绿僵菌第五类Ser/Thr蛋白磷酸酶基因的克隆及表达特征分析

【目的】克隆绿僵菌第五类Ser/Thr蛋白磷酸酶(PP5)基因,了解该基因及其编码产物的结构特征和两种产孢模式(微循环产孢和正常产孢)中的表达特征。【方法】通过绿僵菌中PP5基因EST序列与全基因组数据库比对,获得PP5基因DNA序列;通过同源蛋白比对预测PP5基因的DNA结构并设计引物,PCR扩增获取PP5全长cDNA序列;通过在线分析工具及生物软件进行蛋白结构分析。采用实时荧光定量PCR检测PP5基因在两种产孢模式中的表达特征。【结果】PP5基因长2100 bp,含7个外显子和6个内含子;cDNA开放阅读框长为1428 bp(GenBank登录号HQ317137),编码475个氨基酸;一级、二级及三级结构分析均显示较保守的蛋白磷酸酶结构特征。实时荧光定量PCR分析表明,PP5基因在绿僵菌微循环产孢的不同阶段表达水平具有显著性差异,特别是在孢子接种16、24、32 h高表达,而在正常产孢模式下表达量非常少。【结论】克隆了绿僵菌的PP5基因,详细了解了该基因及其编码产物的结构特征,发现了该基因在微循环产孢孢子形成后期高表达的重要特征,为进一步研究该基因在微循环产孢中的功能奠定了基础。     

Natural Variation in Grain Selenium Concentration of Wild Barley, <Emphasis Type="Italic">Hordeum spontaneum</Emphasis>, Populations from Israel

Wild barley (Hordeum spontaneum), the progenitor of cultivated barley, is an important genetic resource for cereal improvement. Selenium (Se) is an essential trace mineral for humans and animals with antioxidant, anticancer, antiarthropathy, and antiviral effects. In the current study, the grain Se concentration (GSeC) of 92 H. spontaneum genotypes collected from nine populations representing different habitats in Israel was investigated in the central area of Guizhou Province, China. Remarkable variations in GSeC were found between and within populations, ranging from 0 to 0.387 mg kg⁻¹ among the 92 genotypes with an average of 0.047 mg kg⁻¹. Genotype 20_C from the Sede Boqer population had the highest GSeC, while genotype 25_1 from the Atlit population had the lowest. The mean value of GSeC in each population varied from 0.010 to 0.105 mg kg⁻¹. The coefficient of variation for each population ranged from 12% to 163%. Significant correlations were found between GSeC and 12 ecogeographical factors out of 14 studied. Habitat soil type also significantly affected GSeC. The wild barley exhibited wider GSeC ranges and greater diversity than its cultivated counterparts. The higher Se grain concentrations found in H. spontaneum populations suggest that wild barley germplasm confer higher abilities for Se uptake and accumulation, which can be used for genetic studies of barley nutritional value and for further improvement of domesticated cereals.     

Control of plant growth resides in the shoot,and not in the root,in reciprocal grafts of flacca and wild-type tomato (Lysopersicon esculentum), in the presence and absence of salinity stress

Grafted plants of flacca, an ABA-deficient mutant of tomato (Lycopersicon esculentum), and the wild-type variety Rheinlands Ruhm were grown with and without salinity stress to test the roles of roots and shoots in the regulation of plant growth. Fourteen days after exposure to 200 mM NaCl, shoot and root fresh weight, endogenous ABA concentrations, nitrate concentration, activities of selected enzymes related to nitrogen assimilation, and cation accumulation were determined. Rootstock genotype had little influence on the growth of the grafted plants, whereas grafted plants having wild-type shoots (Ws) produced more biomass than those having flacca shoots (Fs), irrespective of the salinity level. Growth of flacca shoots grafted onto wild-type rootstock (Fs/Wr) was superior to that of flacca shoots grafted onto flacca rootstock (Fs/Fr). The improved growth correlated with enhanced levels of ABA in the flaccashoots of Fs/Wr. In all the graft combinations, ABA content was higher in wild-type shoots than in flacca shoots, with or without salinity. There were no significant differences in root ABA concentrations among the different grafted types. Enhanced growth correlated with higher nitrate levels and higher nitrate reductase activity in the roots and shoots of plants with wild-type shoots and with higher shoot concentrations of ABA in plants with wild-type shoots. There were no significant differences in glutamine synthetase and phosphoenol pyruvate carboxylase activities in the shoots and roots of all the grafted plants, regardless of the salinity level. While shoot genotype determined the accumulation of K⁺ and Na⁺ in grafted plants regardless of salinity, it had no influence on Ca²⁺ concentrations. Regardless of the salinity, the total concentration of cations was the same in all the plants, while salinity decreased Mg²⁺ concentration in roots and shoots of all grafts, with the exception of flacca grafted shoots. The scion genotype – and its ABA level – thus played the major role in the growth of grafted plants, regardless of the rootstock genotype and the salinity of the growth medium.     

中国母草科植物分类及地理分布

对国内外关于母草科(Linderniaceae Borsch,Kai Müller&Eberhard Fischer)系统分类学的研究进展进行了综述,整理和总结了中国母草科植物的分类学变动情况。通过收集中国数字植物标本馆、中国国家标本资源平台、《中国植物志》、Flora of China及《中国生物物种名录》物种分布信息,分析了中国母草科植物的分布格局。结果表明,母草科主要由传统玄参科母草族、腭母草族的类群及原苦苣苔科的侧母草属所构成,共22属220余种,中国分布有8属43种,其中15种为中国特有种。在中国,广西与广东分布的母草科植物最为丰富,新疆、青海和宁夏无母草科植物分布。研究更正了中国母草科植物属的范围及地理分布,以期为母草科植物未来的分类学与生物多样性研究提供参考。     

Circ-U2AF1 promotes human glioma via derepressing neuro-oncological ventral antigen 2 by sponging hsa-miR-7-5p

The prognosis for human glioma, a malignant tumor of the central nervous system, is poor due to its rapid growth, genetic heterogeneity, and inadequate understanding of its underlying molecular mechanisms. Circular RNAs composed of exonic sequences, represent an understudied form of noncoding RNAs (ncRNAs) that was discovered more than a decade ago, function as microRNA sponges. We aimed to assess the relationship between circ-U2AF1 (CircRNA ID: hsa_circ_0061868) and hsa-mir-7-5p and examine their effects on proliferation, apoptosis, and the metastatic phenotype of glioma cells regulated by neuro-oncological ventral antigen 2 (NOVA2). We found that the expression levels of circ-U2AF1 and NOVA2 were upregulated, while hsa-miR-7-5p was downregulated in human glioma tissues and glioma cell lines. Our data and bioinformatic analysis indicated the association of these molecules with glioma grade, a positive correlation between circ-U2AF1 and NOVA2 expression levels and a negative correlation of hsa-miR-7-5p with both circ-U2AF1 and NOVA2, respectively. In addition, silencing of circ-U2AF1 expression resulted in increased hsa-miR-7-5p expression and decreased NOVA2 expression both in vitro and in vivo. Luciferase assay confirmed hsa-miR-7-5p as a direct target of circ-U2AF1 and NOVA2 as a direct target of hsa-miR-7-5p. Functionally, silencing of circ-U2AF1 inhibits glioma development by repressing NOVA2 via upregulating hsa-miR-7-5p both in vitro and in vivo. Thus, we assumed that circ-U2AF1 promotes glioma malignancy via derepressing NOVA2 by sponging hsa-miR-7-5p. Taken together, we suggest that circ-U2AF1 can be a prognostic biomarker and the circ-U2AF1/hsa-miR-7-5p/NOVA2 regulatory pathway may be a novel therapeutic target for treating gliomas.     

Novel Protein Interactions with Endoglin and Activin Receptor-like Kinase 1: Potential Role in Vascular Networks

Endoglin and activin receptor-like kinase 1 are specialized transforming growth factor-beta (TGF-β) superfamily receptors, primarily expressed in endothelial cells. Mutations in the corresponding ENG or ACVRL1 genes lead to hereditary hemorrhagic telangiectasia (HHT1 and HHT2 respectively). To discover proteins interacting with endoglin, ACVRL1 and TGF-β receptor type 2 and involved in TGF-β signaling, we applied LUMIER, a high-throughput mammalian interactome mapping technology. Using stringent criteria, we identified 181 novel unique and shared interactions with ACVRL1, TGF-β receptor type 2, and endoglin, defining potential novel important vascular networks. In particular, the regulatory subunit B-beta of the protein phosphatase PP2A (PPP2R2B) interacted with all three receptors. Interestingly, the PPP2R2B gene lies in an interval in linkage disequilibrium with HHT3, for which the gene remains unidentified. We show that PPP2R2B protein interacts with the ACVRL1/TGFBR2/endoglin complex and recruits PP2A to nitric oxide synthase 3 (NOS3). Endoglin overexpression in endothelial cells inhibits the association of PPP2R2B with NOS3, whereas endoglin-deficient cells show enhanced PP2A-NOS3 interaction and lower levels of endogenous NOS3 Serine 1177 phosphorylation. Our data suggest that endoglin regulates NOS3 activation status by regulating PPP2R2B access to NOS3, and that PPP2R2B might be the HHT3 gene. Furthermore, endoglin and ACVRL1 contribute to several novel networks, including TGF-β dependent and independent ones, critical for vascular function and potentially defective in HHT.Transforming growth factor-β (TGF-β)¹ superfamily ligands, including TGF-βs, activins and bone morphogenic proteins (BMPs), regulate several pathways essential for vascular development and function (1). Responses to these ligands are controlled by type I and II serine kinase receptors, coreceptors and signaling SMAD intermediates. Endothelial cells express the coreceptor, endoglin, and the specialized type I receptor, ACVRL1 (activin receptor-like kinase 1 or ALK1); both molecules are critical for regulation of angiogenesis and vasomotor function by TGF-β superfamily ligands (2, 3).Mutations in ENG and ACVRL1 genes lead to hereditary hemorrhagic telangiectasia (HHT), types 1 and 2, respectively (4). HHT affects 1 in 5000–8000 people worldwide and is characterized by arteriovenous malformations (AVMs) in multiple organs, potentially leading to severe hemorrhages and strokes (4). Haploinsufficiency is the underlying cause of HHT, indicating that reduced levels of functional endoglin or ACVRL1 (ALK1) proteins predispose to endothelial dysfunction and AVMs (5). Although the mechanisms responsible for AVMs remain unclear, the elucidation of how members of the TGF-β superfamily and their molecular networks regulate vascular integrity is vital for future treatments of HHT.We have demonstrated that endoglin interacts with endothelial nitric oxide synthase (NOS3 or eNOS) and regulates its activation (2). NOS3 is a Ca⁺² and calmodulin-regulated enzyme that produces NO● in response to humoral and mechanical stimuli via dynamic interactions with various allosteric regulators such as heat shock protein 90 (HSP90). NOS3 is also regulated by dynamic changes in its phosphorylation status. For example, effects of the vascular endothelial growth factor (VEGF) on angiogenesis, vascular permeability and vasomotor tone are mediated in part through Akt-dependent phosphorylation of NOS3 Ser1177 and by increased NOS3-HSP90 association (6). Although phosphorylation of NOS3 Ser1177 is indicative of agonist-induced activation, it is preceded by dephosphorylation at Thr495. TGF-β1 and -β3 but not -β2 responses can sensitize NOS3 for activation by inducing dephosphorylation at Thr495, and therefore contribute to NOS3 activation and NO-dependent vasorelaxation (7). Endoglin regulates TGF-β1 and -β3 but not -β2 responses, and is required for their induction of NOS3 Thr495 dephosphorylation (7, 8).In the vascular endothelium of HHT patients and in Eng and Alk1 heterozygous mice, impaired association of NOS3 with HSP90 renders the enzyme uncoupled, causing production of superoxide (●O₂⁻) instead of NO● (2, 3, 9) and leading to endothelial damage. Interestingly, TGF-β1 and -β3 do not induce phosphorylation at NOS3 Ser1177, yet NOS3 activation in response to TGF-β1 is abolished in endoglin-deficient cells, impairing vasomotor function (3). ACVRL1 (or ALK1) also interacts with NOS3, and its reduced levels in endothelial cells similarly cause NOS3-derived oxidative stress (3, 9).In view of the crucial roles of endoglin and ACVRL1 in the development and maintenance of the normal vasculature and the definite contribution of their mutated state to HHT, we used the LUMIER high-throughput technology (10) to identify novel protein interactions and molecular networks for these predominantly endothelial receptors. We included TGFBR2 to further define TGF-β protein networks potentially important for vascular function, and attempt to distinguish the TGF-β networks from those associated with BMP9/BMP10 and mediated by ACVRL1 in a complex with BMPR2 and endoglin (11, 12).One of identified proteins interacting with all three receptors was protein phosphatase 2A (PP2A), implicated in multiple pathways. PP2A is a holoenzyme with one structural subunit (PPP2R1A or PPP2R1B) associated with one catalytic subunit (PPP2CA or PPP2CB) and one of 19 regulatory B subunits, the latter conferring specificity to the enzyme by recruiting interacting proteins (13, 14). Of interest, PP2A interacts with NOS3 to regulate Ser1177 phosphorylation and NO● production (15). However, the mechanisms governing recruitment of PP2A to NOS3 and the contribution of TGF-β/BMP receptor complexes are unknown. Recently, the human PPP2R2B gene coding for PPP2R2B protein (also known as PP2A-Bβ regulatory subunit) was mapped to chromosome 5q31-q32, in an interval in linkage disequilibrium with the HHT3 locus (16, 17). We now report that PPP2R2B interacts with the ACVRL1/TGFBR2/endoglin complex and that endoglin governs NOS3 phosphorylation and activation status by hindering PP2A access to NOS3 via the PPP2R2B subunit. Loss of endoglin leads to constitutive reduction in NOS3 phosphorylation and likely to changes in several networks with consequent endothelial dysfunction.     

Microcycle conidiation and the conidial properties in the entomopathogenic fungus Metarhizium acridum on agar medium

Conidiation of the entomopathogenic fungus Metarhizium acridum on agar media was investigated. M. acridum CQMa102 exhibits two different conidiation patterns on agar media: normal conidiation in which conidia are formed on extended hyphae and microcycle conidiation in which conidiation occurs directly after conidia germination. Microcycle conidiation resulted in a mass of conidia produced via budding by accelerated development at the inoculation site. The mean total conidial yield (conidiation at day 10) was 4–5-fold greater after microcycle conidiation than during normal conidiation. Insect pathology assays indicated that microcycle conidia produced on SYA agar were as effective as normal aerial conidia against the locust. Ultraviolet (UV)-resistance tests showed no significant differences between the two types of cell propagules. However, microcycle conidia were more heat resistant than normal aerial conidia, and accumulated higher levels of trehalose in response to heat induction compared to normal aerial conidia.     

Establishment and characterization of a novel ‘double‐hit’ follicular lymphoma cell line,FL‐SJC

About 5 per cent of follicular lymphoma (FL) cases are double‐hit (DH) lymphomas. Double‐hit follicular lymphoma (DHFL) cell lines can improve our understanding and drug development on FL. But there are only few DHFL cell lines. Here, we established a new MYC/BCL2 DHFL cell line, FL‐SJC. The cells were obtained from the hydrothorax of a patient with MYC/BCL2 DHFL and cultured for 140 passages in vitro. FL‐SJC cells demonstrated CD19⁺⁺, CD20⁺, CD22⁺⁺, HLA‐DR⁺, CD10⁺, CD38⁺, Lambda⁺ CD23^‐, CD5^‐ and Kappa^‐. The chromosome karyotypic analysis confirmed the co‐existence of t(8;22)(q24;q11) and t(14;18)(q32;q21), as well as additional abnormalities involving chromosomes 2 and 3. Fluorescence in situ hybridization analysis (FISH) showed IGH/BCL2 fusion gene and the MYC rearrangement. In addition, the FL‐SJC cells displayed KMT2D/MLL2 and CREBBP gene mutations. After subcutaneous inoculation of FL‐SJC cells, the SCID mice developed solid tumour masses within 6‐8 weeks. FL‐SJC cells were proven to be free of Epstein‐Barr (EB) virus infection and be multidrug‐resistant. In a conclusion, the FL‐SJC cell line has been identified as a novel MYC/BCL2 double‐hit follicular lymphoma that can be used as a potentially available tool for the clinical and basic research, together with the drug development for MYC/BCL2 DHFL.     

Isolation of High-Quality RNA from <Emphasis Type="Italic">Reaumuria soongorica</Emphasis>, a Desert Plant Rich in Secondary Metabolites

RNA isolation is a prerequisite for the study of the molecular mechanisms of stress tolerance in the desert plant Reaumuria soongorica, an extreme xeric semi-shrub. However, R. soongorica that contains high levels of secondary metabolites that co-precipitate with RNA, making RNA isolation difficult. Here the authors propose a new protocol suitable for isolating high-quality RNA from the leaves of R. soongorica. Based on a CTAB method described by Liu et al., the protocol has been improved as follows: the samples were ground with PVPP to effectively inhibit the oxidation of phenolics, contaminating DNA was removed with DNase I, and NaAc was used along with ethanol for precipitation to enhance the RNA yield and shorten the precipitation time. Gel electrophoresis and spectrophotometric analysis indicated that this isolation method provides RNA with no DNA contamination. Moreover, the yield (183.79 ± 40.36 μg/g) and quality were superior to those using the method of Liu et al., which yields RNA with significant DNA contamination at 126.30 ± 29.43 μg/g. Gene amplification showed that the RNA obtained using this protocol is suitable for use in downstream molecular procedures. This method was found to work equally well for isolating RNA from other desert plants. Thus, it is likely to be widely applicable.     

The mechanism of the mycoinsecticide diluent on the efficacy of the oil formulation of insecticidal fungus

Adverse conditions, including low humidity, UV irradiation, and high temperature, appreciably affect the efficacy of mycoinsecticides. Oil formulation increased the virulence of Metarhizium anisopliae var. acridum (Ascomycota: Hypocreales) against locusts and grasshoppers by reducing the dependence on saturated water. A mycoinsecticide diluent (a water-in-oil emulsion) has been widely used to dilute the oil formulation of M. anisopliae in China. The aim of our study was to elucidate the mechanism by which the mycoinsecticide diluent improves the virulence of M. anisopliae. We investigated the effects of the mycoinsecticide diluent on the virulence, invasion speed, and viability of the conidia under various adverse conditions. The results demonstrated that the mycoinsecticide diluent significantly improved the virulence of conidia at low humidity (68, 75, and 84%). In particular, at an RH of 68%, the LT₅₀ for locusts treated with the emulsion was 5.4 days and was 31.6% lower than the value for locusts treated with an oil formulation. In addition, the concentration of the hyphal bodies found in the haemolymph of locusts treated with emulsion was about 27-fold higher than that in locusts treated with oil formulation four days after inoculation. This result was further confirmed by determining the concentration of M. anisopliae var. acridum DNA in locust haemolymph using quantitative PCR. The percentage germination of conidia in the emulsion was also significantly higher than that in oil at 68% RH. There was no significant difference in percentage germination between conidia treated with the emulsion and oil when exposed to irradiation with ultraviolet-B (UV-B) or high temperature. These results demonstrate that the mycoinsecticide diluent enhances the virulence of M. anisopliae formulated in oil at low humidity by providing adequate water for germination without interfering with the UV tolerance and thermotolerance of the conidia.