The mechanism of sex-specific splicing at the doublesex gene is different between Drosophila melanogaster and Bombyx mori

We have previously reported that Bmdsx, a homologue of the sex-determining gene, doublesex (dsx), was found to be sex-specifically expressed in various tissues at larval, pupal, and adult stages in the silkworm, Bombyx mori, and was alternatively spliced to yield male- and female-specific mRNAs. To reveal sex-specific differences in splicing patterns of Bmdsx pre-mRNA, the genomic sequence was determined and compared with male- and female-specific Bmdsx cDNA sequences. The open reading frame (ORF) consisted of five exons. Exons 3 and 4 were specifically incorporated into the female type of Bmdsx mRNA. On the other hand, exon 2 was spliced to exon 5 to produce the male type mRNA of Bmdsx. As in the case of Drosophila dsx, the OD2 domain was separated by a female-specific intron into sex-independent and sex-dependent regions. Sex-specific splicing occurred in equivalent positions in the Drosophila dsx gene. However, unlike Drosophila dsx, the female-specific introns showed no weak 3′ splice sites, and the TRA/TRA-2 binding site related sequences were not found in the female-specific exon, nor even in any other regions of the Bmdsx gene. Moreover, an in vitro splicing reaction consisting of HeLa cell nuclear extracts showed that the female-type of Bmdsx mRNA represented the default splicing. These findings suggest that the structural features of the sex-specific splicing patterns of Bmdsx pre-mRNA are similar to those of Drosophila dsx but the regulation of sex-specific alternative splicing of Bmdsx pre-mRNA is different.     

鹅掌楸LcUGE基因的克隆和组织表达分析

为了解鹅掌楸(Liriodendron chinense)的UGE基因功能,采用RACE和EPIC-PCR技术克隆到2个UGE基因,命名为LcUGE1和LcUGE2。结果表明,LcUGE1基因的c DNA全长为1 531 bp,包含1 050 bp的开放阅读框,编码349个氨基酸, gDNA长度为11 920 bp;LcUGE2基因的c DNA长度为1 378 bp,包含1 056 bp的开放阅读框,编码351个氨基酸,g DNA长度为6544 bp。LcUGE1和LcUGE2基因均含有9个外显子和8个内含子,且外显子长度和内含子剪切位点序列几乎一致,但内含子片段长度存在显著差异。编码的LcUGE1和LcUGE2蛋白高度保守,保守性达到82%。LcUGE1基因在雄蕊中表达量最高,而LcUGE2基因则在花萼中表达量最高。这表明LcUGEs基因可能参与鹅掌楸的生殖发育过程。     

Natural occurrence of Wolbachia-infected and uninfected Trichogramma species in tomato fields in Portugal

Minute egg parasitoids of the genus Trichogramma (Hymenoptera; Trichogrammatidae) are promising candidates for biological control of lepidopteran pests in tomato in Portugal. This certainly applies to native Trichogramma strains that have thelytokous reproduction, i.e., produce only daughters. In Trichogramma wasps, thelytoky is mostly induced by the intracellular bacterium Wolbachia. In this study, we carried out a field survey of native Trichogramma species in four locations in Ribatejo, the main processing tomato region of Portugal, and determined the prevalence of Wolbachia in those species. Five Trichogramma species were found to emerge from lepidopteran eggs collected in the field, namely Trichogramma bourarache, Trichogramma cordubensis, Trichogramma evanescens, Trichogramma pintoi, and Trichogramma turkestanica. T. evanescens and T. pintoi were by far the dominating species representing, respectively, 64.9 and 26.4% of the trichogrammatids collected. Total natural parasitism rates of the collected lepidopteran eggs by Trichogramma wasps ranged from 28.2 to 64.6%. Three Trichogramma species were found to be infected with Wolbachia, namely T. cordubensis, T. evanescens, and T. turkestanica. All the wasp broods belonging to T. cordubensis were infected, whereas low infection rates were found in T. evanescens (0.9% of the broods) and T. turkestanica (4.5% of the broods). The latter represents the first record of a Wolbachia infection in T. turkestanica. Sequencing of the Wolbachia surface protein, wsp, revealed this Wolbachia infection to be related to other Wolbachia infections in Trichogramma wasps. As Wolbachia-infected thelytokous strains exist for T. evanescens, the most abundant Trichogramma species naturally occurring in the tomato fields of the Ribatejo region, this species offers interesting and powerful options for biological control of lepidopteran pests in processing tomato in this region.     

麻竹miR172a靶基因DlAP2的克隆及其表达

为了解开花麻竹(Dendrocalamus latiflorus)的Dl AP2基因功能,采用RT-PCR和RACE技术克隆了mi R172a靶基因AP2同源序列c DNA全长,命名为Dl AP2。结果表明,Dl AP2基因c DNA全长为1729 bp,包含5′端非编码区81 bp、开放阅读框1464 bp、3′端非编码区160 bp和24个碱基的Poly A尾巴,在编码框靠近3′端130 bp处有1个高度匹配mi R172a的结合位点(CTGCAGCATCATCAGGATTCT)。Dl AP2编码487个氨基酸的蛋白,具有两个AP2结构域,属于AP2/ERF家族AP2亚家族的AP2组,与来自其它单子叶植物的AP2蛋白均有较高同源性。RLM-5′RACE分析表明,mi R172a主要在靶序列的第11~12个碱基之间剪切靶基因Dl AP2的mi RNA。q RT-PCR结果表明,麻竹花芽中Dl AP2基因的表达规律与mi R172a表达变化正好相反,证明mi R172a对Dl AP2基因的表达具有调控作用。     

Phylogenetically distinct Wolbachia gene and pseudogene sequences obtained from the African onchocerciasis vector Simulium squamosum

Wolbachia are intracellular bacteria mostly found in a diverse range of arthropods and filarial nematodes. They have been classified into seven distinct ‘supergroups’ and other lineages on the basis of molecular phylogenetics. The arthropod-infecting Wolbachia are usually regarded as reproductive parasites because they manipulate their host species’ sexing system to enhance their own spread, and this has led to their investigation as potential agents of genetic control in medical entomology. We report 12 partial Wolbachia gene sequences from: aspC, aspS, dnaA, fbpA, ftsZ, GroEL, hcpA, IDA, rpoB, rpe, TopI and wsp as well as a single ftsZ pseudogene sequence, which have all been PCR-amplified from Simulium squamosum (Diptera: Simuliidae). To our knowledge this is the first such report from Simuliidae. Uninterrupted open-reading frame sequences were obtained from all 12 genes, covering ∼6.2 kb of unique DNA sequence. Phylogenetic analyses with the different coding genes gave consistent results suggesting that the Wolbachia sequences obtained here do not derive from any of the known Wolbachia supergroups or lineages. Consistent with a unique genetic status for the S. squamosumWolbachia, the hypervariable regions of the Wolbachia-specific wsp gene were distinct from all previous records in both sequence and length. As well as potential implications for newly emerging Wolbachia-based disease control methods, the results may be relevant to some problems experienced in the laboratory colonisation of Simulium damnosum sensu lato and why it is such a diverse species complex.     

Microbial diversity in the predatory mite Metaseiulus occidentalis (Acari: Phytoseiidae) and its prey, Tetranychus urticae (Acari: Tetranychidae)

Microorganisms associated with the predatory mite Metaseiulus (=Typhlodromus or Galendromus) occidentalis (Nesbitt) and its prey, the two-spotted spider mite Tetranychus urticae (Koch), were assessed using a high-fidelity polymerase chain reaction (PCR) protocol and primers designed to identify Eubacteria, Archaeabacteria, iridoviruses, Helicosporidia, Cytophaga-like microorganisms, Wolbachia and its bacteriophage WO, fungi and yeast-like organisms. Sequences from four bacterial species related to Wolbachia (α-Proteobacteria), Cardinium, Bacteroidetes, and Enterobacter (γ-Proteobacteria) were obtained from M. occidentalis, and three sequences related to Wolbachia, Rickettsia, and Caulobacter (α-Proteobacteria) were obtained from T. urticae. No nucleotide differences were detected between the 16S rRNA, wspA or wspB Wolbachia sequences obtained from M. occidentalis and T. urticae, which suggest that horizontal transfer of Wolbachia could have occurred. Southern blot analyses of genomic DNA from both M. occidentalis and T. urticae using wspA probes were negative, indicating that this Wolbachia sequence is not integrated into the nuclear genome of either species. Two of the T. urticae colonies tested contained the WO bacteriophage, but none of the six M. occidentalis populations were infected. New M. occidentalis-specific forward and reverse 16S rRNA primers based on the Wolbachia, Cardinium, Bacteroidetes, and Enterobacter sequences obtained were designed and used to amplify PCR products from each of two laboratory and four field-collected samples of M. occidentalis females and eggs, indicating that these infections are widespread. Likewise, species-specific primers for T. urticae were designed for the Wolbachia, Rickettsia, and Caulobacter sequences obtained and used to evaluate T. urticae from strawberries, wine grapes, hops, almonds, and cherries from California, Washington, and Florida; all were positive for Wolbachia and Caulobacter but two of the six were negative for Rickettsia. None of the M. occidentalis colonies tested were positive for the microsporidium Oligosporidium occidentalis, which previously had been associated with a pathogenic condition in some of our laboratory colonies. The Gainesville colonies of M. occidentalis and T. urticae were negative for iridovirus, Archaeabacteria, fungi, Helicosporidia, and yeast-like organisms. So far, Wolbachia is the only symbiont that is shared by this predator and its prey.     

Characterization of an Azospirillum brasilense Sp7 gene homologous to Alcaligenes eutropbus pbbB and to Rbizobium meliloti nodG

Summary A 4 kb SalI fragment from Azospirillum brasilense Sp7 that shares homology with a 6.8 kb EcoRI fragment carrying nodGEFH and part of nodP of Rhizobium meliloti 41 was cloned in pUC18 to yield pAB503. The nucleotide sequence of a 2 kb SalI-SmaI fragment of the pAB503 insert revealed an open reading frame, named ORF3, encoding a polypeptide sharing 40% identity with R. mehloti NodG. The deduced polypeptide also shared 60% identity with the Alcaligenes eutrophus NADPH-dependent acetoacetyl-CoA (AA-CoA) reductase, encoded by the pbbB gene and involved in poly--hydroxybutyrate (PHB) synthesis. Northern blot analysis and promoter extension mapping indicated that ORF3 is expressed as a monocistronic operon from a promoter that resembles the Escherichia coli ⁷⁰ consensus promoter. An ORF3-lacZ translational fusion was constructed and was very poorly expressed in E. coli, but was functional and constitutively expressed in Azospirillum. Tn5-Mob insertions in ORF3 did not affect growth, nitrogen fixation, PHB synthesis or NAD(P)H-linked AA-CoA reductase activity. An ORF3 DNA sequence was used to probe total DNA of several Azospirillum strains. No ORF3 homologues were found in A. irakense, A. amazonense, A. halopraeferens or in several A. lipoferum strains.     

Differential fluctuation in virulence and VOC profiles among different cultures of entomopathogenic fungi

Insect-passaged cultures of entomopathogenic fungi grown on potato dextrose agar media have been shown to have altered virulence and profiles of volatile compounds. The present study demonstrated the pathogenic status of FS₀ (in vitro) and FS₁ and FS₂ (insect-passaged cultures grown on PDA) cultures of Metarhizium anisopliae (strains 406 and 02049) and Beauveria bassiana by a non-choice assay, in which filter paper was inoculated with fungal spores at a concentration of 1 × 10⁷ spores/ml. The FS₁ and FS₂ cultures of M. anisopliae strain 02049 and B. bassiana produced conidia with high virulence, and the volatile profiles of these conidia comprised relatively lower percentages of branched-alkanes than conidia from the FS₀ cultures. In contrast, the conidia from an FS₀ culture of M. anisopliae strain 406 had somewhat elevated virulence levels, but their volatile profile had <2% branched-alkanes. The FS₁ and FS₂ cultures of M. anisopliae strain 406 did not gain virulence, and these cultures showed a decline in virulence along with major alteration of their volatile profiles. Their volatile profiles mainly comprised branched-alkanes. The volatile profiles of the FS₁ and FS₂ cultures lacked n-tetradecane, which was an important component of all the virulent cultures. Four compounds, 2-phenylpropenal, 2,5,5-trimethyl-1-hexene, n-tetradecane and 2,6-dimethylheptadecane, were detected only from the virulent cultures, suggesting that low LT₅₀ values were probably due to the production of these compounds. This is the first report to characterize volatiles from FS₀, FS₁ and FS₂ cultures of entomopathogenic fungi; its utility in different aspects opens an interesting area for further investigations.     

In vitro cytogenetic effects of Andrographis paniculata (kalmegh) on arsenic

In vitro effects of arsenic in human peripheral lymphocytes (HPL) at three different doses – 3.6×10⁻⁴, 1.4×10⁻³ and 0.72×10⁻³ μM for 24 h before harvesting on sister chromatid exchanges (SCE), Cell cycle proliferative index/replicative index (CCPI/RI), %M₁, %M₂ and %M₃, population doubling time (PDT) and average generation time (AGT) were examined. Andrographis paniculata (commonly referred to as ‘kalmegh’) has been used for centuries in traditional Indian and Chinese herbal medicine as a safe, natural folk remedy for assorted health concerns. In the present study, kalmegh (0.01 μg/7 ml culture media) was used along with the highest dose of arsenic; the results showed that arsenic induced increase in these genotoxic endpoints were fairly diminished by kalmegh. In addition, mutagenic in vitro effect of ethyl methanesulphonate (EMS) was used as a positive control in this study. It is thus concluded from this study that Andrographis has a protective role in arsenic toxicity.     

苦荞转录因子基因FtbHLH4的克隆及其对非生物胁迫的应答

根据苦荞花期转录组数据,克隆得到1个苦荞bHLH类转录因子基因,命名为FtbHLH4。采用实时荧光定量PCR技术,分析了非生物胁迫对苦荞芽期FtbHLH4基因表达的影响。序列分析表明,苦荞FtbHLH4基因DNA全长1 852bp,由7个外显子和6个内含子构成,符合GT-AG剪接原则;cDNA序列包含1个1 062bp的开放阅读框,编码353个氨基酸,具有bHLH类蛋白典型的螺旋-环-螺旋保守结构域。在脱落酸(ABA)、NaCl和PEG模拟干旱胁迫下,苦荞芽期FtbHLH4基因表达量均持续上升,至48h时达最大,分别为胁迫前的11.3倍、12.0倍和6.1倍。而在冷胁迫和UV-B胁迫下,苦荞芽期FtbHLH4基因表达量迅速下降,分别于6h和12h降低至胁迫前的24%和23%。研究推测FtbHLH4基因以不同机制参与了苦荞对非生物胁迫的应答过程。     

Physiological adaptation of the Asian corn borer Ostrinia furnacalis to chemical defenses of its host plant, maize

A number of gramineous plants such as maize contain cyclic hydroxamic acids (cHx) that are toxic to many herbivores. Among the Ostrinia species found in Japan, the Asian corn borer Ostrinia furnacalis is the only one that utilizes maize, a gramineous plant. We used O. furnacalis and two congeners, Ostrinia scapulalis and Ostrinia latipennis, to obtain insights into the physiological adaptation of O. furnacalis to cHx. When an artificial diet containing a low concentration (0.3 mg/g diet) of cHx was fed to the larvae of O. furnacalis and O. scapulalis, larval growth and survival were significantly less affected in O. furnacalis than O. scapulalis. An artificial diet containing a high level (0.7 mg/g diet) of cHx was found to severely retard the growth of both species, albeit to different degrees. In an assay in vitro, homogenate of the digestive tract of O. furnacalis larvae degraded cHx more rapidly than that of O. scapulalis or O. latipennis. The degradation was found to be enzymatic and dependent on a cofactor, UDP-glucose, suggesting that UDP-glucosyltransferase or other UDP-glucose-dependent enzymes were involved. This enzymatic adaptation probably has enabled O. furnacalis to utilize plants containing cHx.     

牛支原体LRR5蛋白原核表达及其在牛支原体入侵宿主细胞过程中的作用分析

致病性支原体具有入侵宿主细胞的能力,这是其发挥致病作用的关键。介导支原体入侵宿主细胞的自身功能蛋白可能是一种潜在的药物或疫苗靶标。【目的】克隆表达牛支原体(Mycoplasmabovis) MBOVPG45_0564基因编码蛋白(命名为LRR5蛋白),并探究其在M. bovis入侵宿主细胞过程中的作用。【方法】利用NCBI数据库对MBOVPG45_0564基因进行同源性分析,用Discovery Studio Client系统对LRR5蛋白进行蛋白结构预测;原核表达LRR5蛋白并制备其小鼠多克隆抗体,利用免疫电镜对LRR5蛋白进行亚细胞定位;通过平板计数、激光共聚焦显微镜观察LRR5抗体封闭后M. bovis对胎牛肺(embryonic bovine lung, EBL)细胞入侵率的变化;将LRR5蛋白偶联至荧光微球表面后,以激光共聚焦显微镜及高内涵活细胞成像系统观察微球进入EBL细胞情况。【结果】MBOVPG45_0564基因在牛支原体属中为保守基因,其编码蛋白LRR5为膜相关蛋白,空间构象呈典型的月牙状,多个重复的亮氨酸基序以超螺旋方式组装并形成螺线管蛋白质结构单元。LRR5抗体封闭后,M. bovis对EBL细胞的入侵率显著降低(P<0.05),荧光微球偶联LRR5蛋白后,荧光微球可成功进入EBL细胞。【结论】MBOVPG45_0564基因编码的LRR5蛋白定位在M. bovis膜上,在M. bovis入侵宿主细胞过程中发挥着重要作用。