根癌农杆菌介导的白腐丝状真菌——黄孢原毛平革菌的转化

利用农杆菌介导的方法成功地对黄孢原毛平革菌（Phanerochaete chrysosporium）进行了遗传转化。将含有潮霉素磷酸转移酶融合基因的双元质粒pCH61300转入根癌农杆菌（Agrobacterium tumefaciens）208中,然后用该转化菌分别感染黄孢原毛平革菌的分生孢子和原生质体,获得16株可能的转化子,经复筛,共获得6株潮霉素抗性水平为100μg/mL的稳定转化子,分生孢子和原生质体的转化频率没有明显差别。PCR检测结果显示,抗性基因已导入黄孢原毛平革菌细胞中;Southern杂交表明,TDNA以单拷贝形式整合到黄孢原毛平革菌基因组中。其中的一个转化子菌落形态与原野生型菌株相比有所不同,菌丝稀薄,分生孢子较少。利用分生孢子转化更为简便易行,无需特殊的设备和制备原生质体,此方法为深入开展该菌的遗传转化研究奠定了基础。     

根癌农杆菌介导丝状真菌遗传转化的研究进展

根癌农杆菌介导的丝状真菌遗传转化是近年建立起来的一种新方法,该方法和以往的真菌转化体系相比具有转化方法简单、材料易得、效率高以及转化子中T-DNA单拷贝插入比例高等特点。就根癌农杆菌转化的丝状真菌种类、转化的具体过程以及影响转化效率的因素等方面进行了综述,并展望了该方法的应用前景。     

根癌农杆菌介导丝状真菌遗传转化因素的研究进展

根癌农杆菌介导的丝状真菌遗传转化体系,是当前研究真菌基因组学的有力工具,广泛运用于真菌随机插入突变体库的构建和基因打靶。概述了根癌农杆菌介导的丝菌转化的大致过程,转化效率的影响因素。     

根癌农杆菌介导真菌遗传转化的研究进展

根癌农杆菌介导的真菌遗传转化是近年来发展的一种新方法 ,与其它方法相比 ,该方法具有操作简便、转化效率高和易得到稳定转化子等特点。目前 ,在根癌农杆菌介导下已实现了多个属种真菌的遗传转化 ,显示出良好的应用前景。综述了根癌农杆菌介导真菌遗传转化的转化机理和T DNA在真菌细胞中的存在方式等方面的研究结果 ,并展望这一方法的应用前景。     

根癌农杆菌介导的黑曲霉遗传转化体系的建立及优化

基于根癌农杆菌介导的遗传转化方法的独特优点,研究黑曲霉转化过程中各主要影响因素,建立高效的黑曲霉遗传转化方法。构建双元载体pBI-hph,通过电转导入农杆菌LBA4404中,以黑曲霉TCCC41056为受体菌株,利用潮霉素B基因作为筛选标记,对影响转化效率的孢子悬液的新鲜程度及浓度、农杆菌菌液浓度、共培养时间、共培养温度这五个条件进行分析,建立根癌农杆菌介导的黑曲霉遗传转化体系。实验结果表明,上述条件对黑曲霉的转化效率有较大的影响,通过优化,黑曲霉转化效率可达83个转化子/10⁷分生孢子;整合到黑曲霉基因组的外源基因可以稳定遗传,在转接10代后遗传性能仍保持稳定,并在众多转化子中筛选得到了糖化酶活力提高18%的黑曲霉突变株。根癌农杆菌介导的黑曲霉转化体系的建立,为进一步研究黑曲霉的功能基因以及开发黑曲霉表达系统提供了有力的手段。     

一种适于丝状真菌基因RNA干扰方法的研究

目的：获得一种适于丝状真菌基因研究用的、由根癌农杆菌介导的RNA干扰方法。方法采用基因重组及菌株干扰体系筛选的方法。结果获得适于根癌农杆菌转染的重组载体PCB309?pfgrt,并将其成功用于对孢子丝菌双组份信号组氨酸蛋白激酶DRK1基因的干扰中。结论该方法优化了根癌农杆菌的转化体系,解决了丝状真菌RNA干扰载体构建困难及干扰效率低下的难题,在丝状真菌基因功能及遗传转化研究方面具有广泛的应用前景。     

根癌农杆菌介导里氏木霉转化体系的建立和条件优化

目的:采用根癌农杆菌介导的转化方法实现丝状真菌里氏木霉的遗传转化,并优化转化条件.方法:构建含潮霉素抗性基因(hph)的双元载体pCAM-hph后,转化根癌农杆菌LBA4404获得转化菌株.将根癌农杆菌的转化菌株和里氏木霉的分生孢子共培养后在含100μg/mL潮霉素的抗性平板上筛选里氏木霉转化子,并采用PCR扩增和序列测定对转化子中的插入片段进行了分析.结果:使用根癌农杆菌介导的转化方法转化里氏木霉,每106个分生孢子可获得25.8个转化子.最佳的转化条件为:农杆菌初始浓度为OD660约为0.8,孢子数为106个,共培养时间为48h,pH为5.0～5.5,培养温度为28℃.结论:建立了根癌农杆菌介导的里氏木霉转化方法,并获得了最佳的转化条件.     

根癌农杆菌在丝状真菌转化中的应用

原生质体转化法是丝状真菌转化的传统方法,但其过程繁琐且转化效率低。根癌农杆菌介导的转化方法原本是进行植物遗传转化的标准方法,但近年来发现该方法还可用于丝状真菌的转化。根癌农杆菌介导的丝状真菌转化具有操作简便、转化效率高、重复性好等优点,从而可以解决丝状真菌转化难的问题。在本文中,就其转化机制、特点和转化条件优化等方面的最新研究进展进行了综述。     

纤维素降解球毛壳菌NK-102的高效遗传转化方法

摘要:【目的】在球毛壳菌(Chaetomium globosum)NK-102 中,建立菌株特异性转化体系。【方法】构建新的抗性标记pUCATPH-Pgap,转化效率优于pUCATPH 和pCM768。建立了PEG-原生质体和根癌农杆菌(Agrobacterium tumefaciens)EHA105 介导的两种转化方法。【结果】原生质体转化效率为30－50 个转化子/10 μg DNA,抗性标记pUCATPH-Pgap 效率最高。EHA105 介导转化率达到3.2×102 转化子/107 孢子。Sout     

影响根癌农杆菌介导的木霉菌遗传转化因素分析*

采用根癌农杆菌介导的转化方法,以木霉菌分生孢子为受体材料,对影响转化效率的主要因素进行了分析。结果表明,农杆菌菌株类型、初始菌液量、分生孢子浓度、共培养时间以及乙酰丁香酮的诱导等因素对转化效率都具有重要的影响。通过对这些因素的分析,基本得出了根癌农杆菌转化系统对木霉菌遗传转化的特点和规律,为将该转化系统用于其它丝状真菌的遗传转化提供了重要参考。     

Agrobacterium tumefaciens-mediated transformation in Colletotrichum falcatum and C. acutatum

Agrobacterium tumefaciens-mediated transformation (ATMT) is becoming an effective system as an insertional mutagenesis tool in filamentous fungi. We developed and optimized ATMT for two Colletotrichum species, C. falcatum and C. acutatum, which are the causal agents of sugarcane red rot and pepper anthracnose, respectively. A. tumefaciens strain SK1044, carrying a hygromycin phosphotransferase gene (hph) and a green fluorescent protein (GFP) gene, was used to transform the conidia of these two Colletotrichum species. Transformation efficiency was correlated with cocultivation time and bacterial cell concentration and was higher in C. falcatum than in C. acutatum. Southern blot analysis indicated that about 65% of the transformants had a single copy of the T-DNA in both C. falcatum and C. acutatum and that T-DNA integrated randomly in both fungal genomes. T-DNA insertions were identified in transformants through thermal asymmetrical interlaced PCR (TAIL-PCR) followed by sequencing. Our results suggested that ATMT can be used as a molecular tool to identify and characterize pathogenicity-related genes in these two economically important Colletotrichum species.     

根癌农杆菌介导的轮枝镰孢菌遗传转化及T-DNA插入突变体的筛选

建立并优化了农杆菌介导转化轮枝镰孢菌Fusarium verticillioides获得T-DNA插入突变体的体系,在镰孢菌孢子浓度106个/mL、农杆菌OD600=0.15-0.20、乙酰丁香酮浓度为200μmol/mL的条件下共培养36h转化率最高,可达60-120个/106个孢子。共获得转化子1000多个,连续转接5代能够稳定遗传。PCR验证潮霉素B抗性基因已整合进转化子基因组DNA中,部分转化子表现为生长和形态异常。该转化体系的建立为研究该菌的致病机制和功能基因分析奠定了基础。     

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.     

农杆菌介导的出芽短梗霉遗传转化及高效筛选聚苹果酸高产菌株

建立根癌农杆菌介导的出芽短梗霉遗传转化方法及T-DNA突变库,高效筛选聚苹果酸高产菌株及功能基因。通过含潮霉素和草铵磷抗性基因的农杆菌转化出芽短梗霉,抗性压力筛选及PCR验证建立根癌农杆菌介导的出芽短梗霉遗传转化方法,结合发酵液p H与聚苹果酸含量响应变化,微孔板高效筛选高产聚苹果酸的T-DNA插入突变株,基因组步移确定T-DNA插入位点及功能基因。结果获得遗传稳定的抗性基因菌株,每107个细胞可获得80-120个转化子,出芽短梗霉H27号T-DNA突变株聚苹果酸摇瓶发酵产量提高24.5%,基因组步移证实糖酵解途径磷酸甘油酸变位酶基因被破坏。成功建立了根癌农杆菌介导的出芽短梗霉遗传转化方法和T-DNA插入突变库,结合高效筛选方法为聚苹果酸合成功能基因挖掘及高产机制解析奠定基础。     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-<Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of<Emphasis Type="Italic"> Helminthosporium turcicum</Emphasis>, the maize leaf-blight fungus

Agrobacterium tumefaciens has the ability to transfer its T-DNA to plants, yeast, filamentous fungi, and human cells and integrate it into their genome. Conidia of the maize pathogen Helminthosporium turcicum were transformed to hygromycin B resistance by a Agrobacterium-tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) and the enhanced green fluorescent protein (EGFP) genes controlled by the gpd promoter from Agaricus bisporus and the CaMV 35S terminator. Agrobacterium-tumefaciens-mediated transformation yielded stable transformants capable of growing on increased concentrations of hygromycin B. The presence of hph in the transformants was confirmed by PCR, and integration of the T-DNA at random sites in the genome was demonstrated by Southern blot analysis. Agrobacterium-tumefaciens-mediated transformation of Helminthosporium turcicum provides an opportunity for advancing studies of the molecular genetics of the fungus and of the molecular basis of its pathogenicity on maize.     

Agrobacterium tumefaciens-Mediated Transformation for Investigating Pathogenicity Genes of the Phytopathogenic Fungus Colletotrichum sansevieriae

Agrobacterium tumefaciens-mediated transformation (AtMT) has become a common technique for DNA transformation of yeast and filamentous fungi. In this study, we first established a protocol of AtMT for the phytopathogenic fungus Colletotrichum sansevieriae. Binary T-DNA vector containing the hygromycin B phosphotransferase gene controlled by the Aspergillus nidulans gpdA promoter and the trpC terminator was constructed with pCAMBIA0380 and used with three different strains LBA4404, GV3101, and GV2260 of A. tumefaciens. Transformants were most effectively obtained when GV2260 and C. sansevieriae Sa-1-2 were co-cultivated; there were about 320 transformants per 10(6) spores. When 1,048 transformants were inoculated on Sansevieria trifasciata, three transformants were found to have completely lost their pathogenicity and two transformants displayed reduced pathogenicity. All of the five transformants had a single copy of T-DNA in their genomes. The three pathogenicity-deficient transformants were subjected to thermal asymmetric interlaced polymerase chain reaction and the reaction allowed us to amplify the sequences flanking the left and/or right borders. The flanking sequences of the two transformants, M154 and M875, showed no homology to any sequences in databases, but the sequences of M678 contained motifs of alpha-1,3-glucan synthase, suggesting that the gene might contribute to the pathogenicity of C. sansevieriae. This study describes a useful method for investigating pathogenicity genes in C. sansevieriae.     

Agrobacterium tumefasciens-mediated transformation of the aquatic fungus Blastocladiella emersonii

Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting model of study of cellular function and differentiation. We constructed binary T-DNA vectors containing hygromycin phosphotransferase (hph) or enhanced green fluorescent protein (egfp) genes, under the control of Aspergillus nidulans trpC promoter and terminator sequences. 24 h of co-cultivation in induction medium (IM) agar plates, followed by transfer to PYG-agar plates containing cefotaxim to kill Agrobacterium tumefsciens and hygromycin to select transformants, resulted in growth and sporulation of resistant transformants. Genomic DNA from the pool o resistant zoospores were shown to contain T-DNA insertion as evidenced by PCR amplification of hph gene. Using a similar protocol we could also evidence the expression of enhanced green fluorescent protein (EGFP) in zoospores derived from transformed cells. This protocol can also open new perspectives for other non-transformable closely related fungi, like the Chytridiomycete class.     

A critical assessment of Agrobacterium tumefaciens-mediated transformation as a tool for pathogenicity gene discovery in the phytopathogenic fungus Leptosphaeria maculans

We evaluated the usefulness and robustness of Agrobacterium tumefaciens-mediated transformation (ATMT) as a high-throughput transformation tool for pathogenicity gene discovery in the filamentous phytopathogen Leptosphaeria maculans. Thermal asymmetric interlaced polymerase chain reaction allowed us to amplify the left border (LB) flanking sequence in 135 of 400 transformants analysed, and indicated a high level of preservation of the T-DNA LB. In addition, T-DNA preferentially integrated as a single copy in gene-rich regions of the fungal genome, with a probable bias towards intergenic and/or regulatory regions. A total of 53 transformants out of 1388 (3.8%) showed reproducible pathogenicity defects when inoculated on cotyledons of Brassica napus, with diverse altered phenotypes. Co-segregation of the altered phenotype with the T-DNA integration was observed for 6 of 12 transformants crossed. If extrapolated to the whole collection, this indicates that 1.9% of the collection actually corresponds to tagged pathogenicity mutants. The preferential insertion into gene-rich regions along with the high ratio of tagged mutants renders ATMT a tool of choice for large-scale gene discovery in L. maculans.     

High frequency of single-copy T-DNA transformants produced by floral dip in CRE-expressing Arabidopsis plants

For genetic transformation of plants, floral dip with Agrobacterium often results in integration of multiple T-DNA copies at a single locus and frequently in low and unstable transgene expression. To obtain efficient single-copy T-DNA transformants, two CRE/ loxP recombinase-based simplifying strategies for complex T-DNA loci were compared. A T-DNA vector with oppositely oriented loxP sites was transformed into CRE -expressing and wild-type control Arabidopsis thaliana plants. Of the primary CRE -expressing transformants, 55% harboured a single copy of the introduced T-DNA, but only 15% in the wild-type plants. However, 73% of the single-copy transformants in the CRE background showed continuous somatic inversion of the DNA segment between the two loxP sites. To avoid inversion of the loxP -flanked T-DNA segment, two T-DNA vectors harbouring only one loxP site were investigated for their suitability for CRE/ loxP recombinase-mediated resolution upon floral-dip transformation into CRE -expressing plants. On average, 70% of the transformants in the CRE background were single-copy transformants, whereas the single-copy T-DNA frequency was only 11% for both vectors in the wild-type background. Both resolution strategies yielded mostly Cre transformants in which the 35S-driven transgene expression was stable and uniform in the progeny and remarkably, also in Cre transformants with multiple T-DNA copies. Therefore, a role is proposed for the CRE recombinase in preventing inverted T-DNA repeat formation or modifying the locus chromatin structure, resulting in a reduced sensitivity for silencing.