鸭圆环病毒(DuCV)感染能够引起鸭的免疫抑制,引起多种病原的继发性感染,在我国鸭群中广泛流行.为了解DuCV的遗传变异情况,对采自山东、江苏和贵州3个省份的19个鸭场的样品进行了 PCR测定,并对DuCV的ORF-C1基因进行测序和遗传进化分析.PCR测定结果显示,19个鸭场中有9个鸭场的样品为DuCV阳性,总体阳性率为47.4％;ORF-C1基因序列测定结果显示,9株DuCV之间的核苷酸序列和氨基酸序列同源性分别为93.4％～100％和82.2％～100％,与NCBI中已公布的DuCV毒株的核苷酸序列和氨基酸序列同源性分别为77.8％～99.5％和71.5％～-99.6％;遗传进化分析显示,9株DuCV全部属于DuCV-1b亚型,其中有7株在同一分支上,与D11-JW-001等韩国毒株遗传距离较近,另外2株则与山东、广西的毒株遗传距离较近;此外,与已公布的毒株相比,9株DuCV都在158位发生了氨基酸突变,由E/D变为L/P.上述结果表明,DuCV-1仍然为我国鸭群中的优势流行基因型,但病毒的基因也在不断地发生变异,应该对其进行持续监测.

黄病毒RNA复制子在病毒致病机制、新型疫苗和药物研发上应用广泛,而坦布苏病毒(Tembusu virus,TMUV)是我国新发现的以引起水禽产蛋量下降为主要特征的黄病毒属成员.为构建TMUV RNA复制子,本研究以TMUV JXSP株感染性克隆为基础,利用Overlap PCR分别对病毒基因组进行CPrME、CPrM、PrME和PrM等4种方式结构蛋白基因的缺失,并在缺失部位插入绿色荧光蛋白报告基因,报告基因下游连接口蹄疫病毒2A蛋白基因序列,获得的基因组全长PCR融合产物纯化后,体外转录成mRNA,并转染至BHK-21细胞,采用荧光显微镜观察不同时间点绿色荧光蛋白的表达;利用构建的TMUV RNA复制子,将绿色荧光蛋白基因替换为H9亚型禽流感病毒HA基因,mRNA转染细胞72 h后Western blotting检测蛋白表达.结果显示,4种TMUV RNA复制子转染细胞的胞浆和胞核内均能观察到明显的绿色荧光信号;Western blotting能检测到HA蛋白的表达.结果表明,本研究成功构建了 ΔCPrME、ΔCPrM、ΔPrME和ΔPrM等4种能有效表达外源基因的TMUV RNA复制子.

Additional file 10: GO analysis information of DEGs significantly enriched in 244 biological processes.

Duck hepatitis A virus type 3 (DHAV-3) is an important pathogen that causes substantial losses in the Chinese duck industry. DHAV-3 is highly fatal to ducklings and there is no licensed vaccine in China available to reduce DHAV-3 infection. Our goal was to develop a live attenuated vaccine candidate against DHAV-3. A field isolated strain, SD, was attenuated by serially passaging in specific-pathogen-free (SPF) chicken embryos, and it lost its pathogenicity after 40 passages. The 70th passaged strain (SD70), which achieved good growth capacity in chicken embryos with a viral titer of 107.5 ELD50/mL, was chosen to be the live attenuated vaccine candidate. The SD70 strain did not cause clinical signs of disease or mortality in 1-day-old ducklings and showed no virulence reversion after seven rounds of in vivo back passages. The minimum effective dose of SD70 was determined to be 102.5 ELD50 via the vaccination route of subcutaneous inoculation. A single dose of the SD70 provided good protection to susceptible ducklings against the lethal DHAV-3 strain. Compared with the genomic sequence of the parent SD strain, the SD70 had 12 amino acid substitutions, some of which may play a role in virulence attenuation. This study demonstrated that the attenuated SD70 strain is a promising vaccine candidate for the prevention of DHAV-3 infection in China. It exhibited safety, good stability and excellent protection.

BACKGROUND:Duck hepatitis A virus type 3 (DHAV-3) is one of the most harmful pathogens in the duck industry. However, the molecular mechanism underlying DHAV-3 infection in ducklings remains poorly understood. To study the genetic regulatory network for miRNA-mRNA and the signaling pathways involved in DHAV-3 infection in ducklings, we conducted global miRNA and mRNA expression profiling of duckling liver tissues infected with lethal DHAV-3 by high-throughput sequencing.RESULTS:We found 156 differentially expressed miRNAs (DEMs) and 7717 differentially expressed genes (DEGs) in livers of mock-infected and DHAV-3-infected duckling. A total of 19,606 miRNA-mRNA pairs with negatively correlated expression patterns were identified in miRNA-mRNA networks constructed on the basis of these DEMs and DEGs. Moreover, immune-related pathways, including the cytokine-cytokine receptor interaction, apoptosis, Toll-like receptor, Jak-STAT, and RIG-I-like receptor signaling pathway, were significantly enriched through analyzing functions of mRNAs in the network in response to DHAV-3 infection. Furthermore, apl-miR-32-5p, apl-miR-125-5p, apl-miR-128-3p, apl-miR-460-5p, and novel-m0012-3p were identified as potential regulators in the immune-related signaling pathways during DHAV-3 infection. And some host miRNAs were predicted to target the DHAV-3 genome.CONCLUSIONS:This is the first integrated analysis of miRNA and mRNA in DHAV-3-infected ducklings. The results indicated the important roles of miRNAs in regulating immune response genes and revealed the immune related miRNA-mRNA regulation network in the DHAV-3-infected duckling liver. These findings increase our knowledge of the roles of miRNAs and their target genes in DHAV-3 replication and pathogenesis. They also aid in the understanding of host-virus interactions.

Ethylene-responsive transcription factors (ERFs) belonging to the AP2/ERF family have diverse functions in plants. However, the functions of LeERF-1, a member of ERF family from the medicinal plant Lithospermum erythrorhizon, remain unclear. In this study, by cloning the promoter of LeERF-1, we found that the promoter region contained a number of potential regulatory motifs related to drought and pathogen resistances. Further transgenic studies showed that the heterologous overexpression of LeERF-1 in Arabidopsis displayed phenotypes of higher survival ratio, lower root inhibition rate, slow water loss in leaf discs, and smaller stomatal apertures under drought stress, compared with wild type (WT) of Arabidopsis. LeERF-1 transgenic Arabidopsis also displayed fewer chlorotic symptoms, lower incidence rates, and lower levels of bacterial proliferation on leaves after the inoculation of bacterial pathogen compared with WT. These results suggested that LeERF-1 can also confer drought and pathogen resistances. Our work provided a candidate gene with remarkable potential use in genetic engineering for stress resistance improvement in plants.

Background: Duck hepatitis A virus type 3 (DHAV-3) is one of the most harmful pathogens in the duck industry. However, the molecular mechanism underlying DHAV-3 infection in ducklings is remain poorly understood. To elucidate the genetic regulatory network for miRNA-mRNA and the signaling pathways involved in DHAV-3 infection in ducklings, we conducted global miRNA and mRNA expression profiling of duckling liver tissues infected with lethal DHAV-3 using high-throughput sequencing. Results: We found 156 differentially expressed miRNAs (DEMs) and 7717 differentially expressed miRNAs (DEGs) between mock-infected and DHAV-3-infected duckling livers. A total of 19,606 miRNA-mRNA pairs with negatively correlated expression patterns were identified in miRNA-mRNA networks constructed on the basis of these DEMs and DEGs. Moreover, immune-related pathways including the cytokine-cytokine receptor interaction, apoptosis, Toll-like receptor, Jak-STAT, and RIG-I-like receptor signaling pathway were significantly enriched through analyzing functions of mRNAs in the network in response to DHAV-3 infection. Besides, apl-miR-32-5p, apl-miR-125-5p, apl-miR-128-3p, apl-miR-460-5p, and novel-m0012-3p were identified as potential regulators in the immune-related signaling pathways during the DHAV-3 infection. Conclusions: To our knowledge, this is the first report on integrated analysis of miRNA-seq and mRNA-seq in DHAV-3-infected ducklings. The results indicated the important roles of miRNAs in regulating immune response genes and revealed the immune related miRNA-mRNA regulation network in the DHAV-3-infected duckling liver. Our findings may provide valuable information to further investigate the roles of miRNAs and their target genes in DHAV-3 replication and pathogenesis; additionally, they may offer clues for further understanding host-virus interactions.

The synthetic growth hormone2,4-dichlorophenoxyacetic acid (2,4-D) significantly inhibits shikonin and its derivatives biosynthesis in the medicinal plant Lithospermum erythrorhizon. However, the molecular mechanism of this regulation remains unclear.In this study, we attempt to address this issue by comparing the transcriptome of 2,4-D treated cell cultures with that of the control (CK). A total of 216 up-regulated and 269 down-regulated genes by 2,4-D were discovered. Gene ontology (GO) enrichment analysis revealed that differentially expressed genes (DEGs) were statistically significantly related to the metabolic process. Pathway classification enrichment analysis further confirmed that the DEGs were mainly related to the secondary metabolism. More importantly, 2,4-D has no effect on the two key enzyme genes ACS and ACO for ethylene biosynthesis. However,2,4-D significantly down-regulated the genes directly involved in ethylene signal transduction pathway (ESTP), including IllvL45, EIN3, and ORCA3. 2,4-D also down-regulated two indirect genes, LOX and GA2ox, thereby conferring the jasmonate (JA) and GA biosynthesis, which regulates ESTP by targeting EIN3. By blocking ESTP, 2,4-D fmally down regulated the phenylpropanoid formation-related genes, thereby conferring shikonin and its derivatives biosynthesis. Our fmdings provide new hints for the deep understanding of the molecular mechanism of shikonin formation.

Shikonin and its derivatives extracted from Lithospermeae plants' red roots have current applications in food and pharmaceutical industries. Previous studies have cloned some genes related to shikonin biosynthesis. However, most genes related to shikonin biosynthesis remain unclear, because the lack of the genome/transcriptome of the Lithospermeae plants. Therefore, in order to provide a new understanding of shikonin biosynthesis, we obtained transcriptome data and unigenes expression profiles in three shikonin-producing Lithospermeae plants, i.e., Lithospermum erythrorhizon, Arnebia euchroma and Echium plantagineum. As a result, two unigenes (i.e., G10H and 12OPR) that are involved in "shikonin downstream biosynthesis" and "methyl jasmonate biosynthesis" were deemed to relate to shikonin biosynthesis in this study. Furthermore, we conducted a Lamiids phylogenetic model and identified orthologous unigenes under positive selection in above three Lithospermeae plants. The results indicated Boraginales was more relative to Solanales/Gentianales than to Lamiales.

BACKGROUND:Shikonin is a naphthoquinone secondary metabolite with important medicinal value and is found in Lithospermum erythrorhizon. Considering the limited knowledge on the membrane transport mechanism of shikonin, this study investigated such molecular mechanism.RESULTS:We successfully isolated an ATP-binding cassette protein gene, LeMDR, from L. erythrorhizon. LeMDR is predominantly expressed in L. erythrorhizon roots, where shikonin accumulated. Functional analysis of LeMDR by using the yeast cell expression system revealed that LeMDR is possibly involved in the shikonin efflux transport. The accumulation of shikonin is lower in yeast cells transformed with LeMDR-overexpressing vector than that with empty vector. The transgenic hairy roots of L. erythrorhizon overexpressing LeMDR (MDRO) significantly enhanced shikonin production, whereas the RNA interference of LeMDR (MDRi) displayed a reverse trend. Moreover, the mRNA expression level of LeMDR was up-regulated by treatment with shikonin and shikonin-positive regulators, methyl jasmonate and indole-3-acetic acid. There might be a relationship of mutual regulation between the expression level of LeMDR and shikonin biosynthesis.CONCLUSIONS:Our findings demonstrated the important role of LeMDR in transmembrane transport and biosynthesis of shikonin.

MicroRNAs (miRNAs) play an important role in plant growth, development, and response to environment. For identifying and comparing miRNAs and their targets in seed development between two maize inbred lines (i.e. PH6WC and PH4CV), two sRNAs and two degradome libraries were constructed. Through high-throughput sequencing and miRNA identification, 55 conserved and 24 novel unique miRNA sequences were identified in two sRNA libraries; moreover, through degradome sequencing and analysis, 137 target transcripts corresponding to 38 unique miRNA sequences were identified in two degradome libraries. Subsequently, 16 significantly differentially expressed miRNA sequences were verified by qRT-PCR, in which 9 verified sequences obviously target 30 transcripts mainly involved with regulation in flowering and development in embryo. Therefore, the results suggested that some miRNAs (e.g. miR156, miR171, miR396 and miR444) related reproductive development might differentially express in seed development between the PH6WC and PH4CV maize inbred lines in this present study.

The phytohormone ethylene (ET) is a crucial signaling molecule that induces the biosynthesis of shikonin and its derivatives in Lithospermum erythrorhizon shoot cultures. However, the molecular mechanism and the positive regulators involved in this physiological process are largely unknown. In this study, the function of LeACS-1, a key gene encoding the 1-aminocyclopropane-1-carboxylic acid synthase for ET biosynthesis in L. erythrorhizon hairy roots, was characterized by using overexpression and RNA interference (RNAi) strategies. The results showed that overexpression of LeACS-1 significantly increased endogenous ET concentration and shikonin production, consistent with the up-regulated genes involved in ET biosynthesis and transduction, as well as the genes related to shikonin biosynthesis. Conversely, RNAi of LeACS-1 effectively decreased endogenous ET concentration and shikonin production and down-regulated the expression level of above genes. Correlation analysis showed a significant positive linear relationship between ET concentration and shikonin production. All these results suggest that LeACS-1 acts as a positive regulator of ethylene-induced shikonin biosynthesis in L. erythrorhizon hairy roots. Our work not only gives new insights into the understanding of the relationship between ET and shikonin biosynthesis, but also provides an efficient genetic engineering target gene for secondary metabolite production in non-model plant L. erythrorhizon.

The revelation of mechanisms of photodynamic therapy (PDT) at the cellular level as well as singlet oxygen (1O2) as a second messengers requires the quantification of intracellular 1O2. To detect singlet oxygen, directly measuring the phosphorescence emitted from 1O2 at 1270 nm is simple but limited for the low quantum yield and intrinsic efficiency of 1O2 emission. Another method is chemically trapping 1O2 and measuring fluorescence, absorption and Electron Spin Resonance (ESR). In this paper, we used indocyanine green (ICG), the only near-infrared (NIR) probe approved by the Food and Drug Administration (FDA), to detect 1O2 in vitro. Once it reacts with 1O2, ICG is decomposed and its UV absorption at 780 nm decreases with the laser irradiation. Our data demonstrated that ICG could be more sensitive and accurate than Singlet Oxygen Sensor Green reagent® (SOSG, a commercialized fluorescence probe) in vitro, moreover, ICG functioned with Eosin Y while SOSG failed. Thus, ICG would reasonably provide the possibility to sense 1O2 in vitro, with high sensitivity, selectivity and suitability to most photosensitizers.

BACKGROUND:The phytohormone ethylene (ET) is a key signaling molecule for inducing the biosynthesis of shikonin and its derivatives, which are secondary metabolites in Lithospermum erythrorhizon. Although ETHYLENE INSENSITIVE3 (EIN3)/EIN3-like proteins (EILs) are crucial transcription factors in ET signal transduction pathway, the possible function of EIN3/EIL1 in shikonin biosynthesis remains unknown. In this study, by targeting LeEIL-1 (L. erythrorhizon EIN3-like protein gene 1) at the expression level, we revealed the positive regulatory effect of LeEIL-1 on shikonin formation.RESULTS:The mRNA level of LeEIL-1 was significantly up-regulated and down-regulated in the LeEIL-1-overexpressing hairy root lines and LeEIL-1-RNAi hairy root lines, respectively. Specifically, LeEIL-1 overexpression resulted in increased transcript levels of the downstream gene of ET signal transduction pathway (LeERF-1) and a subset of genes for shikonin formation, excretion and/or transportation (LePAL, LeC4H-2, Le4CL-1, HMGR, LePGT-1, LeDI-2, and LePS-2), which was consistent with the enhanced shikonin contents in the LeEIL-1-overexpressing hairy root lines. Conversely, LeEIL-1-RNAi dramatically repressed the expression of the above genes and significantly reduced shikonin production.CONCLUSIONS:The results revealed that LeEIL-1 is a positive regulator of the biosynthesis of shikonin and its derivatives in L. erythrorhizon hairy roots. Our findings gave new insights into the molecular regulatory mechanism of ET in shikonin biosynthesis. LeEIL-1 could be a crucial target gene for the genetic engineering of shikonin biosynthesis.

MicroRNAs (miRNAs) are a class of noncoding small RNAs (sRNAs) that play many roles in plant growth, development, and the stress response.

Novel shikonin derivatives were synthesised and probed as anticancer agents. Compound 40 showed the best anticancer activity with an IC50 of 1.26 μM, could induce apoptosis and cause cell cycle arrest at the G2/M phase via the P21 p-CDC2 (Tyr15) pathway independent of P53.

植物次生代谢产物是人类重要的药物及化工原料来源,其产生与植物正常的生长发育及对环境的适应密切相关,并受到多种因素的调控.乙烯作为一种植物内源激素,广泛参与植物的生长、发育、抗逆和次生代谢产物合成等重要生理过程的调控.该文综述了乙烯的信号转导机制及其调控作用;重点归纳了乙烯对植物次生代谢产物形成所表现出的双重调控效应,即在一定浓度范围内,乙烯对植物次生代谢产物的合成起促进作用,低于或超过该浓度范围则起抑制作用;并对今后该领域的研究方向进行了展望.