Molecular characterization and functional analysis of USP-1 by RNA interference in the Asian gypsy moth Lymantria dispar

Ecdysteroids play an important role in regulating diverse physiological processes in arthropods,such as molting,metamorphosis,reproduction and diapause.Ecdysteroids mediate the response by binding to a heterodimeric complex of two nuclear receptors:the ecdysone receptor and the ultraspiracle(USP).To investigate the role of USP in development of the Asian gypsy moth(Lymantria dispar),a USP cDNA was obtained from the transcriptome of L.dispar and verified by PCR.In-depth profiling of transcript levels of L.dispar USP-1(LdUSP-1)at different developmental stages and over time in thirdinstar larvae and different tissues isolated during the thirdinstar stage of L.dispar was then carried out.Transcript levels of LdUSP-1 were relatively high before 72 h in the third-instar larvae after ecdysis and in the adult male.The function of LdUSP-1 in molting was analyzed by knockdown of LdUSP-1 in third instar larvae using RNA interference.Silencing of LdUSP-1 significantly downregulated the transcript level of E75,an ecdysone-inducible gene,and of Sad,a Halloween gene.In addition,the duration of the third-instar stage was slightly shortened and larval mortality increased after the LdUSP-1 knockdown.

Interference of CsGPA1,theα-submit of G protein,reduces drought tolerance in cucumber seedlings

The G proteinα-subunit,GPA1,is an integral component of several signaling pathways in plants,including response to abiotic stress.However,the molecular mechanism behind these processes remains largely unknown in the cucumber plant(Cucumis sativus L.).In order to further understand the role of CsGPA1 in cucumber under drought stress,changes in plant growth,physiological parameters,and gene expression of CsAQPs were all measured under water stress induced by polyethylene glycol(PEG)using wild type(WT)and CsGPA1-interference(RNAi)cucumber seedlings.Our results demonstrated that the RNAi plants had lower drought tolerance,displaying seriously withered leaves,lower relative growth rate,lower root-shoot ratio,and lower root activity under drought stress compared to WT plants.Physiological studies indicated that the suppression of CsGPA1 weakened drought stress tolerance due to higherwater loss rate in the leaves,higher levels of hydrogen peroxide(H2O2),increased malondialdehyde(MDA)content,lower free proline content,lower soluble sugar content,lower soluble protein content,and decreased antioxidant enzyme activities.qRT-PCR analysis demonstrated that the interference of CsGPA1 up-regulated the expression of most AQP genes(except for CsPIP2;3 in leaves)and down-regulated the expression of CsPIP1;2,CsPIP1;4,CsPIP2;1,and CsPIP2;4 in roots under drought stress when compared to WT plants.Our results demonstrated that CsGPA1 could function as a positive regulator in drought stress response by decreasing the accumulation of reactive oxygen species(ROS),improving permeable potentials,and reducing water loss in cucumber plants.

Construction and characterization of a recombinant fowlpox virus containing HIV-1 multi-epitope-p24 chimeric gene in mice

The epidemic of HIV/AIDS is sweeping across the world. It is of great importance to figure out new ways to curb this disease. Epitope-based vaccine is one of these solutions. In this study, a chimeric gene was obtained by combination of a designed HIV-1 multi-epitope gene (MEG) and HIV-1 p24 gene. A re- combinant plasmid pUTA2-MEGp24 was then constructed by inserting MEGp24 gene into the down- stream of the promoter (ATI-P7.5×20) of fowlpox virus (FPV) transfer vector pUTA2. The recombinant plasmid and wild-type FPV 282E4 strain were then co-transfected into CEF cells and homologous re- combination occurred. A recombinant virus expressing HIV-1 protein MEGp24 was screened by ge- nome PCR and Western blot assay. Large scale preparation and purification of the recombinant fowl- pox virus (rFPV) were then carried out. BALB/c mice were immunized intramuscularly with the rFPV for three times on day 0, 14 and 42. Mice were executed and sampled one week after the third inoculation. Anti-HIV-1 antibody in serum and Th1 cytokines in the supernatant of cultured spleen cells were as- sayed by ELISA. The count of T lymphocyte subsets and the CTL activity of spleen lymphocytes were analyzed by flow cytometry and lactate dehydrogenase (LDH) release assay, respectively. The results showed that HIV-1 specific antibody in serum and increased T lymphocyte subsets (CD4+ T, CD8+ T) were detected in the immunization group. CTL target-killing activity and higher secretion of Th1 cyto- kines (IFN-γ and IL-2) of spleen lymphocytes stimulated by H-2d-restricted CTL peptide were observed in immunized mice. We concluded that the rFPV may induce HIV-1 specific immunity especially cellular immunity in mice.

Transcriptomics and metabolomics reveal the possible mechanism by which 1-methylcyclopropene regulates the postharvest senescence of Zizania latifolia

To understand the mechanism governing the postharvest senescence of Zizania latifolia(Z.latifolia),and the regulatory mechanism induced by 1-methylcyclopropene(1-MCP)during storage at 25℃,physiobiochemical and conjoint analyses of the transcriptome and metabolome were performed.The results indicated that 1-MCP treatment engendered changes in the expression of genes and metabolites during the postharvest storage of Z.latifolia.The 1-MCP treatment maintained a good visual appearance,preserved the cell structure and membrane integrity of Z.latifolia by keeping the expression of membrane-related lipolytic enzymes(and related genes)low and the amount of phosphatidylethanolamine high.Compared to the control group,1-MCP treatment enhanced the activities of antioxidant enzymes,resulting in a decrease of reactive oxygen species and malondialdehyde contents,and thus inhibition of oxidative damage and loss of membrane integrity.In addition,1-MCP treatment retarded the senescence of Z.latifolia by downregulating the expression of ethylene biosynthesis-related genes and promoting upregulation of brassinosteroid insensitive 1 kinase inhibitor 1,calmodulin,glutathione reductase,jasmonate amino acid synthase,and mitogen-activated protein kinase-related genes.Moreover,1-MCP retarded Z.latifolia senescence by inducing the activity of adenosine triphosphate(ATP)biosynthesis-related genes and metabolites.Our findings should facilitate future research on the postharvest storage of Z.latifolia,and could help delay senescence and prolong the storage time for commercial applications.

Immune responses of a designed HIV-1 DNA vaccine on rhesus monkeys

An effective HIV-1 vaccine will be the ultimate solution for the prevention of HIV/AIDS, though HAART plays important roles in treating the disease. In this study, a large-scale recombinant DNA plasmid containing a designed HIV-1 multi-epitope- p24 chimeric gene was prepared and purified. Rhesus monkeys were then inoculated muscularly with the plasmid for four times in week 0, 4, 8 and 18. Whole blood was collected two weeks after the third and fourth inoculation, followed by serum and pe- ripheral blood mononuclear cell (PBMC) separation. The CTL activity and proliferation of PBMCs stimu- lated by macaque MHC-I-restricted HIV-1 CTL epi- tope peptide were analyzed by MTT and LDH release assay, respectively. Th1 cytokines in supernatant of cultured PBMC stimulated by HIV-1 CTL epitope peptide and anti-HIV-1 antibody in serum were as- sayed by ELISA. The results showed that increased CTL target-killing activity, higher secretion of Th1 cytokines (IFN-γ and IL-2) and promoted proliferative reaction of monkey PBMCs stimulated by HIV-1 CTL epitope peptide were detected in the immunization group inoculated by the recombinant DNA vaccine for three times, which were further enhanced by the fourth inoculation. At the same time, HIV-1 specific antibody in serum of immunized monkeys was higher than that in controls. We concluded that the designedHIV-1 DNA vaccine may induce HIV-1 specific cellular and humoral immunity on monkeys.