monocytic DC precursors to investigate how overexpression of HIV-1 Nef influences the processes of differentiation and maturation of dendritic cells. In striking contrast to negative controls, our results showed that morphological and phenotypical changes (CD11c, CD14, CD40, CD80, CD83, CD86, and HLA-DR) occurred on recombinant THP-1 expressing HIV-1 Nef (short for Nef) upon co-stimulation of GM-CSF/IL-4 or GM-CSF/IL-4/TNF-α/ionomycin. Moreover, CD4, CCR5, and CXCR4 were also down-regulated on Nef. It might be hypothesized that Nef prevents superinfection and signal transduction in HIV-1 infected monocytes. Collectively, our study demonstrates that long-lasting expression of Nef at high levels indeed retards differentiation and maturation of dendritic cells in terms of phenotype and morphology. We are hopeful that potentially, stable expression of intracellular Nef in vivo may function as a subtle mode to support long-lasting HIV-1 existence.

Genes expression analyses of sea-island cotton (Gossypium barbadense L.) during fiber development.

Sea-island cotton (Gossypium barbadense L.) is one of the most valuable cotton species due to its silkiness, luster, long staples, and high strength, but its fiber development mechanism has not been surveyed comprehensively. We constructed a normalized fiber cDNA library (from -2 to 25 dpa) of G. barbadense cv. Pima 3-79 (the genetic standard line) by saturation hybridization with genomic DNA. We screened Pima 3-79 fiber RNA from five developmental stages using a cDNA array including 9,126 plasmids randomly selected from the library, and we selected and sequenced 929 clones that had different signal intensities between any two stages. The 887 high-quality expressed sequence tags obtained were assembled into 645 consensus sequences (582 singletons and 63 contigs), of which 455 were assigned to functional categories using gene ontology. Almost 50% of binned genes belonged to metabolism functional categories. Based on subarray analysis of the 887 high-quality expressed sequence tags with 0-, 5-, 10-, 15-, and 20-dpa RNA of Pima 3-79 fibers and a mixture of RNA of nonfiber tissues, seven types of expression profiles were elucidated. Furthermore our results showed that phytohormones may play an important role in the fiber development.