Apparent mRNA instability in Aspergillus nidulans and Aspergillus terreus of a heterologous cDNA encoding the major capsid antigen of Rotavirus

Two expression plasmids designed to produce the rotaviral VP6 protein in Aspergillus nidulans and Aspergillus terreus have been constructed. In one of these plasmids the inducible A. terreus Gla1 glucoamylase gene promoter and Gla1 signal sequence are fused to the VP6 cDNA to enable induction and extracellular secretion of the final protein product; in the other, the strong, constitutive A. nidulans gpdA gene promoter has been employed. A. nidulans and A. terreus transformants containing intact copies of these plasmids have been obtained but neither intra- nor extra-cellular VP6 protein was detectable. Northern analysis indicated specific degradation of the VP6 mRNA. This lack of VP6 mRNA stability may be related to fundamental differences between the general structure of Aspergillus mRNA and that of rotavirus, including codon usage and AU/GC ratio.     

Transformation of Aspergillus terreus with the hygromycin B resistance marker from Escherichia coli

Aspergillus terreus was transformed to hygromycin B resistance using a bacterial resistance gene under the control of Aspergillus nidulans regulatory sequences. Southern hybridization of transformants indicated that in most of the cases the vector DNA was integrated into the recipient chromosome in the form of tandem arrays. Transformants were mitotically stable in both selective and non-selective medium and retained their capacity to produce xylanase or glucoamylase activities.     

Features of integration of recombinant cosmids, containing Aspergillus terreus DNA, in the Saccharomyces cerevisiae genome

A genome clonotheque of 25-40 kb Sau3A fragments of Aspergillus terreus DNA was constructed in the episomal cosmid vector pES33 containing the ARG4 gene of yeast. 23 independently originated stable Arg+ transformants were selected after transformation of the cir0 yeast strain ESH-O with pooled cosmid molecules. Both genetic and Southern analysis showed that 39% of these stable transformants occurred due to recombination between DNA sequences from A. terreus and Saccharomyces cerevisiae chromosome XII which took place most likely in the regions of homology within the ribosomal clusters. The data present the first evidence of in vivo recombination between foreign sequences and their S. cerevisiae counterparts.     

稻瘟净（Blasticidin S）脱氨酶基因的克隆

从一株抗稻瘟净（ＢＳ）的Ａｓｐｅｒｇｉｌｌｕｓ ｔｅｒｒｅｕｓ菌中克隆到一个ｂｌａｓｔｉｅｉｄｉｎＳ脱氨酶基因,命名为ｂｓｒＡＳ。ＤＮＡ序列分析表明ｂｓｒＡＳ不含内含子。编码区长３９０ｂｐ,编码１３０个氨基酸。将ｂｓｒＡＳ转化到稻瘟菌中,能使受体菌表达出ＢＳ脱氨酶的活性,从而产生抗药性。该基因可作为抗药标记基因使用,建立稻瘟菌的基因转化系统。     

Transformation of Aspergillus niger using the argB gene of Aspergillus nidulans

A mutant of Aspergillus niger defective in ornithine transcarbamylase function was transformed with plasmids carrying a functional copy of the argB gene of Aspergillus nidulans after treatment of spheroplasts in the presence of polyethylene glycol and calcium ions. The plasmid pDG3 gave stable transformants at a frequency of 4 per microgram of input DNA. Southern blot analysis of DNA from transformants showed that pDG3 DNA had integrated into the A. niger chromosomes at a variety of locations. The transformants were phenotypically stable for many mitotic divisions. This procedure may potentially be used to insert any gene into the genome of A. niger. A cosmid shuttle vector, pDG1, for cloning in Aspergillus was also constructed.     

Transformation of Aspergillus nidulans by the orotidine-5'-phosphate decarboxylase gene of Neurospora crassa

Relief of an auxotrophic requirement for uridine in Aspergillus nidulans strain G191 has been achieved by transformation with a segment of Neurospora crassa DNA containing the corresponding gene coding for orotidine-5'-phosphate decarboxylase. The mitotic stability of such transformants suggests that the DNA has integrated into the genome. Southern hybridisation analysis of DNA isolated from transformants revealed the presence of pBR322 sequences which have integrated into the host genome along with the N. crassa DNA.     

Integrative Transformation of Aspergillus oryzae with a Plasmid Containing the Aspergillus nidulans argB Gene

The transformation of Aspergillus oryzae has been achieved with a plasmid carrying the Aspergillus nidulans argB gene coding for ornithine carbamoyltransferase (OCTase). The frequency of transformation was relatively low (0.7 transformants/μg DNA) but the transformed phenotype was extremely stable for many generations without selective pressure.

Southern blot analysis revealed that transformation had occurred by integration of multiple tandem copies of plasmid DNA into the host genome through non-homologous recombination. There was no evidence of the existence of free plasmid in the transformants. The number of integrated copies of the plasmid ranged from 15 to 60. The specific activity of OCTase in the cell- free extract was proportional to the copy number of the plasmid, indicating that most of the integrated argB gene was expressed.     

Transformation of Aspergillus alliaceus using the Aspergillus niger niaD gene encoding nitrate reductase

Abstract A heterologous transformation system for Aspergillus alliaceus based on the Aspergillus niger nitrate reductase structural gene ( niaD ) has been developed. Two mutants of A. alliaceus (M3 and M17), each carrying an niaD mutation were isolated by screening UV-irradiated cells for the inability to grow on nitrate as sole nitrogen source. Using plasmid pSTA 10, transformation frequencies of 4 and 200 per μg DNA respectively were obtained for these two strains. All the niaD ⁺ transformants tested were mitotically stable. Southern hybridisation analyses showed that the vector DNA sequences were present.     

RNAi沉默lovF基因实现土曲霉一步法发酵生产辛伐他汀

辛伐他汀是重要的降胆固醇处方药物。莫那可林J是辛伐他汀合成过程的关键中间体,也是洛伐他汀生物合成的中间产物。为获得莫那可林J并通过一步法发酵生成辛伐他汀,构建了洛伐他汀生物合成关键基因lov F基因的RNAi载体p MHJ137,通过农杆菌介导的方法转化土曲霉F001,筛选整合到染色体的阳性菌,并在阳性菌株MJ1-24的发酵过程中加入了DMB-S-MMP验证其直接合成辛伐他汀的效率。结果显示MJ1-24不再产生洛伐他汀,产物中仅有莫那可林J累积。如果在发酵过程中加入前体物质,可得到产物辛伐他汀。综上,RNAi技术能够有效实现土曲霉基因沉默。此项技术推进了一步法发酵生产辛伐他汀的工艺开发。     

Onychomycosis caused by Aspergillus terreus.

Four cases of onychomycosis caused by Aspergillus terreus are presented. The clinical characteristics consisted of spotted and striated leuconychia, dark spots and fragility of the nails. The mycology is described, and is shown that A. terreus has an inhibitory effect on the growth of dermatophytes and Scopulariopis brevicaulis. Topical treatment with 1% Pimafucin in a mixture of 60% dimethylsulfoxide (DMSO) and 40% water as well as 9% aqueous solution of sodium benzoate were both effective. The literature concerning onychomycosis caused by A. terreus is summarized.     

Cloning of the nitrate reductase gene (niaD) of Aspergillus nidulans and its use for transformation of Fusarium oxysporum

An heterologous transformation system for the phytopathogenic fungus Fusarium oxysporum has been developed based on the use of the Aspergillus nidulans nitrate reductase gene (niaD). F. oxysporum nia- mutants were easily selected by chlorate resistance. The A. nidulans niaD gene was isolated from a gene library by complementation of an A. nidulans niaD mutant. The cloned gene is capable of transforming F. oxysporum nia- mutants at a frequency of up to ten transformants per microgram of DNA. Southern analysis of the DNA of the F. oxysporum transformants showed that transformation resulted in integration of one or more copies of the vector DNA into the genome.     

Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori

Many transformation methods have been developed to introduce DNA into filamentous fungi. One of these methods is Agrobacterium-mediated transformation (AMT). Here, we describe an efficient protocol for AMT of Aspergillus awamori. This protocol has been used to determine the function of Agrobacterium virulence genes during AMT, to identify factors influencing transformation frequencies, to generate insertional mutants and to generate A. awamori gene knockout transformants. This protocol in not only applicable to A. awamori, but can be used as a more general guideline for AMT of other filamentous fungi. Conidiospores are incubated with induced Agrobacterium, and, after a cocultivation and selection period, hygromycin-resistant transformants are obtained with a frequency of 200-250 transformants per 1 x 10(6) conidiospores. Using this protocol, transformants can be obtained within 10-12 d.     

Aspterric acid and 6-hydroxymellein,inhibitors of pollen development in Arabidopsis thaliana,produced by Aspergillus terreus

Aspterric Acid, 6-Hydroxymellein, Arabidopsis thaliana, Aspergillus terreus Aspterric acid (1) and 6-hydroxymellein (2), inhibitors of pollen development in Arabidopsis thaliana, have been isolated from the fungus Aspergillus terreus. 1 and 2 inhibited the pollen development at concentrations of 38 and 52 microM, respectively. The microscopic examination of pollen development suggested that the inhibition by the treatment with 1 caused at meiosis and the inhibition by the treatment with 2 caused at microspore stage. 1 and 2 could be useful agents for the molecular investigation of anther and pollen development in higher plants.     

Genetic Transformation of an argB Mutant of Aspergillus oryzae

An argB mutant of Aspergillus oryzae NRRL 492 has been genetically transformed with the Aspergillus nidulans argB gene. Protoplasts were generated with a combination of Novozyme 234 and β-glucuronidase and regenerated on sucrose-stabilized minimal medium without arginine as described for A. nidulans. A frequency of 5 to 10 transformants per μg of DNA was obtained; however, most transformants appeared abortive. The A. nidulans argB gene and vector sequences appeared to be integrated into the A. oryzae chromosome.     

Detection of Aspergillus in lung and other tissue samples using the MycAssay Aspergillus real-time PCR kit

The MycAssay? Aspergillus real-time PCR kit was tested on tissues from patients with invasive fungal infections. Tissue samples from nine organ transplant recipients and 33 patients with haematological malignancy were from lung (n = 30), skin (n = 4), and others. Samples were preprocessed with proteinase K and lyticase, followed by DNA extraction and real-time PCR. For all samples, the sensitivity of the MycAssay Aspergillus test was 82% and specificity 79% relative to microscopy and 90% and 64%, respectively, compared with Aspergillus culture. The positive predictive value and negative predictive values compared with culture were 69% and 88% and were 88% and 69% compared with microscopy, respectively. The MycAssay Aspergillus test detected tissue invasive infections with Aspergillus fumigatus , Aspergillus flavus , and Aspergillus terreus.     

Transformation of Aspergillus sp. and Trichoderma reesei using the pyrithiamine resistance gene (ptrA) of Aspergillus oryzae

A pyrithiamine (PT) resistance gene (ptrA) was cloned from a PT resistant mutant of Aspergillus oryzae and was useful as a dominant selectable marker for transformation of all A. oryzae wild type strain as well as A. nidulans. For further study, we examined whether or not ptrA could be used as the transformation marker in other species of filamentous fungi. Two types of plasmid, which contain ptrA as a selectable marker, were constructed, and the transformation experiments were done with them. One is an integrative plasmid, pPTRI, and another is the autonomously replicating plasmid pPTRII, which contains AMA1. PT-resistant transformants were obtained in the cases of A. kawachii, A. terreus, A. fumigatus, and Trichoderma reesei as hosts with pPTRI and pPTRII. Furthermore, a beta-glucuronidase (GUS) gene was introduced into A. kawachii and A. fumigatus using pPTRII. Almost all the transformants turned blue on GUS assay plates. These results indicate that ptrA can also be used for some other filamentous fungi besides A. oryzae and A. nidulans.     

曲霉生物膜的形成过程与结构特征

目的 研究曲霉生物膜的形成过程和结构特征.方法 我们利用一个曲霉生物膜体外模型研究其形成过程和结构特征.将200 μL浓度为1×10<'5>孢子/mL的受试曲霉(烟曲霉AF293,黄曲霉BMU03940,土曲霉BMU00802,黑曲霉BMU04689)的孢子悬液加到24孔组织培养板中的无菌塑料细胞培养盖玻片上,37℃孵育不同时间(0、2、4、8、10、12、16、18、24、48、72 h),加入25 μmol/L的FUN-1室温避光染色后,用波长488 nm激光激发,通过共聚焦激光扫描显微镜观察曲霉生物膜的形成过程;再用波长为488 am和633 am激光同时激发,将两个波长下的图像叠加后观察曲霉生物膜的活力;利用:XYZ轴成像观察其结构特征.在上述不同的时间点用钙荧光白染色后,用波长为405 nm的紫外光激发,观察曲霉生物膜细胞外基质的产生.结果 烟曲霉AF293在第4 h即开始有散在的孢子黏附于盖玻片上;8 h时孢子开始萌芽,10～12 h菌丝延长形成单细胞层;16～20 h菌丝缠绕形成多层立体结构;24 h形成一个具有复杂的三维立体结构特征的多细胞菌落,菌丝有序排列,细胞外基质弥散的分布在菌丝的周围;48～72 h生物膜逐渐成熟.成熟的烟曲霉生物膜是由细胞外基质包裹的有序排列的菌丝形成的复杂立体结构.黄曲霉BMU03940、土曲霉BMU00802、黑曲霉BMU04689与烟曲霉AF293有类似的生物膜发育阶段,包括黏附、孢子萌芽、菌丝延长、菌丝有序排列形成三维立体结构.结论 烟曲霉、黄曲霉、土曲霉和黑曲霉在体外都能形成典型的生物膜,它的形成过程和结构特征与其他真菌生物膜类似.