农杆菌介导的小孢拟盘多毛孢遗传转化及突变体筛选

本研究通过农杆菌EHA105介导的方法,以含潮霉素抗性基因和GFP基因的双元载体pCAMBgfp为转化载体,对小孢拟盘多毛孢Pestalotiopsis microspora KFRD-2菌株进行遗传转化。基于潮霉素及GFP荧光抗性进行转化子的初步筛选,随后,进一步对转化子的菌落特征、菌丝生长速率、产孢量、荧光稳定性及致病力进行验证。结果获得阳性转化子100余株,转化效率达200个转化子/10⁶个孢子。大部分转化子与野生型菌株无明显形态及致病力差异。同时,获得了14株菌丝形态、产孢量或致病力与野生型存在明显差异的突变株,可用于小孢拟盘多毛孢关键致病基因的挖掘验证及致病机理等研究。     

球孢白僵菌不同分离株的生物学及对小猿叶甲成虫致病性测定

球孢白僵菌Beauveriabassiana是一种寄主范围广、致病性强、适应性广的昆虫病原真菌。对采自广东阳山小猿叶甲Phaedonbrassicae的球孢白僵菌 6个分离株 (A ,B ,C ,D ,E ,F)的菌落生长速率、产孢量、分生孢子萌发率进行了实验 ,测定和比较了这 6个分离株对小猿叶甲成虫的致病力。结果表明 :各分离株在菌落生长速率上没有显著差异 ,但各分离株的孢子萌发率有显著差异 ,其中C ,D ,E分离株产孢量明显高A ,F分离株。用 5× 1 0 7分生孢子 mL的悬浮液处理小猿叶甲成虫 ,分离株间的致病力有显著的差异 ,以D分离株对小猿叶甲成虫的感染死亡率最高 ,为 86 67%。综合比较 ,产孢量和致病性较高的D分离株可作为进一步研究的材料。     

灰葡萄孢分生孢子产生相关基因的克隆及功能分析

[目的]克隆灰葡萄孢分生孢子产生相关基因,并研究其功能,为进一步研究灰葡萄孢分生孢子产生机理和灰葡萄孢侵染及致病机理奠定基础.[方法]通过筛选灰葡萄孢ATMT突变体库,获得一株不能产生分生孢子的突变菌株BCt78,采用PCR和Southern Blotting技术,对突变菌株BCt78进行分子鉴定.利用TAIL-PCR技术获得T-DNA插入位点的侧翼序列;将所获得侧翼序列与灰葡萄孢基因组数据库中的已知基因序列进行BLAST分析,推测出T-DNA的插入位点;通过PCR进一步验证T-DNA的插入位点,利用RT-PCR技术确定突变基因;最后对突变菌株的菌落形态、生长速度、胞壁降解酶活力、粗毒素的生物活性、对番茄叶片的致病能力及部分致病相关基因的表达情况进行研究.[结果]TAIL-PCR结果证实T-DNA插入到灰葡萄孢BCIG 12707.1基因的ATG起始密码子区;RT-PCR结果证实突变基因为BCIG_12707.1,该基因DNA全长为135 bp,编码一个44个氨基酸的假定蛋白(Hypothetical protein).突变菌株在PDA培养基上菌落呈灰白色,生长速度减慢,不能产生分生孢子及菌核;对番茄叶片的致病性增强,且胞壁降解酶(PG、PMG和Cx)活力增强;突变菌株中参与细胞壁降解的角质酶基因cutA和多聚半乳糖醛酸酶基因Bepg1,信号转导途径基因(PKA1、PKA2、Bac、Bmp3),产毒素基因BcBOT2(Sesquiterpene synthase),漆酶基因Lac1,跨膜蛋白基因Btp1表达都增强.[结论]BC1G_ 12707.1基因在灰葡萄孢分生孢子产生、菌核形成及致病力等方面起重要作用.     

稻瘟菌MgORP1基因敲除突变株的构建及其表型分析

[目的]了解稻瘟病菌中氧固醇结合蛋白(oxysterol-binding proteins related proteins,缩写为ORPs)家族成员组成情况,构建MgORP1基因缺失突变株和互补株,对MgORP1基因功能进行初步研究.[方法]以ORPs家族的典型结构域"ORD"为靶标,对稻瘟病菌基因组数据库进行BlastP搜索.通过同源重组的策略,构建MgORP1基因缺失突变体,再通过重新导入该基因全长片段获得互补株.然后对野生型、突变体和互补株进行菌落、分生孢子和附着胞形态或形成情况、以及致病力进行比较分析.[结果]稻瘟病菌基因组中含有6个可能的ORPs族蛋白,其中MgORP1基因的破坏降低了稻瘟菌在完全培养基上的菌落生长速率和产孢量.但对菌丝、分生孢子和附着胞的形态,以及在水稻上的致病力没有明显影响.[结论]MgORP1基因可能与稻瘟病菌的菌落生长和产孢量相关.     

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。     

AsSlt2基因对茄链格孢细胞壁完整性的调控

【背景】由茄链格孢(Alternaria solani)引起的马铃薯早疫病被普遍认为是马铃薯生产上的第二大叶部病害,在马铃薯各产区普遍发生,给马铃薯生产造成了巨大的经济损失。【目的】明确AsSlt2基因对茄链格孢细胞壁完整性的影响。【方法】在含有刚果红、细胞壁降解酶和十二烷基硫酸钠(sodiumdodecylsulfate,SDS)等细胞壁胁迫的培养基上观察ΔAsSlt2缺失突变株的生长情况,计算相对生长抑制率;通过实时荧光定量PCR (RT-qPCR)方法检测ΔAsSlt2菌株中细胞壁合成相关基因的表达情况;进一步检测ΔAsSlt2细胞壁中几丁质的含量及胞外酶活性。【结果】ΔAsSlt2缺失突变株对SDS、刚果红、细胞壁降解酶等细胞壁胁迫的敏感性增强,在加入细胞壁降解酶后突变株原生质体释放量显著增多;ΔAsSlt2对外源氧胁迫更敏感,突变株胞外过氧化物酶和漆酶活性均显著降低;进一步研究发现,ΔAsSlt2细胞壁中几丁质含量减少,几丁质合成相关基因与漆酶合成相关基因的表达量均明显降低。【结论】AsSlt2基因在茄链格孢细胞壁的完整性及抵御外界胁迫方面发挥重要作用。     

橡胶树胶孢炭疽菌NADPH氧化酶功能研究

由病原真菌胶孢炭疽菌引起的炭疽病害是造成海南省橡胶减产的主要原因之一。研究橡胶树胶孢炭疽菌致病力的分子机制能为其病害防治提供理论依据。采用同源重组原理、运用原生质体转化法构建了橡胶树胶孢炭疽菌NADPH氧化酶编码基因的敲除突变株,并对其生长发育及致病力等生理表型进行分析。结果表明,在橡胶树胶孢炭疽菌中,存在3个编码NADPH氧化酶的基因CgNoxA、CgNoxB、CgNoxC,以及一个编码NADPH氧化酶调节蛋白的基因CgNoxR。PCR分析表明成功构建了这4个基因的敲除突变株。与野生型菌株相比,这4个敲除突变株的产孢能力、致病力均有不同程度的变化。其中CgNoxA敲除突变株的产孢能力严重受损;CgNoxR敲除突变株的孢子萌发过程明显异常;CgNoxB敲除突变株对橡胶树叶片的致病力显著下降。染色观察表明,CgNoxA和CgNoxB参与调控胶孢炭疽菌菌丝中超氧阴离子代谢过程。上述结果表明,NADPH氧化酶在调控胶孢炭疽菌的孢子萌发及致病力过程中起着重要作用。     

费氏链霉菌中子辐射诱变菌株对新霉素的生物转化

对新霉素产生菌费氏链霉菌进行中子辐射诱变和突变株筛选, 获得不产新霉素的突变株。以突变株为转化菌种, 以新霉素为底物, 对转化发酵液进行高效液相色谱分析, 研究了不同转化条件对新霉素转化的影响。结果表明, 底物浓度、底物添加时间、底物添加方式、接种量、培养基装量、转化时间、碳源、氮源、pH、温度对新霉素转化具有不同程度的影响。以转化条件优化参数进行转化大量培养, 转化液经4步离子交换层析进行分离纯化, 薄层层析检测纯化样品为单一斑点。采用薄层生物自显影对获得的4个转化产物分离样品做生物活性检测, 发现4个样品对金黄色葡萄球菌和姜青枯假单孢杆菌都具有抑制活性, 1个样品对大白菜软腐样品具有明显的抑制活性。     

维生素C二步发酵中巨大芽孢杆菌对氧化葡萄糖酸杆菌生长和产酸的影响

通过测定氧化葡萄糖酸杆菌转化L－山梨糖中成ZKGA的细胞酶活性、摇瓶发酵及中长变化,研究了Vc:步发酵中巨大茅孢杆菌对氧化葡萄糖酸杆菌生长和产酸作用的影响。结果显示：巨大芽孢杆菌胞外液和胞内液均可促进氧化葡萄糖酸杆菌的增殖,主要表现为缩短其中长周期中的延迟期;巨大芽孢杆菌通过所产生的部分生物活性物质增强氧化葡萄糖酸杆菌产酸的细胞酶活性,促进氧化葡萄糖酸杆菌转化L一山梨糖生成2KGA.     

玉米弯孢叶斑病菌REMI白化突变体的筛选与致病性测定

旨在探讨玉米弯孢叶斑病菌致病机制及遗传变异特性,利用限制性内切酶介导的基因整合技术(REMI)对Curvularia lunata进行了遗传转化,共获得109个稳定的突变株。PCR检测结果表明,质粒p UC-JS已成功导入突变株中,转化子既有单拷贝也有多拷贝,白化菌株单拷贝插入频率为40%。此外,白化玉米弯孢叶斑病菌REMI突变株菌落颜色与野生菌株差异明显,并且在致病性上也明显弱于野生菌株;质粒拯救、测序与blast分析推测插入可能导致FAD3基因功能丧失。     

莱氏野村菌紫外线诱变抗敌敌畏菌株的生理特性

[背景]夜蛾科害虫易对化学杀虫剂产生高抗性,但一些化学农药可以对部分虫生真菌的毒力作用效果起增幅作用,目前缺乏对莱氏野村菌(Nomuraea rileyi)的该方面研究.[目的]探究对常用有机磷杀虫剂敌敌畏具有较强耐药性的紫外线诱变莱氏野村菌突变菌株的生理特性,包括菌丝生长、产孢情况和产几丁质酶活性.[方法]在紫外线诱...     

Transformation and mutagenesis of the nematode-trapping fungus Monacrosporium sphaeroides by restriction enzyme-mediated integration (REMI)

In this study, the nematode-trapping fungus, Monacrosporium sphaeroides, was transformed with a plasmid harboring the hygromycin B phosphotransferase gene, via restriction enzyme-mediated integration (REMI). Frequencies of up to 94 transformants microg(-1) per linearized plasmid DNA were obtained by optimizing the PEG concentration, as well as the category and quantity of the added restriction enzyme. 90% of the transformants were determined to be stable for drug resistance when 20 randomly selected transformants were tested. Southern analyses revealed that the transforming DNA was integrated into the M. sphaeroides genome either with or without rearrangement. Five mitotic stable mutant strains were obtained using this approach, all of which had been altered with regard to sporulation capacity and pathogenicity toward nematodes. Southern blot analyses of the five mutants revealed that foreign plasmid DNA had integrated into the genome. Three of the mutants, Tms2316, Tms3583 and Tms1536, exhibited integration at a single location, whereas the remaining two, Tms32 and Tms1913, manifested integration at double or multiple locations. Our results suggest that the transformation of M. sphaeroides via REMI will facilitate insertional mutagenesis, the functional analysis of a variety of genes, and the tagging or cloning of genes of interest.     

棉花枯萎病菌突变体库的构建及分析

[背景]棉花枯萎病逐渐成为威胁新疆海岛棉产业发展的主要病害,但关于棉花枯萎病菌的致病力、产孢量、生长速度及颜色变化等相关功能基因目前还不是十分明确.[目的]通过构建绿色荧光蛋白(green fluorescent protein,GFP)标记棉花枯萎病菌突变体库,筛选出由于T-DNA的随机插入而导致性状发生变异的突变体...     

Disruption of Botrytis cinerea class I chitin synthase gene Bcchs1 results in cell wall weakening and reduced virulence

To get a better insight into the relationship between cell wall integrity and pathogenicity of the fungus Botrytis cinerea, we have constructed chitin synthase mutants. A 620 bp class I chitin synthase gene fragment (Bcchs1) obtained by PCR amplification was used to disrupt the corresponding gene in the genome. Disruption of Bcchs1 occurred at a frequency of 8%. Nine independent mutants were obtained and the Bcchs1 mutant phenotype compared to that of transformants in which the gene was not disrupted. These disruption mutants were dramatically reduced in their in vitro Mg2+, Mn2+, and Co2+-dependent chitin synthase activity. Chitin content was reduced by 30%, indicating that Bcchs1p contributes substantially to cell wall composition. Enzymatic degradation by a cocktail of glucanases revealed cell wall weakening in the mutant. Bcchs1 was transcribed at a constant level during vegetative exponential growth, suggesting that it was necessary throughout hyphal development. Bcchs1 mutant growth was identical to undisrupted control transformant growth, however, the mutant exhibited reduced pathogenicity on vine leaves. It can be assumed that disruption of Bcchs1 leads to cell wall weakening which might slow down in planta fungal progression.     

The role of a fadA ortholog in the growth and development of Colletotrichum graminicola in vitro and in planta

A transposon-based split-marker protocol was used to produce insertional mutations in the fadA ortholog of the maize anthracnose pathogen Colletotrichum graminicola. The mutants grew more slowly in culture, produced fewer oval spores, produced fusiform rather than falcate phialospores, lost their normal clockwise spiral growth pattern in culture, and were significantly reduced in their pathogenicity to maize stalks and leaves. The differential effect of the fadA mutation on oval spore versus phialospore production suggests that there are differences in the signaling pathways that regulate these two types of sporulation. It has been suggested that oval spores function in anthracnose lesion extension. In maize stalks, production of oval spores appeared to be relatively unaffected in the mutant strains, but production of vegetative hyphae and elongation of primary lesions were both reduced. This suggests that vegetative hyphae play a more important role than oval spores in primary lesion development. However, production of discontinuous secondary lesions in maize stalks infected by mutant strains did not appear to be seriously affected, and thus oval spores may play a more important role in that process.     

Construction of a Tn5-Tagged Mutant Library of Xanthomonas oryzae pv. oryzicola as An Invaluable Resource for Functional Genomics

To genome-widely mine pathogenesis-related genes of Xanthomonas oryzae pv. oryzicola (Xoc), which is the casual agent of bacterial leaf streak resulting in significant yield loss and poor quality in rice, a Tn5 transposon-mediated mutation library was generated. Twenty-five thousand transformants were produced by using Tn5 transposome, appropriately corresponding to 5 × ORF coverage of the genome, and inoculated into rice and tobacco, individually and respectively, for screening candidate virulence genes. Southern blot and thermal asymmetric interlaced polymerase chain reaction analysis of Tn5 insertion sites of randomly selected mutants suggested a random mode of transposition and a saturation library. Characterization of extracellular polysaccharides, extracellular protease activity, and pigment production of individual mutants in the growth media revealed that 11 mutants enhanced in growth, 12 reduced extracellular polysaccharide production, 12 lost extracellular protease activity completely or partially, and 21 were pigment deficient. In planta pathogenicity assays revealed 253 mutants reduced virulence in rice, but kept triggering hypersensitive response in tobacco; 49 lost the ability to elicit HR in tobacco and pathogenicity in rice; and 3 still induced hypersensitive response in tobacco, but lost pathogenicity in rice. The achieved mutant library of Xoc is of high-quality and nearly saturated and candidate virulence mutants provided a strong basis for functional genomics of Xoc.     

Analysis of Sporulation Mutants II. Mutants Blocked in the Citric Acid Cycle

Sporulation mutants that were unable to incorporate uracil during the developmental period recovered this capacity with the addition of ribose and in most cases with the addition of glutamate. Of the mutants that responded to both ribose and glumate, all but three also responded to citrate, and all but five responded to acetate. One of the exceptional strains was deficient in aconitase and another one in aconitase and isocitrate dehydrogenase; both required glutamate for growth. For the mutants which did not respond to glutamate, the products made from (14)C-glutamate were determined by thin-layer chromatography. Significant differences were found which enabled the identification of mutant blocks. The deficiency of the corresponding enzyme activity was verified. Several mutants were deficient in alpha-ketoglutarate dehydrogenase, and one lacked succinic dehydrogenase. These mutants could still grow on glucose as sole carbon source, but not on glutamate. The intact Krebs cycle is therefore not required for vegetative growth of aerobic Bacillis subtilis, but it is indispensable for sporulation.     

Bacteriocin-resistant mutants of Erwinia chrysanthemi: possible involvement of iron acquisition in phytopathogenicity.

A series of bacteriocin-resistant mutants of Erwinia chrysanthemi 3937JRH were unable to elicit soft-rot symptoms on saintpaulia plants. The loss of pathogenicity was correlated with the disappearance of one to three outer membrane polypeptides (molecular weights, about 80,000 to 90,000) whose production in wild-type strains was greatly enhanced under iron-limited growth conditions. The mutants did not exhibit altered extracellular pectinolytic or cellulolytic activities.     

clap1, a gene encoding a copper-transporting ATPase involved in the process of infection by the phytopathogenic fungus Colletotrichum lindemuthianum

A screen for insertional mutants of Colletrichum lindemuthianum, the causative agent of common bean anthracnose, led to the identification of a non-pathogenic, lightly colored transformant. This mutant is unable to induce disease symptoms on intact or wounded primary leaves of seedlings and plantlets of Phaseolus vulgaris. In vitro, it exhibits normal vegetative growth, sporulation and conidial germination, but the cultures remain beige instead of becoming black. Microscopic examination revealed that this mutant forms fewer appressoria than the wild-type strain, and these are misshapen and poorly melanized. Molecular analyses indicated that the mutagenic plasmid had targeted clap1, a gene encoding a putative copper-transporting ATPase sharing 35% identity with the human Menkes and Wilson proteins and the product of the CCC2 gene of Saccharomyces cerevisiae. Complementation of the non-pathogenic beige mutant with a wild-type allele of clap1 restored both pathogenicity and pigmentation. Conversely, replacement of the wild-type allele with a disrupted clap1 gene gave rise to non-pathogenic beige transformants. Compared with the wild-type strain, extracts from clap1 mutants were found to have very low levels of phenol oxidase activity. These observations suggest that the clap1 gene product may be involved in the pathogenicity of C. lindemuthianum strains because of its role in delivering copper to secreted cuproenzymes, such as the phenol oxidases that mediate the polymerization of 1,8-dihydroxynaphthalene to melanin.