Physical mapping across an avirulence locus of Phytophthora infestans using a highly representative, large-insert bacterial artificial chromosome library

The oomycete plant pathogen Phytophthora infestans is the causal agent of late blight, one of the most devastating diseases of potato worldwide. As part of efforts to clone avirulence (Avr) genes and pathogenicity factors from P. infestans, we have constructed a bacterial artificial chromosome (BAC) library from an isolate containing six Avr genes. The BAC library comprises clones with an average insert size of 98 kb and represents an estimated 10 genome equivalents. A three-dimensional pooling strategy was developed to screen the BAC library for amplified fragment length polymorphism (AFLP) markers, as this type of marker has been extensively used in construction of a P. infestans genetic map. Multiple positive clones were identified for each AFLP marker tested. The pools were used to construct a contig of 11 BAC clones in a region of the P. infestans genome containing a cluster of three avirulence genes. The BAC contig is predicted to encompass the Avr11 locus but mapping of the BAC ends will be required to determine if the Avr3 and Avr10 loci are also present in the BAC contig. These results are an important step towards the positional cloning of avirulence genes from P. infestans, and the BAC library represents a valuable resource for largescale studies of oomycete genome organisation and gene content.     

Screening and identification of insertion mutants from Bipolaris eleusines by mutagenesis based on restriction enzyme-mediated integration

Ophiobolin A is sesterterpenoid-type phytotoxin and may be an important candidate for development of new crop protection and pharmaceutical products. The restriction enzyme-mediated integration (REMI) method was used to introduce the plasmid pSH75 into the ophiobolin A-producing filamentous fungus Bipolaris eleusines. A total of 323 stable transformants were obtained, all of which were capable of growing on potato-dextrose agar medium containing 200?μg?mL(-1) hygromycin B. The transformation frequency was about 4-5 transformants?μg(-1) plasmid DNA. An ophibolin A-deficient transformant (B014) was assessed and the presence of the hph gene in this transformant was confirmed by PCR. The cell-free cultural filtrates of this transformant showed significantly less inhibition on mycelial growth of the fungal pathogen Rhizoctoni solani but little effect on barnyard grass as opposed to that of the wild-type B.?eleusines. There was no detectable amount of ophiobolin A in B014 samples measured with HPLC. This research suggests REMI as a potential approach for improving the production of ophiobolin A by B.?eleusines via genetic engineering to upregulate certain genes responsible for desired biosynthetic pathways.     

Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences

Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There were no indications that the tomato DNA inserts conferred autonomous replication on the plasmids. Instead, Southern blot hybridization analysis of seven kanamycin resistant calli revealed the presence of at least one kanamycin resistance locus per transformant integrated in the tomato nuclear DNA. Generally one to three truncated plasmid copies were found integrated into the tomato nuclear DNA, often physically linked to each other. For one transformant we have been able to use the bacterial ampicillin resistance marker of the vector plasmid pUC9 to rescue a recombinant plasmid from the tomato genome. Analysis of the foreign sequences included in the rescued plasmid showed that integration had occurred in a non-repetitive DNA region. Calf-thymus DNA, used as a carrier in transformation procedure, was found to be covalently linked to plasmid DNA sequences in the genomic DNA of one transformant. A model is presented describing the fate of exogenously added DNA during the transformation of a plant cell. The results are discussed in reference to the possibility of isolating DNA sequences responsible for autonomous replication in tomato.     

根癌农杆菌介导的Pr1基因在贵阳腐霉的组成性表达

【目的】构建组成性表达贵阳腐霉Pr1基因的载体pCambia-hph-Pr1,转化贵阳腐霉,以获得高毒力的基因工程转化菌株。【方法】以双元载体pCambia-Pnos-PNN-hph为基本骨架,将Pr1基因置于组成型启动子PgpdA和终止子TtrpC之间,获得含Pr1基因的表达元件。以贵阳腐霉菌丝体为受体材料,利用根癌农杆菌介导的遗传转化法转化贵阳腐霉,观察转化株的生物学特征和灭蚊毒力。【结果】转化株的菌丝生长速度和游动孢子产量与野生株无显著性差异。生物测定表明转化株对蚊幼虫平均致死率达到32.9%,比野生株提高了约一倍,并且使受试蚊幼虫生长速度明显减缓。【结论】利用根癌农杆菌介导的遗传转化获得了遗传稳定的高毒力菌株。     

基于PEG介导原生质体转化构建粉红聚端孢荧光标记

粉红聚端孢是多种植物的重要病原菌。本研究通过酶解粉红聚端孢幼嫩菌丝细胞壁获得原生质体,用PEG介导原生质体转化将携带GFP基因和博来霉素抗性基因的外源DNA随机插入粉红聚端孢基因组,共获得博来霉素抗性菌株90株,转化效率达18个/μg。选取22株转化子荧光观察,发现均可表达荧光,菌株间荧光强度不同,其中10株转化子荧光表达较强。与野生型相比,突变菌株TR45的菌落生长、产孢量和致病力等生物学特性均未改变,在不含博来霉素的培养皿中继代培养10代荧光仍能稳定表达。本研究构建的高效原生质体制备和PEG介导粉红聚端孢遗传转化方法,可用于该菌基因功能研究,绿色荧光标记菌株可用于病菌侵入、田间监测、侵染循环等发生规律研究。     

A reliable transformation method and heterologous expression of beta-glucuronidase in Lentinula edodes

A simple and reliable mushroom transformation procedure based on electroporation of basidiospores or mycelial fragments was developed. This method eliminated the problem of protoplast preparation, the transformation efficiency were 30-150 transformants per mug DNA and the hygromycin resistant marker gene and gus were expressed in Lentinula edodes successfully. No false positive antibiotic-resistant cultures were detected by PCR amplification and the beta-glucuronidase (GUS) expression was maintained stable during mitotic cell division without selection pressure for more than 6 months. Southern analysis of transformants indicated the integration of gene might occur by non-homologous recombination. Using the glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter with the first intron of gpd gene, the average GUS activity in L. edodes reached 144.6+/-3.9 U mg(-1) soluble protein, while only 30.1+/-0.7 U mg(-1) soluble protein was detected for those without the intron. The percentage of GUS in total soluble protein was 5.67 x 10(-4) (0.06%) for the transformant with the highest GUS activity. This rapid and convenient electroporation procedure offers a new approach for the genetic manipulation and tool to tag genes of important edible mushroom species.     

The Escherichia coli C homoprotocatechuate degradative operon: hpc gene order,direction of transcription and control of expression

The processing of DNA molecules during transformation was characterized in the oomycete Phytophthora infestans. Linear and circular forms of nonreplicating transformation vectors supported similar rates of stable transformation. Remarkably, digestion of plasmids within the selectable marker genes neomycin phosphotransferase (npt) or hygromycin phosphotransferase (hpt) had little effect on the recovery of drug-resistant transformants, and the cleaved sites were shown to be reconstituted in the transformants. An assay for the transient expression of β-glucuronidase (GUS) in protoplasts treated with partial or disrupted GUS genes demonstrated that active genes could be reconstituted through intramolecular and/or intermolecular ligation between compatible ends, while incompatible ends were inefficiently joined. Stable transformation studies also demonstrated that complementing portions of incomplete npt or hpt genes joined through homologous recombination. Based on the indication of efficient ligation between DNA molecules during transformation, an efficient procedure for cotransformation was developed. The frequency of cotransformation between vectors expressing selected genes (npt or hpt) and nonselected sequences (GUS, β-galactosidase, or streptomycin phosphotransferase) approached unity when the plasmids were linearized with the same restriction enzyme before transformation. In contrast, cotransformation between circular plasmids or those cut with different enzymes occurred infrequently (10%). Hybridization analysis of DNA from cotransformants demonstrated that linearized plasmids became colocalized within genomic DNA, while circular plasmids typically inserted at unliked sites.     

Improvement on genetic transformation in the nematode-trapping fungus Arthrobotrys oligospora and its quantification on dung samples

An improved DNA-mediated transformation system for nematode-trapping fungus Arthrobotrys oligospora based on hygromycin B resistance was developed. The transformation frequency varied between 34 and 175 transformants per μg linearized DNA and 93% of the transformants were stable for drug resistance when tested 100 randomly selected transformants. More than 2000 transformants were obtained by transformation of the fungus with pBChygro in the presence of HindIII and among them, one, YMF1.00110, which lost its ability of forming predacious structure, was isolated. Southern analysis showed that the plasmid DNA had integrated into the genome of all tested transformants (including YMF 1.00110) except one. The transformant tagged with hph gene could be re-isolated and quantified from dung samples based on the resistance of hygromycin B. All the results suggested that the method of restriction enzyme mediated integration (REMI) should facilitate not only the insertional mutagenesis for tagging and analysis genes of interest but also the ecological investigation of tagged fungi in a given environment.     

Advances in Pollen Mediated Genetic Transformation

植物遗传转化技术是植物科学基础理论与应用研究的有力武器,已成为植物遗传改良的重要途径之一。但是、目前遗传转化所采用的受体系统,大都需要体外培养和植株再生过程,才能获得转基因植株。其中、基因型限制和遗传变异是该技术不可逾越的两大障碍。花粉管通道法可省去转化体的离体培养,不过、多数植物受花器结构的限制而难以经花柱注射ＤＮＡ,只能向子房注射,并不是真正的“花粉管通道”。又由于此法外源基因的导入发生在授粉之后,因此该方法亦不属于花粉遗传转化。利用小孢子胚胎发生体系进行遗传转化与利用花粉作为外源ＤＮＡ的媒介,继而、通过授粉受精获得转基因种子,是目前花粉遗传转化的两个重要方面和活跃的研究方向。前者仍需要离体再生系统,后者则可以利用植物自身的再生机制,本文称之为花粉介导法（ｐｏｌｅｎ－ｍｅｄｉａｔｅｄｔｒａｎｓｆｏｒｍａｔｉｏｎ）。该方法通过自然的胚胎发育过程获得转基因子代,避免了组织培养过程中的遗传变异和转基因植株的嵌合现象。可望成为简便快速的植物遗传转化体系。目前对花粉介导的遗传转化进行专门评述的文献较少,本文对该领域的研究分三个层次进行了综述。一、外源基因转化方法小孢子或由小孢子形成的胚状体是很有潜力的遗传转化受体     

Inheritance and structure of foreign DNA in progenies of transgenic tobacco obtained by direct gene transfer

Summary One of the transformed tobacco plants obtained by direct DNA transformation possessed two marker genes, a chimeric aminoglycoside phosphotransferase and nopaline synthase genes. Selfed progenies of this plant (T3-d) showed stable inheritance of these two genes. The minimum size of foreign DNA integrated into tobacco genome was estimated to be 5.4 kbp. A deleted nopaline synthase gene co-existed with an intact gene. The linkage analysis indicated that two transformants, T1-b and T3-c, possessed foreign DNA inserted in different chromosomes or in different sites of the same chromosome that recombine freely.     

Construction of a bacterial artificial chromosome library of Phytophthora infestans and transformation of clones into P. infestans

A bacterial artificial chromosome (BAC) library of Phytophthora infestans was constructed in a derivative of pBELOBACII that had been modified by adding a npt selectable marker gene for transforming P. infestans. A total library of 8 genome equivalents was generated and 16,128 clones with inserts averaging 75 kb (4.9 genome equivalents) were individually picked and stored as an arrayed library in microtiter plates. This coverage was confirmed by screening the library for 11 DNA loci by colony hybridization and by polymerase chain reaction of DNA pools. Transformation of P. infestans with BAC clones containing inserts of 93 to 135 kb was demonstrated. The efficiency of transformation with most BACs was noticeably higher than that with smaller plasmids. Detailed analyses of transformants obtained with a 102-kb BAC indicated that entire inserts were present in about one-quarter of the transformants.