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 Vinca protoplasts mediated by Agrobacterium spheroplasts

Summary Vinca rosea protoplasts and Agrobacterium tumefaciens spheroplasts harboring octopine-type Ti plasmid were mixed and treated with polyethylene glycol or polyvinyl alcohol, which facilitated the introduction of spheroplasts into plant protoplasts. After the protoplasts had been kept at 40° C for 4 days, bacteria were found to be completely eliminated from the medium. Among treated protoplasts 1–2 per 1,000 formed colonies on the Murashige and Skoog medium (1962) lacking hormones. When the colonies were isolated and subcultured, they could be maintained as clones. Octopine, an amino acid specific to crown gall, was detected in half of these clones. The phenotypic features of these putative transformants were compared but did not show any coincidental tendencies in relation to color, hardness, form, growth rate, or octopine production. The significance of this system in transformation of higher plant cells is discussed.     

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。     

Genetic transformation of Trichosporon cutaneum with a plasmid, pAN 7–1, from filamentous fungi

Genetic transformation of a biological oxygen demand (BOD)-sensing yeast, Trichosporon cutaneum IFO 10466 was carried out using a plasmid shuttle vector, pAN 7–1, between Escherichia coli and Aspergillus niger and Aspergillus nidulans. The hygromycin B-resistant transformants were obtained with the plasmid by using the spheroplasts. The transformation frequency was up to 70 colonies per g of plasmid DNA. Southern-blot analysis and transformation test of E. coli using DNA extracts from hygromycin B-resistant transformants of IFO 10466 indicated that pAN 7–1 existed independently in the cells without integration into the chromosome. These results suggest that pAN 7–1 has potential availability for introduction and expression of the external gene such as a bioluminescence gene, lux or luc, to create a luminous yeast for BOD sensing. © Rapid Science Ltd. 1998     

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.     

HAL1 mediate salt adaptation in Arabidopsis thaliana

INTRODUCTIONSalinity is a major environmental stress that isa substantial constraint to crop production both fordry land and irrigated agriculture. The detrimental impact of this stress is perpetuated and exacerbated by management practices used to facilitatehigh-output crop production. To overcome theselimitations and improve production efficiency in theface of a burgeoning world population, more salt tolerant crops must be developed. In contrast with traditional breeding, the direct ill…     

Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the symbiotic ectomycorrhizal fungus Hebeloma cylindrosporum

We transformed haploid mycelium of Hebeloma cylindrosporum via Agrobacterium tumefaciens and optimised the procedure to develop a new tool for insertional mutagenesis in this fungus. Southern blot analysis of 83 randomly selected transformants showed that they all contained plasmid inserts. Each of them showed a unique hybridisation pattern, suggesting that integration was random in the fungal genome. Sixty percent of transformants obtained in the presence of bacteria pre-treated with acetosyringone integrated a single transferred DNA copy. Thermal asymmetric interlaced polymerase chain reaction allowed us to recover the left border and the right border junctions in 85% and 15% of transformants analysed, respectively. Results show that A. tumefaciens-mediated transformation may be a powerful tool for insertional mutagenesis in H. cylindrosporum.     

Insertional mutagenesis of Arabidopsis thaliana: increase of germinating seeds transformation efficiency after sonication

An efficient procedure of transforming Arabidopsis thaliana germinating seeds was elaborated on the basis of the method of Feldmann and Marks. The procedure involves microdamaging T1 seeds by sonication before culturing with a vector Agrobacterium strain and yields more than 1% T2 transformants. Germinating seeds were transformed with Agrobacterium timefaciens hypervirulent strain A281 carrying plasmid pLD3 derived from pBI121. A collection of 54 A. thaliana T2 transformants was obtained; some of them showed marked morphological alterations. The transgenic nature of plants that acquired resistance to a marker antibiotic was confirmed histochemically and by PCR amplification of a T-DNA region.     

The T-DNA integration pattern in Arabidopsis transformants is highly determined by the transformed target cell

Transgenic loci obtained after Agrobacterium tumefaciens -mediated transformation can be simple, but fairly often they contain multiple T-DNA copies integrated into the plant genome. To understand the origin of complex T-DNA loci, floral-dip and root transformation experiments were carried out in Arabidopsis thaliana with mixtures of A. tumefaciens strains, each harboring one or two different T-DNA vectors. Upon floral-dip transformation, 6–30% of the transformants were co-transformed by multiple T-DNAs originating from different bacteria and 20–36% by different T-DNAs from one strain. However, these co-transformation frequencies were too low to explain the presence of on average 4–6 T-DNA copies in these transformants, suggesting that, upon floral-dip transformation, T-DNA replication frequently occurs before or during integration after the transfer of single T-DNA copies. Upon root transformation, the co-transformation frequencies of T-DNAs originating from different bacteria were similar or slightly higher (between 10 and 60%) than those obtained after floral-dip transformation, whereas the co-transformation frequencies of different T-DNAs from one strain were comparable (24–31%). Root transformants generally harbor only one to three T-DNA copies, and thus co-transformation of different T-DNAs can explain the T-DNA copy number in many transformants, but T-DNA replication is postulated to occur in most multicopy root transformants. In conclusion, the comparable co-transformation frequencies and differences in complexity of the T-DNA loci after floral-dip and root transformations indicate that the T-DNA copy number is highly determined by the transformation-competent target cells.     

蓖麻毒蛋白A链基因RNAi转化研究

通过基因沉默技术调控蓖麻毒蛋白A链基因的表达,以期获得低毒蓖麻新材料.利用基因克隆技术获得蓖麻毒蛋白A链基因762 bp片段,命名为RTA基因.进一步利用该基因构建了植物RNAi表达载体pBI-RTA-S-AS,通过农杆菌介导法转化蓖麻子叶节,用卡那抗性筛选转化再生植株,PCR进一步鉴定转基因植株.结果表明:克隆得到目的基因长762 bp,与预期结果一致;卡那抗性筛选和PCR鉴定结果显示,获得了3株转基因阳性植株.     

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.     

Homologous recombination in plant cells after Agrobacterium-mediated transformation.

A single amino-acid change in the acetolactate synthase (ALS) protein of tobacco confers resistance to the herbicide chlorsulfuron. A deleted, nonfunctional fragment from the acetolactate synthase gene, carrying the mutant site specifying chlorsulfuron resistance plus a closely linked novel restriction site marker, was cloned into a binary vector. Tobacco protoplasts transformed with Agrobacterium tumefaciens carrying this vector yielded chlorsulfuron-resistant colonies. DNA gel blot analysis of DNA from these colonies suggested that in three transformants homologous recombination had occurred between the endogenous ALS gene and the deleted ALS gene present in the incoming T-DNA. Plants were regenerated from these chlorsulfuron-resistant colonies, and in two of the transformants, genetic analysis of their progeny showed that the novel gene segregated as a single Mendelian locus. Possible models for the generation of these recombinant plants are discussed.     

In planta transformation of Arabidopsis thaliana

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure. In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7–8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci. The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny. The small numbers of transformants recovered from individual T₁ plants and the fact that none of the T₂ progeny were homozygous for a specific T-DNA insert suggest that transformation occurs late in floral development.     

Direct repeats of T-DNA integrated in tobacco chromosome: characterization of junction regions

Plant transformation via Agrobacterium frequently results in formation of multiple copy T-DNA arrays at one target site of the chromosome. The T-DNA copies are arranged in repeats, direct or inverted around one of the T-DNA borders. A Ti plasmid-derived transformation vector has been constructed enabling direct selection of transformants carrying at least two linked copies of T-DNA in the same orientation. The selection is based on expression of a promoterless neomycin phosphotransferase gene on one T-DNA copy from a promoter located on the other T-DNA copy. After co-cultivation of tobacco protoplasts with Agrobacterium, as many as 30% of regenerated transformed plants carried directly repeated T-DNA copies. The junction regions between two T-DNAs were amplified and 13 amplified fragments were cloned and sequenced. The involvement of T-DNA left and right border sequences in direct repeat junctions was determined. In some junctions, additional filler DNA was detected. The length of filler DNA varied from a few up to almost 300 bp. The longer filler DNAs from two clones were found to be T-DNA fragments in direct or reverse orientation. We discuss the recently suggested models for T-DNA integration and propose that the formation of direct repeats in genomes does not necessarily result from ligation of intermediates (i.e. T-strands), but more likely from the co-integration of several intermediates into one target site.     

Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens

A simple, highly efficient, and reliable Agrobacterium tumefaciens-mediated transformation method was developed for the insect pathogenic fungus Metarhizium anisopliae. Expression of the green fluorescent protein gene, egfp, and the benomyl resistance gene, benA3, were used as markers in transformed M. anisopliae. Transformation efficiencies were dependent on the strain of A. tumefaciens used. With strain AGL-1, 17.0 +/- 1.4 transformants per plate could be obtained using conidial concentrations of 10(6) conidia/mL and a 2 day co-cultivation in the presence of 200 micromol/L acetosyringone. On the other hand, transformations using strain LBA4404 were unsuccessful. Ten transformants were tested by Southern analysis and found to contain a single copy T-DNA. Twenty transformants were subcultured for five generations on nonselective media, and 95% of the transformants were mitotically stable. Agrobacterium tumefaciens-mediated transformation of M. anisopliae can serve as a useful tool to investigate genes involved in insect pathogenicity.     

Comparison of genetic transformation in <Emphasis Type="Italic">Morus alba</Emphasis> L. via different regeneration systems

Three different regeneration systems, viz. direct regeneration of adventitious shoot buds from explant, regeneration through callus cultures and somatic embryos were compared to see their effect on transfer of neomycin phosphotransferase (nptII) and β-glucuronidase (GUS) reporter gene (gus) to Morus alba clone M5, through Agrobacterium tumefaciens mediated transformation. Pre-conditioning and co-cultivation durations had a marked effect on transformation frequency. The highest transformation frequency of 18.6% was obtained using direct induction of adventitious shoot buds. Expression and presence of transgene were assayed histochemically and through polymerase chain reaction. Southern analysis of GUS and PCR positive transformants confirmed stable integration of transgenes with two to four copy numbers. The selected transformants showed normal phenotype under in vitro and field conditions.     

根癌农杆菌介导的球毛壳菌遗传转化及T-DNA插入突变体的获得

根癌农杆菌介导的遗传转化系统是植物基因工程常用方法,目前已将这一转化系统应用到酵母、丝状真菌以及人类细胞的转化。利用这一转化系统,成功地实现了丝状真菌球毛壳菌（Chaetomium globosum）的遗传转化,转化率约为60～180个转化子/10.7个孢子 。通过对转化子的PCR检测和Southern 杂交分析表明,TDNA已整合进毛壳菌基因组中,而且在所检测的转化子中都是以单拷贝的形式整合,转化子都能够稳定遗传。根癌农杆菌介导的遗传转化具有转化率高、低拷贝、遗传稳定、操作简便等优点,因此有可能成为丝状真菌遗传转化和功能基因组研究的有力工具。     

根癌农杆菌介导的哈茨木霉菌遗传转化的研究

利用根癌农杆菌EHA105进行了哈茨木霉Th-33的转化,在优化的转化条件下,EHA105对木霉菌的转化效率约45-100个转化子/106个孢子。本实验室利用该方法已建立了含4000多个转化子的木霉T-DNA插入突变体库。随机挑选24个转化子进行遗传稳定性分析,结果显示转化子经过5代无选择压力连续转接后都能在选择平板上正常生长;潮霉素抗性基因的PCR扩增和Southern blot杂交分析表明,木霉转化子含有该基因,Southern blot杂交进一步表明转化子多为单拷贝随机插入。该转化体系的改进将有利于木霉菌生防功能基因的克隆和作用机制的研究。     

根癌农杆菌介导转化红曲菌及转化子发酵性能的研究

利用根癌农杆菌介导转化技术成功将潮霉素抗性基因转入发白红曲菌中,优化了抗生素浓度,发白红曲菌孢子浓度,根癌农杆菌浓度,共培养温度及时间,以及乙酰丁香酮浓度等转化条件,最终转化效率可达52个转化子/105个红曲孢子.将转化子在含有潮霉素B的培养基继代培养5代,得到了多株稳定的转化子,对部分转化子进行PCR鉴定,结果进一步...