多核丝状真菌的选育及遗传稳定性

细胞中多核体的存在 ,对其隐性突变体的分离及优良形状的稳定遗传产生了相当大的障碍。通过采用大剂量的NTG诱变多核单细胞丝状真菌三孢布拉霉JMβ817的孢子 ,再经紫外线复合照射该孢子制备成的原生质体 ,使其高产菌株———超黄突变体的隐性基因CarS和CarD得以表达 ,从中分离到一株CarS隐性突变株JMβ2 87,β -胡萝卜的产量达到 2 36g/l,比出发菌株提高了 30 %。并通过孢子的亚培养进行定向选育 ,使高产性状得以稳定遗传 ,为促进工业化发酵生产天然 β -胡萝卜提供了必要的手段。

被子植物核型胚乳的细胞化

文章介绍被子植物胚乳多核体的产生及其意义、初始垂周壁和初始平周壁的产生以及微管在初始垂周壁和初始平周壁产生中的作用、胚乳发育及胚乳核细胞化的分子机制以及胚乳细胞壁的物质来源和组成的研究进展。

Polypyrimidine Tract-Binding Protein Enhances Zika Virus Translation by Binding to the 5'UTR of Internal Ribosomal Entry Site

Objectives To identify the 5'untranslated region of Zika virus(ZIKV 5'UTR)RNA-binding proteins and to investigate the impact of the binding protein on the activity of internal ribosomal entry site(IRES)located in ZIKV 5'UTR and virus production.Methods Interacting proteins in U251 cells were captured using tRSA-tagged ZIKV 5'UTR RNA and tRSA-ZIKV 5'UTR RNA-binding proteins were visualized by SDS-PAGE silver staining,Subsequently,liquid chromatographytandem mass spectrometry(LC-MS/MS),bioinformatics analysis,and Western blot were used to identify the candidate proteins binding to ZIKV 5'UTR.Dicistronic expression assay and plaque forming assay were performed to analyze the effect of the binding protein on ZIKV IRES activity and ZIKV production,respecitvely.Results tRSA RNA pull-down assay,LC-MS/MS,and Western blot analysis showed that polypyrimidine tractbinding protein(PTB)bound to the ZIKV 5'UTR.Furthermore,dual luciferase reporter assay revealed that overexpression of PTB significantly enhanced the IRES activity of ZIKV(t=10.220,P<0.001),while PTB knockdown had the opposite effect(t=4.897,P<0.01).Additionally,virus plaque forming assay demonstrated that up-regulation of PTB expression significantly enhanced viral titer(t=6.400,P<0.01),whereas reducing PTB expression level weakened virus infectivity(t=5.055,P<0.01).Conclusion PTB positively interacts with the ZIKV 5'UTR and enhances IRES activity and virus production.

Generation of Antibodies Against DMRT1 and DMRT4 of Oreochromis aurea and Analysis of Their Expression Profile in Oreochromis aurea Tissues

Sex determination is composed of somatic and germ-line sex differentiation hierarchies whose interaction is poorly understood. A single gene known to control somatic sex determination, the DM-domain containing （Doublesex/Mab-3 DNA-binding motif） gene, is highly conserved across species. Vertebrate DMRT1 （DM-related transcription factor 1） expression occurs predominantly in the testis. Here, however, isolated two distinct DM-domain cDNA from Oreochromis aurea ovary and testis have been named DMRT4 （DM-related transcription factor 4） and DMRT1 by BLAST, respectively. Despite high homology in the DM-domain there is little similarity outside the DM-domain.To better understand the structure, function, and possible roles of DMRT4 and DMRT1 as potential candidates for sex differentiation and sex determination, the intact regions encoding DMRT4 and DMRT1 obtained by PCR were sub-cloned into the vector pMAL-c2x and introduced into the Escherichia coli TB1 cell for efficient fusion expression. After purification and cleavage, DMRT4 and DMRT1 proteins were used to immunize adult rabbits following standard protocols. Consequently, it was found by using Western blot analysis that polyclonal antibodies against DMRT4 and DMRT1 had high specificity. The relative expression levels of DMRT4 and DMRT1 mRNA were determined by fluorescent Real-time RT-PCR in female and male Oreochromis aurea with 13-actin as the internal standard. DMRT1 was expressed only in testis, whereas DMRT4 was over expressed in the ovary, but in both female and male, a slight expression in the brain was also detected. Statistical analysis showed that in the brain, mean DMRT4 mRNA levels in female were significantly higher than in male. Meanwhile, the expression of DMRT4 and DMRT1 protein was also analyzed using the purified antibodies through Western blot and immunohistochemistry. It was found that DMRT4 was exclusively expressed in the ovary and DMRT1 in the testis. Study on DMRT4 and DMRT1 expression facilitated the elucidation of their roles and the understanding of sex differentiation of fish.

水霉病防治案例分析

1水霉病水霉病病原体为水霉科(Saprolegniaceae),水霉属(Saprolegnia)和绵霉属(Achlya)的一些种类。一般由内外2种菌丝组成,菌丝为管状,为没有横隔的多核体。内菌丝像根一样附着在水产动物的损伤处,分枝多而纤细,有固着和吸收营养物质的作用;外菌丝较粗壮,分枝较少,伸出鱼体组织,菌丝长度可达3 cm,形成肉眼能见的灰白色棉絮状物质[1],引起组织发炎、坏死,并最终致其死亡。

水霉病防治案例与分析

1.病原体病原体为水霉科(Saprolegniaceae)、水霉属(Saprolegnia)和绵霉属(Achlya)的一些种类。一般有内外两种丝状的菌丝组成,菌丝为管状,为没有横隔的多核体。内菌丝像根样附着在水产动物的损伤处,分枝多而纤细,有固着和吸收营养物质的作用;外菌丝较粗壮,分枝较少,伸出于鱼体组织之外,可长达3cm,形成肉眼能见的灰白色棉絮状物。

Application of Spodoptera litura Nucleopolyhedrovirus for Crop Pest Control

The laboratory bioassay and field control efficacy of Spodoptera litura nucleopolyhedrovirus（Spli NPV） Chenzhou strain were preliminarily examined. The efficient artificial propagation method was to feed the host larvae with virus suspension,and the average mortality of the insects was 65.0%. The death peak of the pests appeared 4-8 d after virus infection. The high temperature, high humidity and poor light could help the virus infection and propagation. Filed control efficacy of Chenzhou strain was 86.6% in laboratory, which was better than of another commercial strain. The corrected control efficacy of this strain was 88.4% the field, which was higher than that of avermectin pesticide significantly. It was detected that the occlusion body（OB） concentration of the initial virus＇ s stock solution was 1.03×1011OBs/ml,and it was a strong SpliNPV strain, as it showed an excellent efficacy to control the pest Spodoptera litura, and thus there will be a good prospect of application and development of this SpliNPV strain.

Expression and Purification of E.coli NrfA Protein and Preparation of Polyclonal Antibody against NrfA

[Objective] This study aimed to clone the E. coil NrfA gene and construct the pET-28a （＋）-NrfA prokaryotic expression vector for preparation of polyclonal anti- body against E. coil NrfA. [Method] E. coil NrfA gene was cloned from the E. coli genome DNA by PCR and inserted into the vector pET-28a（＋） to construct prokary- otic expression vector pET-28a （＋）-NrfA. E. coil NrfA protein was expressed by IPTG induction and purified. Polyclonal antibody against NrfA protein was prepared by im- munizing rabbit with routine method. The specificity and titer of polyclonal antibody was confirmed by ELISA and Western Blotting. [Result] The constructed prokaryotic expression vector pET-28a（＋）-NrfA was induced by IPTG, the recombinant NrfA pro- tein could be expressed effectively. The titer of rabbit anti-NrfA polyclonal antibody obtained by immunization and purification was about 1：204 900. Western Blotting anal- ysis indicated that the obtained polyclonal antibody against E. coil NrfA protein had high titer and high specificity. [Conclusion] E. coil NrfA gene was cloned and the prokaryotic expression vector pET-28a （＋）-NrfA was constructed successfully, poly- clonal antibody with high titer and high specificity was prepared, which laid the foun- dation for the study of NrfA in different strains of bacteria.

Prokaryotic Expression and Polyclonal Antibody Preparation of SDG711 C-terminal from Rice

[Objective] This study aimed to conduct prokaryotic expression of rice SDG711 C-terminal and to prepare its polyclonal antibody. [Method] C-terminal of rice SDG711 containing relatively intensive antigen determinants was selected for prokaryotic expression, prokaryotic expression vector pET28a-711C was constructed and the recombinant plasmid was transformed into Escherichia coli BL21 （DE3） competent cells. The recombinant fusion protein was induced by IPTG and purified to immunize a New Zealand white rabbit as the antigen, and polyclonal antibody was obtained and confirmed by Western-blot analysis. [Result] The polyclonal anti- body was successfully prepared and could efficiently detect the expressed antigen. [Conclusion] This study laid the foundation for further investigating the functions of SDG711 protein in rice.

Encyclopedia of Autographa californica Nucleopolyhedrovirus Genes

The Autographa californica multiple capsid nucleopolyhedrovirus (AcMNPV) was the first baculovirus for which the complete nucleotide sequence became known. Since then 15 years lapsed and much research has been performed to elucidate putative functions of the annotated open reading frames of this virus and this endeavour is still ongoing. AcMNPV is the most well-known and well-studied baculovirus species, not in the least for its application as a vector for the high-level expression of foreign genes in insect cells. This article is the first monograph of a single baculovirus and gives a current overview of what is known about the 151 AcMNPV ORFs, including (putative) function and temporal and spatial presence of transcripts and protein. To date 60 ORFs have a proven function, another 19 ORFs have homologs for which functions are known in other baculoviruses and 72 ORFs are still enigmatic. This paper should assist the reader in quickly finding the essentials of AcMNPV.

Baculovirus per os Infectivity Factors Are Involved in HearNPV ODVs Infection of HzAM1 Cells in vitro

Baculoviruses produce two viral phenotypes, the budded virus （BV） and the occlusion-derived virus （ODV）. ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the lack of an in vitro system, the molecular mechanism of ODV infection is still unclear. Here we present data demonstrating that Helicoverpa armigera nucleopolyhedrovirus （HearNPV） ODV infected cultured Hz-AM1 cells in a pH dependent manner. The optimal pH for ODV infection was 8.5, which is same to that in the microvilli of midgut epithelial cells, the ODV native infection sites. Antibodies neutralization analysis indicated that four HearNPV oral infection essential genes p74, pif-l, pif-2 and pif-3 are also essential for HearNPV ODV infection in vitro. Thus, HearNPV-HzAM1 system can be used to analyze the mechanism of ODV entry.

Involvement of Lipid Rafts and Cellular Actin in AcMNPV GP64 Distribution and Virus Budding

GP64 is the major envelope glycoprotein associated with the budded virus (BV) of Autographa californica nucleopolyhedrovirus (AcMNPV) and is essential for attachment and budding of BV particles. Confocal microscopy and flotation assays established the presence of lipid raft domains within the plasma membranes of AcMNPV-infected Sf9 cells and suggested the association of GP64 with lipid rafts during infection. GP64 and filamentous actin (F-actin) were found to co-localise at the cell cortex at 24 and 48 hpi and an additional restructuring of F-actin during infection was visualised, resulting in a strongly polarised distribution of both F-actin and GP64 at the cell cortex. Depletion of F-actin, achieved by treatment of Sf9 cells with latrunculin B (LB), resulted in the redistribution of GP64 with significant cytoplasmic aggregation and reduced presence at the plasma membrane. Treatment with LB also resulted in reduced production of BV in Sf9 cells. Analysis of virus gene transcription confirmed this reduction was not due to decreased trafficking of nucleocapsids to the nucleus or to decreased production of infectious progeny nucleocapsids. Reduced BV production due to a lack of GP64 at the plasma membrane of AcMNPV-infected Sf9 cells treated with LB, suggests a key role for F-actin in the egress of BV.

The Protamine-like DNA-binding Protein P6.9 Epigenetically Up-regulates Autographa californica Multiple Nucleopolyhedrovirus Gene Transcription in the Late Infection Phase

Protamines are a group of highly basic proteins first discovered in spermatozoon that allow for denser packaging of DNA than histones and will result in down-regulation of gene transcription^l~. It is well recognized that the Autographa californica multicapsid nucleopolyhedrovirus （AcMNPV） encodes P6.9, a protamine-like protein that forms the viral subnucleosome through binding to the viral genome[29]. Previous research demonstrates that P6.9 is essential for viral nucleocapsid assembly, while it has no influence on viral genome replication1311. In the present study, the role of P6.9 in viral gene transcription regulation is characterized. In contrast to protamines or other protamine-like proteins that usually down-regulate gene transcription, P6.9 appears to up-regulate viral gene transcription at 12-24 hours post infection （hpi）, whereas it is non-essential for the basal level of viral gene transcription. Fluorescence microscopy reveals the P6.9＇s co-localization with DNA is temporally and spatially synchronized with P6.9＇s impact on viral gene transcription, indicating the P6.9-DNA association contributes to transcription regulation. Chromatin fractionation assay further reveals an unexpected co-existence of P6.9 and host RNA polymerase II in the same transcriptionally active chromatin fraction at 24 hpi, which may probably contribute to viral gene transcription up-regulation in the late infection phase.

Nematode Diversity of Qingdao Coast Inferred from the 18S Ribosomal RNA Gene Sequence Analysis

The 18S ribosomal DNA gene （18S rDNA） sequences （approxtmately 1300 bp in length） were amplified from the DNA extracted from the free-living marine nematodes collected from the inter-tidal sediment of Qingdao coast in bulk with nematode specific primers. The PCR products were cloned, re-amplified, digested with Rsa I and Hin61 restriction endonucleases and separated in agarose gel. Among 17 restriction fragment length types, types 1, 2 and 6 covered 61.2%, 14.4% and 9.3% of the clones analyzed, respectively, while the remaining 14 only covered 21 clones, which accounted for 15.1% of the total. Twenty-four representative clones were sequenced and phylogenetically analyzed by referring to those currently available in RDP and GenBank databases. Although it was hard to assign these sequences to known species or genera due to the lack of the 18S rDNA sequence data of known marine free-living nematodes, the obtained sequences were assigned to the nematodes of Adenophorea. Among them, twelve sequences were close to Pontonema vulgate and Adoncholaimus sp., four to Daptonema procerus and two （identical） to Enoplus brews. Our results showed that free-living marine nematode diversities could be determined by PCR retrieving and analysis of the 18S rDNA sequences and an 18S rDNA sequence could be assigned to a species or a genus only if the 18S rDNA sequences of the free-living marine nematodes were accumulated to some extent.

Upper gastrointestinal bleeding etiology score for predicting variceal and non-variceal bleeding

AIM： To identify clinical parameters, and develop an Upper Gastrointesinal Bleeding （UGIB） Etiology Score for predicting the types of UGIB and validate the score. METHODS： Patients with UGIB who underwent endoscopy within 72 h were enrolled. Clinical and basic laboratory parameters were prospectively collected. Predictive factors for the types of UGIB were identified by univariate and multivariate analyses and were used to generate the UGIB Etiology Score. The best cutoff of the score was defined from the receiver operating curve and prospectively validated in another set of patients with UGIB. RESULTS： Among 261 patients with UGIB, 47 （18%） had variceal and 214 （82%） had non-variceal bleeding. Univariate analysis identified 27 distinct parameters significantly associated with the types of UGIB. Logistic regression analysis identified only 3 independent factors for predicting variceal bleeding; previous diagnosis of cirrhosis or signs of chronic liver disease （OR 22.4, 95% CI 8.3-60.4, P 〈 0.001）, red vomitus （OR 4.6, 95% CI 1.8-11.9, P = 0.02）, and red nasogastric （NG） aspirate （OR 3.3, 95% CI 1.3-8.3, P = 0.011）. The UGIB Etiology Score was calculated from （3.1× previous diagnosis of cirrhosis or signs of chronic liver disease） ＋ （1.5× red vomitus） ＋ （1.2× red NG aspirate）, when 1 and 0 are used for the presence and absence of each factor, respectively. Using a cutoff ≥ 3.1, the sensitivity, specificity, accuracy, positive predictive value （PPV）, and negative predictive value （NPV） in predicting variceal bleeding were 85%, 81%, 82%, 50%, and 96%, respectively. The score was prospectively validated in cases （46 variceal and 149 another set of 195 UGIB non-variceal bleeding）. The PPV and NPV of a score ≥ 3.1 for variceal bleeding were 79% and 97%, respectively. CONCLUSION： The UGIB Etiology Score, composed of 3 parameters, using a cutoff ≥ 3.1 accurately predicted variceal bleeding and may help to guide the choice of initial therapy for UGIB before endoscopy.

Exotic Structures of Odd-A Carbon Isotopes in the Deformed Relativistic Mean-FieldTheory

We study contributions of the pion meson and spatial component of the omega meson in the odd-A carbon isotopes. The pion and spatial omega provide small attractions in odd-A nuclei, giving rise to considerable influences on the single-particle energies rather than the bulk properties such as total binding energies, and root-mean-square (rms) radii. The ±? (spin) splittings, arising from the spatial omega, are large in 11C and 13C and drop as the isospin rises in odd-A carbon isotopes. As an isovector, the pion can shift slightly the relative potential depth of neutron and proton, contrary to the role of the rho meson. There is a general trend that both the pion and spatial omega fields reduce with the rise of isospin in the isotopic chain. From the normal nucleus to halo nucleus, an abnormal drop of the pion or spatial omega field may occur, as can be seen in 19C, 15C, and 21C.

Expression and Purification of Enterovirus Type 71 Polyprotein P1 using Pichia pastoris system

Enterovirus type 71（EV71） causes severe hand-foot-and-mouth disease （HFMD） resulting in hundreds of deaths of children every year; However, currently, there is no effective treatment for EV71. In this study, the EV71 poly-protein （EV71-P1 protein） gene was processed and cloned into the eukaryotic expression vector pPIC9k and then expressed in Pichia pastoris strain GSll5. The EV71 P1 protein with a molecular weight of 100 kD was produced and secreted into the medium. The soluble EV71 P1 protein was purified by column chromatography with a recovery efficiency of 70%. The result of the immunological analysis showed that the EV71 P1 protein had excellent immunogenicity and could stimulate the production of EV71-VP1 IgG antibody in injected rabbits. We suggest that EV71-P1 protein is an ideal candidate for an EV71 vaccine to prevent EV71 infection.

Application of Computed Tomographic Colonography in Diagnosis of Colonic Polyps

Objective To assess the clinical values of computed tomographic colonography （CTC） in diagnosis of colonic polyps. Methods Forty-two patients who were clinically suspicious of colonic polyps or underwent colonic polyps screening received examinations with both CTC and conventional colonoscopy. Sixteen- or 64-slice spiral computed tomography and professional imaging processing techniques were used for evaluation. Per-polyp and per-patient results were analyzed. Those by per-polyp were subsequently divided into ≥10 mm group, 5-10 mm group, and ≤5 mm group. Sensitivity, positive predictive value （PPV）, specificity, negative predictive value （NPV）, and accuracy were calculated using statistical method for diagnostic studies, with conventional colonoscopy as a gold standard. Results Ninety and 61 polyps were found by CTC and conventional colonoscopy, respectively. The per-polyp sensitivity/PPV were 80.3%/55.6% in total, and 100%/92.9%, 93.8%/65.2%, and 68.8%/ 41.5% in the ≥10 mm group, 5-10 mm group, and ≤5 turn group, respectively. The per-patient sensitivity, PPV, specificity, NPV, and accuracy were 97.1%, 89.5%, 42.9%, 75.0%, and 88.1%, respectively. Conclusion CTC can clearly reveal the morphology of colonic polyps and be used as a routine monitoring method for the clinical diagnosis of polyps.

Specificity of Developmental Resistance in Gypsy Moth (Lymantria dispar) to two DNA-Insect Viruses

Gypsy moth （Lymantria dispar） larvae displayed marked developmental resistance within an instar to L dispar M nucleopolyhedrovirus （LdMNPV） regardless of the route of infection （oral or intrahemocoelic） in a previous study, indicating that in gypsy moth, this resistance has a systemic component. In this study, gypsy moth larvae challenged with the Amsacta moorei entomopoxvirus （AMEV） showed developmental resistance within the fourth instar to oral, but not intrahemocoelic, inoculation. In general, gypsy moth is considered refractory to oral challenge with AMEV, but in this study, 43% mortality occurred in newly molted fourth instars fed a dose of 5×10^6 large spheroids of AMEV; large spheroids were found to be more infectious than small spheroids when separated by a sucrose gradient. Developmental resistance within the fourth instar was reflected by a 2-fold reduction in mortality （18%-21%） with 5 X 106 large spheroids in larvae orally challenged at 24, 48 or 72 h post-molt. Fourth instars were highly sensitive to intrahemocoelic challenge with AMEV; 1PFU produced approximately 80% mortality regardless of age within the instar. These results indicate that in gypsy moth, systemic developmental resistance may be specific to LdMNPV, reflecting a co-evolutionary relationship between the baculovirus and its host.

Prokaryotic Expression and Purification of HIV-1 Vif and hAPOBEC3G, Preparation of Polyclonal Antibodies

To prepare HIV-1 Vif and hAPOBEC3G and to produce their antibodies, the full length gene fragment of HIV-1 vif was amplified by PCR from a plasmid of HIV-1 NL4.3 cDNA, and the APOBEC3G gene was obtained by RT-PCR from the total RNA of H9 cells. The resulting DNA construct was cloned into a prokaryotic expression vector (pET-32a). Recombinant pET-vif and pET-APOBEC3G were expressed respectively in Eserichia coli BL21 (DE3) as an insoluble protein. The vector also contained a six-histidine tag at the C-terminus for convenient purification and detection. To express and purify the HIV-1 Vif and hAPOBEC3G in E.coli cells, the accuracy of inserted gene and specificity of proteins were detected by the two enzyme digestion method, SDS-PAGE, and Western blotting. Rabbits were then immunized by Vif or APOBEC3G protein and serum samples were tested by indirect ELISA to determine the level of antibodies. Immunoenzyme and immunofluorescence assays were performed to identify the specificity of polyclonal antibodies. The titer of the anti-Vif antibodies was 1:204800, and that of the anti-APOBEC3G antibodies was 1:102400. Thus the antibodies could detect the antigen expression in the cells, demonstrating that fusion proteins with high purity and their corresponding polyclonal antibodies with high titer and specificity were achieved.