稻瘟病菌T-DNA插入方法优化及其突变体分析

优化了农杆菌介导转化稻瘟病菌获得T-DNA插入突变的条件,包括选择转化子的潮霉素B用量,抑制农杆菌的抗生素头孢噻肟钠和羧苄青霉素的配比,不同转化阶段培养基的选择等。转化1×10⁶个孢子平均可获得约500个左右的转化子,PCR和TAILPCR检测表明约85%转化子中含T-DNA插入。对1520个突变体进行形态变异观察,发现菌落颜色突变的有15个;随机取58个突变体进行比较,发现产孢量减少的4个,孢子萌发率降低的8个,附着胞形成率降低的9个;还获得对水稻品种C101LAC（Pi-1）和751127（Pi-9）致病的突变体,为进一步克隆相应的无毒基因奠定了基础。     

Genetic basis of carotenoid overproduction in Fusarium oxysporum

The phytopathogenic fungus Fusarium oxysporum is a model organism in the study of plant-fungus interactions. As other Fusarium species, illuminated cultures of F. oxysporum exhibit an orange pigmentation because of the synthesis of carotenoids, and its genome contains orthologous light-regulated car genes for this biosynthetic pathway. By chemical mutagenesis, we obtained carotenoid overproducing mutants of F. oxysporum, called carS, with upregulated mRNA levels of the car genes. To identify the regulatory gene responsible for this phenotype, a collection of T-DNA insertional mutants obtained by Agrobacterium mediated transformation was screened for carotenoid overproduction. Three candidate transformants exhibited a carS-like phenotype, and two of them contained T-DNA insertions in the same genomic region. The insertions did not affect the integrity of any annotated ORFs, but were linked to a gene coding for a putative RING-finger (RF) protein. Based on its similarity to the RF protein CrgA from the zygomycete Mucor circinelloides, whose mutation results in a similar carotenoid deregulation, this gene (FOXG_09307) was investigated in detail. Its expression was not affected in the transformants, but mutant alleles were found in several carS mutants. A strain carrying a partial FOXG_09307 deletion, fortuitously generated in a targeted transformation experiment, exhibited the carS phenotype. This mutant and a T-DNA insertional mutant holding a 5-bp insertion in FOXG_09307 were complemented with the wild type FOXG_09307 allele. We conclude that this gene is carS, encoding a RF protein involved in down-regulation of F. oxysporum carotenogenesis.     

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。     

Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in medicinal fungus Cordyceps militaris

Cordyceps militaris is an insect-born fungus with various biological and pharmacological activities. The mutant library of C. militaris was constructed by improved Agrobacterium tumefaciens-mediated transformation (ATMT), for the ultimate identification of genes involved in isolate degeneration during fruiting body production. Successful transformation of C.?militaris JM4 by A. tumefaciens AGL-1 carrying vector pATMT1 was performed, with efficiency in the range of 30-600 transformants per 1×10(5) conidia. Acetosyringone (AS) supplement in C. militaris ATMT was not necessary during either precultivation or cocultivation. The transformation procedure was optimised based on the ratios between donor A. tumefaciens and recipient conidia, and pH value of cocultivation media. The integration of the hyg gene into C. militaris genome was determined by PCR and Southern blot analysis, suggesting that 67-88% resulting transformants in cultivation conditions with or without AS were inserted by T-DNA and 55-80% were single-copy. Special mutants with altered phenotypes and growth potentials were characterised. The efficient TAIL-PCR approach was established for identifying T-DNA flanking sequences from C. militaris mutants. The successful construction of the mutant library indicated the usefulness of this approach for functional genetic analysis in this important fungus.     

Characteristic analysis of transformants in T-DNA mutation library of <Emphasis Type="Italic">Monascus ruber</Emphasis>

Monascus ruber, a red mold species, has been widely used in the fields of food and medicine. In this research, we transformed Monascus ruber spores using Agrobacterium tumefaciens as a tool for random insertional mutagenesis with the hygromycin phosphotransferase gene as the selected marker. Three types of mutants including citrinin-producing mutants, mutants with abnormal aerial hyphae and pigment change mutants were screened for molecular analysis. Southern blot analysis showed that more than 83.3% of transformants contained single T-DNA insertions. The genomic DNA segments of the transformants flanking the T-DNA could be amplified from their left borders with TAIL-PCR. Homologous comparison using the Blast tool showed that none of the isolated DNA sequences had any similarity to each other, suggesting that the T-DNA was randomly integrated into the fungal genome, which provided the hypothetical reason for the variant phenotypes of the transformants. The successful creation of transformants with a single T-DNA tag insertion may help us to clone functional genes related to the metabolism and differentiation of Monascus spp., which will greatly facilitate the molecular analysis of this important fungus and the improvement of strains at the genetic level.     

Efficient insertional mutagenesis system for the dimorphic pathogenic fungus Sporothrix schenckii using Agrobacterium tumefaciens

Sporothrix schenckii is a dimorphic pathogenic fungus that causes human and animal sporotrichosis globally. Here we developed and optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) system of S. schenckii for insertional mutagenesis. The transformation efficiency reached more than 600 transformants per 10⁶ conidia. Using this protocol enabled us to obtain a large number of T-DNA insertional mutants within a short experimental period. Several mutants with altered phenotypes were obtained during the transformation experiments. The mutants displayed mitotic stability. Transferred DNA (T-DNA) flanking sequences were cloned by thermal asymmetric interlaced PCR (TAIL-PCR). Our results demonstrated that the ATMT system can be an effective tool for insertional mutagenesis in S. schenckii. This is the first report of a suitable mutagenesis system which may provide valuable mutants and information for both forward and reverse genetics research in the future for this medically important fungus.     

农杆菌介导的红曲菌T-DNA插入突变库的构建及色素突变子的性质分析

以农杆菌EHA105为介导,将带有潮霉素抗性基因和gus基因的T-DNA片段转化到红色红曲菌(Monascusruber)中。通过转化条件的优化,成功构建了含有5132个转化子的T-DNA插入突变库。根据菌落颜色与色素分泌情况筛选到50株色素突变子,聚合酶链式反应(PCR)表明上述突变子均有T-DNA片段插入。经5代的继代培养后,94%的突变子具有稳定性(潮霉素抗性)。对突变子产色素和桔霉素能力的分析表明其分泌次生代谢产物的能力发生了较大变化。本方法的建立,为进一步深入研究红曲菌的代谢调节和基因功能奠定了基础。     

农杆菌介导转化黑曲霉分生孢子及产孢缺陷突变子T-DNA插入位点分析

【目的】利用农杆菌(Agrobacterium tumefaciens)T-DNA系统,建立转化黑曲霉(Aspergillus niger)分生孢子的方法,构建T-DNA插入突变子文库,为黑曲霉基因组功能注释研究打下基础。【方法】采用携带二元质粒载体pCAMBIA1301的农杆菌EHA105,诱导转化黑曲霉分生孢子,筛选具有潮霉素抗性的突变子。分析抗性稳定突变子菌株的表型,采用反向PCR方法分析T-DNA插入位点相邻位置的序列,并推测突变基因可能具有的功能。【结果】实验获得具有稳定潮霉素抗性转化子193株,转化率为5.6×102转化子/108分生孢子。部分转化子表型出现较为明显改变,其中一株不能产孢,对其T-DNA插入位点序列分析比对结果显示,突变基因属于超级转运家族(major facilitator superfamily,MFS)。【结论】本研究建立的农杆菌转化黑曲霉分生孢子平台,结合T-DNA插入突变位点分析,可以为黑曲霉基因组功能注释研究提供一种简便有效的途径。     

灰葡萄孢菌（Botrytis cinerea）基因组中T-DNA整合模式分析

摘要：【目的】研究灰葡萄孢菌（Botrytis cinerea）基因组中T-DNA插入位点的整合模式特征。【方法】利用农杆菌（Agrobactirium tumfacience）介导法构建灰葡萄孢菌T-DNA插入突变体库。利用热不对称交错PCR（TAIL-PCR）技术对转化子中T-DNA的旁侧序列进行扩增和克隆,对获得的旁侧序列进行比对分析。【结果】T-DNA插入在灰葡萄孢菌基因组非编码区的占69%,插入在外显子的占30%。T-DNA在插入的过程中发生了碱基缺失、增加等重组现象,其中左边界（left border,LB）整合到基因组碱基缺失较少,有的保持完整,而右边界（right border,RB）及其近邻的T-DNA区域缺失碱基较多。T-DNA的插入位点还发现有额外的序列插入。【结论】对灰葡萄孢菌中插入T-DNA的整合模式的分析为开展该菌的功能基因组学奠定了基础。     

Spectrum of T-DNA integrations for insertional mutagenesis of Histoplasma capsulatum

Agrobacterium-mediated transformation is being increasingly used for insertional mutagenesis of fungi. To better evaluate its effectiveness as a mutagen for the fungal pathogen Histoplasma capsulatum, we analyzed a collection of randomly selected T-DNA insertion mutants. Testing of different T-DNA element vectors engineered for transformation of fungi showed that pBHt2 provides the highest transformation efficiency and the lowest rate of vector backbone carryover. Sixty-eight individual T-DNA integrations were characterized by recovery of T-DNA ends and flanking genomic sequences. The right border (RB) end of the T-DNA is largely preserved whereas the left border (LB) end is frequently truncated. Analysis of T-DNA insertion sites confirms the lack of any integration hotspots in the Histoplasma genome. Relative to genes, T-DNA integrations show significant bias towards promoter regions at the expense of coding sequences. With consideration for potential promoter interruption and the demonstrated efficacy of intronic insertions, 61 % of mapped T-DNA insertions should impair gene expression or function. Mapping of T-DNA flanking sequences demonstrates 67 % of T-DNA integrations are integrations at a single chromosomal site and 31 % of T-DNA integrations are associated with large-scale chromosomal rearrangements. This characterization of T-DNA insertions in mutants selected without regard to phenotype supports application of Agrobacterium-mediated transformation as an insertional mutagen for genome-based screens and functional discovery of genes in Histoplasma.     

Transposon mutagenesis, characterization, and cloning of transformation genes of Haemophilus influenzae Rd.

A plasmid library of PstI fragments of Haemophilus influenzae Rd genomic DNA was mutagenized in Escherichia coli with mini-Tn10kan. The mutagenized PstI fragments were introduced by transformation into the H. influenzae chromosome, and kanamycin-resistant transformants were screened for the transformation-deficient phenotype by a cyclic AMP-DNA plate method. Fifty-four mutant strains containing 24 unique insertions that mapped to 10 different PstI fragments were isolated. Strains carrying unique insertions were tested individually for DNA uptake, transformation efficiency, UV sensitivity, and growth rate. The transformation frequencies of these mutants were decreased by factors of 10(-2) to 10(-6). Five of the mutants had normal competence-induced DNA uptake, and the rest were variably deficient in competence development. Three strains were moderately UV sensitive. All strains but one had doubling times within 50% of that of the wild type. Mutated genes were cloned into an H. influenzae-E. coli shuttle vector, and wild-type loci were recovered by in vivo recombinational exchange. Hybridization of these clones to SmaI genomic fragments separated in pulsed-field gels showed that these insertions were not clustered in a particular region of the chromosome.     

Use of REMI and Agrobacterium-mediated transformation to identify pathogenicity mutants of the biocontrol fungus, Coniothyrium minitans

Restriction enzyme mediated integration (REMI) and Agrobacterium-mediated transformation (ATMT) were used to transform protoplasts or germinated conidia of the mycoparasite Coniothyrium minitans to hygromycin resistance. Using REMI, up to 32 transformants mug DNA(-1) were obtained, while 37.8 transformants 5 x 10(5) germlings(-1) were obtained using ATMT. Single-copy integrations occurred in 8% and 40% of REMI and ATMT transformants, respectively. A novel microtitre plate-based test was developed to expedite screening of 4000 REMI and ATMT C. minitans transformants. Nine pathogenicity mutants that displayed reduced or no pathogenicity on sclerotia of Sclerotinia sclerotiorum were identified.     

A fungal cell wall integrity-associated MAP kinase cascade in Coniothyrium minitans is required for conidiation and mycoparasitism

Coniothyrium minitans is an important biocontrol agent against Sclerotinia diseases. Previously, a conidiation-deficient mutant ZS-1T1000 was screened out from a T-DNA insertional library of C. minitans. CmBCK1, encoding MAP kinase kinase kinase and homologous to BCK1 of Saccharomyces cerevisiae, was disrupted by T-DNA insertion in this mutant. Targeted disruption of CmBCK1 led to the mutants undergoing autolysis and displaying hypersensitivity to the cell wall-degrading enzymes. The △CmBCK1 mutants lost the ability to produce pycnidia and conidia compared to the wild-type strain ZS-1. △CmBCK1 mutants could grow on the surface of sclerotia of Sclerotinia sclerotiorum but not form conidia, which resulted in much lower ability to reduce the viability of sclerotia of S. sclerotiorum. Furthermore, CmSlt2, a homolog of Slt2 encoding cell wall integrity-related MAP kinase and up-regulated by BCK1 in S. cerevisiae, was identified and targeted disrupted. The △CmSlt2 mutants had a similar phenotype to the △CmBCK1 mutants. The △CmSlt2 mutants also had autolytic aerial hyphae, hypersensitivity to cell wall-degrading enzymes, lack of conidiation and reduction of sclerotial mycoparasitism. Taken together, our results suggest that CmBCK1 and CmSlt2 are involved in conidiation and the hyperparasitic activities of C. minitans.     

Agrobacterium-mediated transformation of Aspergillus awamori in the absence of full-length VirD2, VirC2, or VirE2 leads to insertion of aberrant T-DNA structures

Reductions to 2, 5, and 42% of the wild-type transformation efficiency were found when Agrobacterium mutants carrying transposon insertions in virD2, virC2, and virE2, respectively, were used to transform Aspergillus awamori. The structures of the T-DNAs integrated into the host genome by these mutants were analyzed by Southern and sequence analyses. The T-DNAs of transformants obtained with the virE2 mutant had left-border truncations, whereas those obtained with the virD2 mutant had truncated right ends. From this analysis, it was concluded that the virulence proteins VirD2 and VirE2 are required for full-length T-DNA integration and that these proteins play a role in protecting the right and left T-DNA borders, respectively. Multicopy and truncated T-DNA structures were detected in the majority of the transformants obtained with the virC2 mutant, indicating that VirC2 plays a role in correct T-DNA processing and is required for single-copy T-DNA integration.     

Nonmycorrhizal (myc-) mutants of Hebeloma cylindrosporum obtained through insertional mutagenesis

Polyethylene glycol-mediated transformation of protoplasts was used as a method for insertional mutagenesis to obtain mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum impaired in symbiotic ability. Following restriction enzyme-mediated integration or conventional plasmid insertion, a library of 1,725 hygromycin-resistant monokaryotic transformants was generated and screened for the symbiotic defect, using Pinus pinaster seedlings as host plants. A total of 51 transformants displaying a dramatically reduced mycorrhizal ability were identified. Among them, 29 were nonmycorrhizal (myc-), but only 10 of them had integrated one or several copies of the transforming plasmid in their genome. Light and scanning electron microscopy observations of pine roots inoculated with myc- mutants suggested that we selected mutants blocked at early stages of interaction between partners or at the stage of Hartig net formation. Myc- mutants with plasmid insertions were crossed with a compatible wild-type monokaryon and allowed to fruit. Monokaryotic progenies were obtained in three independent crosses and were analyzed for symbiotic activity and plasmid insertion. In all three progenies, a 1:1 myc-:myc+ segregation ratio was observed, suggesting that each myc- phenotype resulted from a single gene mutation. However, for none of the three mutants, the myc- phenotype segregated with any of the plasmid insertions. Our results support the idea that master genes, the products of which are essential for symbiosis establishment, do exist in ectomycorrhizal fungi.     

An efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation method for aflatoxin generation fungus <Emphasis Type="Italic">Aspergillus flavus</Emphasis>

Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ～ 60 positive transformants per 10⁶ conidia using our protocol. A small-scale insertional mutant library (～ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.     

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.     

稻瘟菌突变体T-DNA插入位点的精细定位和插入模式的研究

随机挑取已构建的37个稻瘟菌T-DNA突变株,利用TAIL-PCR技术扩增出T-DNA插入位点的侧翼序列,测序并进行比对分析。结果显示:成功获得扩增产物并测序的序列共有39条,T-DNA边界序列为稻瘟菌序列的有19条,其余20条为载体主干序列。在这有效扩增为稻瘟菌序列的19条中,有10条是T-DNA右侧翼序列与稻瘟菌序列,9条为左侧翼序列加稻瘟菌序列。分析T-DNA剪切位点,10条右侧翼序列中有9条的剪切位点相同,这与农杆菌介导T-DNA转化植物一样。而左边界的剪切位点就没有这种规律性。研究也精细确定了17个不同突变株的T-DNA插入位置,为后续的基因功能研究奠定基础。     

农杆菌介导的紫色红曲霉遗传转化体系的建立和优化

通过优化各种转化因素,建立了根癌农杆菌(Agrobacterium tumefaciens)介导红曲霉(Monascus)的高效转化体系:红曲霉在PDA培养基培养21 d后收集孢子,制备红曲霉孢子悬浮液,浓度为106个/mL,根癌农杆菌浓度为OD600值0.5,诱导剂AS浓度为100μmol/L,农杆菌与红曲霉在25℃共培养3 d。采用此转化体系构建了含有530多个转化子的红曲霉T-DNA插入突变体库。随机选取50株转化子菌株进行分子验证和稳定性检测,证明T-DNA成功插入红曲霉基因组DNA中,并能稳定遗传。最后,通过形态观察筛选出8株变异较大的菌株,为以后的红曲霉基因功能研究奠定了一定的基础。