农杆菌介导的灰葡萄孢T-DNA插入突变体库构建及插入位点分析

【目的】利用农杆菌(Agrobacterium tumefaciens)介导法对灰葡萄孢(Botrytis cinerea)进行转化,构建T-DNA插入突变体库,为从分子水平上认识灰葡萄孢的致病机制打下基础。【方法】以含有pCAMBIA 1390双元载体的农杆菌对灰葡萄孢进行转化,利用潮霉素进行筛选。对抗性稳定的转化子进行生物学和形态学观察,采用离体番茄叶片进行致病性测定。利用TAIL-PCR技术对突变体中T-DNA的旁侧序列进行克隆。【结果】得到了一些突变体,表现为生长速率减缓、产孢能力下降、致病力减弱等。克隆并分析了其中一个突变体中T-DNA插入的位置和旁侧序列。【结论】本实验建立了农杆菌介导的灰葡萄孢转化体系,构建了T-DNA插入的灰葡萄孢突变体库。用TAIL-PCR进行突变体中T-DNA旁侧序列的分析是可行的。     

球孢白僵菌T-DNA突变体库的构建及高温和高渗敏感型突变体的筛选

摘要：【目的】筛选对高温和高渗等逆境胁迫敏感的球孢白僵菌T-DNA随机插入突变体,并克隆相关基因,为研究杀虫真菌适应逆境的分子机理奠定基础。【方法】利用逆境胁迫的方法从球孢白僵菌的T-DNA随机插入突变库中筛选对高温和高渗敏感的突变体,进而利用YADE (Y-shaped adaptor dependent extension)技术克隆相关基因。【结果】筛选得到5个对高温和高渗敏感的突变体。其中2个（212和2550）对高温敏感,在32oC下生长完全受抑;其它3个突变体对高渗胁迫敏感。突变体212的分生孢     

油茶果生炭疽菌小分子GTP酶Rab7的功能研究

【目的】炭疽病是油茶的一种重要病害,果生炭疽菌是油茶炭疽病的主要致病菌。本文对果生炭疽菌小分子GTP酶Rab7进行研究,为油茶炭疽病的防控治理提供依据。【方法】构建CfRAB7基因敲除载体,通过PEG介导的原生质体转化、抗性筛选和PCR电泳验证获得果生炭疽菌突变体菌株△Cfrab7和互补菌株△Cfrab7/CfRAB7。进一步分析CfRAB7基因敲除突变体△Cfrab7的生长、产孢、附着孢的形成、胁迫应答、液泡融合和致病力等生物学表型。【结果】在PDA和MM培养基上,突变体△Cfrab7的菌落直径显著减小,产孢量和附着孢形成率显著降低,且不能穿透玻璃纸;在10mmol/LH2O2条件下,△Cfrab7生长受到明显抑制;进一步研究发现突变体△Cfrab7液泡无法正常融合,在油茶有伤和无伤的幼叶上均不发病。【结论】CfRAB7基因参与调控果生炭疽菌生长产孢、附着孢形成、H2O2胁迫应答、液泡融合和致病力。     

菰黑粉菌T-DNA突变体库的构建及分析

通过建立适用于菰黑粉菌Ustilago esculenta的农杆菌介导遗传转化(Agrobacterium tumefaciens-mediated transformation,ATMT)体系,构建菰黑粉菌T-DNA插入突变体库。针对性地筛选双核菌丝形成缺陷型转化子,并对T-DNA插入位点进行分析,为研究菰黑粉菌二态型转换的分子调控机理打下基础。以构建的菰黑粉菌自融合菌株TSP为出发菌株,以含有遗传霉素(G418)抗性基因(neo)的质粒为载体,通过ATMT构建菰黑粉菌T-DNA突变体库,并对诱导剂乙酰丁香酮(AS)浓度、转化的共培养时间、农杆菌浓度和菰黑粉菌芽孢子浓度等建库影响因素进行单因素条件试验,筛选最优条件;对继代培养的转化子基因组中的遗传霉素抗性基因进行PCR检测,验证转化子遗传稳定性;对突变体库中的转化子双核菌丝生长情况进行观察,测定其双核菌丝形成能力;对上述双核菌丝形成缺陷型转化子进行基因组重测序,分析其T-DNA插入位点。当遗传霉素浓度为75μg/mL时,菰黑粉菌的生长被完全抑制。当AS浓度为100μg/mL、共培养时间为24 h、孢子浓度为1×10⁵     

尖镰孢古巴专化型Focr4-1701突变体的生物学表型研究

由尖镰孢古巴专化型(Fusarium oxysporum f.sp.cubense,Foc)引起的香蕉枯萎病是香蕉生产中的毁灭性病害之一。Foc有4个小种,其中4号小种(Focr4)因其寄主范围广且无高抗病性的香蕉品种,对香蕉产业的危害最大,其致病机制至今尚不清楚。为研究其致病机制,本文对Focr4野生型菌株(Focr4-193-6)、因其T-DNA插入导致致病性严重减弱的突变体Focr4-1701、T-DNA插入标签基因敲除子△Focr4-1701及敲除基因互补子△Focr4-1701-cp-1为材料,从致病性测定、玻璃纸穿透试验、孢子形态观察、产孢量与孢子萌发率测定、粗毒素测定等方面对其与致病性相关的生物学表型进行了测定。结果表明:T-DNA插入突变体Focr4-1701和基因敲除突变体△Focr4-1701接种后的香蕉植株叶片没有表现发病症状,敲除基因互补菌株与野生型菌株一样表现出发病症状;T-DNA插入突变体和基因敲除突变体不能穿透玻璃纸生长,T-DNA插入突变体及基因敲除突变体在产孢量、孢子萌发率和产毒素能力上均极显著低于野生型菌株及互补菌株。这些表型性状差异说明Focr4-1701致病性严重减弱可能是T-DNA插入位点标签基因失活后导致了菌丝体侵染能力下降及产毒素能力降低造成的。     

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。     

大丽轮枝菌（Verticillim dahliae）突变体库的构建与分析

为了更加快捷地筛选大丽轮枝菌致病关键基因,利用聚乙二醇介导的原生质体转化法构建病原菌随机插入突变体库,对部分阳性转化子的生长表型指标和致病力进行分析,筛选生长发育与致病缺陷突变体并进行靶标基因定位。结果表明,本研究共获得13 030多个阳性转化子,随机挑选5个转化子与V991野生型菌株进行比较分析,发现其中一个突变体在PDA和不同碳源培养基上的菌落直径、产孢量以及致病力均显著降低。侧翼序列测序结果结合Blast比对分析表明,该缺陷突变体中的潮霉素抗性基因表达盒定位于大丽轮枝菌3号染色体1 528 782 bp处,属于内切葡聚糖酶1基因（VDAG＿04017）。综上所述,本研究通过大丽轮枝菌插入突变体库构建、突变体生长表型指标和致病力鉴定及靶标基因定位等一系列分子生物学手段,可初步鉴定病原菌致病相关基因,为从全基因组层面深入研究大丽轮枝菌致病机制奠定了一定基础。     

胶孢炭疽菌C2H2型转录因子CgGcp1的生物学功能

【背景】胶孢炭疽菌(Colletotrichum gloeosporioides)可以寄生于多种植物,侵染方式多样,能够引起严重的农业危害。在胶孢炭疽菌中,CgGcp1是一个C2H2型的转录因子,关于其生物学功能的研究未见报道。【目的】明确CgGcp1的生物学功能,为深入解析该病菌的致病机制奠定一定的理论依据。【方法】构建CgGCP1基因的敲除载体,利用同源重组得到敲除突变体。通过表型分析,包括营养生长、胁迫响应、孢子产生、附着胞形成及致病性分析等,明确该基因的生物学功能。【结果】CgGCP1基因敲除突变体生长速率较野生型减慢,对SDS、刚果红、NaCl和甘油更加敏感,孢子产量显著降低,附着胞的形成率降低且侵入能力减弱,在橡胶叶片上的致病力明显下降。【结论】CgGcp1参与调控胶孢炭疽菌营养生长、细胞壁完整性、分生孢子产生、附着胞形成与侵入和致病性。     

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。     

基于CRISPR-Cas9技术构建橡胶树胶孢炭疽菌的基因敲除系统

[背景]CRISPR-Cas9基因组编辑技术为病原真菌的基因敲除、敲入及定点编辑提供了新的思路。[目的]建立适用于橡胶树胶孢炭疽菌的CRISPR-Cas9基因敲除系统。[方法]通过大肠杆菌原核表达系统合成含有细胞核定位信号的Cas9蛋白;以URA5为靶标基因,预测该基因中Cas9的切割位点,并在体外转录合成相应的SgRNA;体外构建Cas9-SgRNA复合体,并将该复合体转入橡胶树胶孢炭疽菌原生质体;通过表型筛选及测序鉴定,筛选URA5的敲除突变体菌株。[结果]体外表达的Cas9蛋白与SgRNA能够形成复合体,并在体外对目标基因URA5的DNA序列进行切割;Cas9-SgRNA复合体能够成功转入橡胶树胶孢炭疽菌原生质体,并完成对URA5的敲除;敲除突变株表现出尿嘧啶缺陷表现型。[结论]建立了适用于橡胶树胶孢炭疽菌的基因敲除系统。     

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。     

Identifying pathogenicity genes in the rubber tree anthracnose fungus Colletotrichum gloeosporioides through random insertional mutagenesis

To gain more insight into the molecular mechanisms of Colletotrichum gloeosporioides pathogenesis, Agrobacterium tumefaciens-mediated transformation (ATMT) was used to identify mutants of C. gloeosporioides impaired in pathogenicity. An ATMT library of 4128 C. gloeosporioides transformants was generated. Transformants were screened for defects in pathogenicity with a detached copper brown leaf assay. 32 mutants showing reproducible pathogenicity defects were obtained. Southern blot analysis showed 60.4% of the transformants had single-site T-DNA integrations. 16 Genomic sequences flanking T-DNA were recovered from mutants by thermal asymmetric interlaced PCR, and were used to isolate the tagged genes from the genome sequence of wild-type C. gloeosporioides by Basic Local Alignment Search Tool searches against the local genome database of the wild-type C. gloeosporioides. One potential pathogenicity genes encoded calcium-translocating P-type ATPase. Six potential pathogenicity genes had no known homologs in filamentous fungi and were likely to be novel fungal virulence factors. Two putative genes encoded Glycosyltransferase family 28 domain-containing protein and Mov34/MPN/PAD-1 family protein, respectively. Five potential pathogenicity genes had putative function matched with putative protein of other Colletotrichum species. Two known C. gloeosporioides pathogenicity genes were also identified, the encoding Glomerella cingulata hard-surface induced protein and C. gloeosporioides regulatory subunit of protein kinase A gene involved in cAMP-dependent PKA signal transduction pathway.     

Transformation of Coniothyrium minitans, a parasite of Sclerotinia sclerotiorum, with Agrobacterium tumefaciens

Coniothyrium minitans is a potential biological control agent of the plant pathogenic fungus Sclerotinia sclerotiorum. In this research, T-DNA insertional transformation of strain ZS-1 of C. minitans mediated by Agrobacterium tumefaciens was obtained, with optimization of spore maturity for transformation. After confirmation by PCR, transformants were subjected to Southern blot analysis, and results showed that more than 82.7% of transformants had single T-DNA insertions, and 12.1% of transformants had two copies T-DNA insertions. The genomic DNA segments of transformants flanking the T-DNA could be amplified from both borders with TAIL-PCR. Four types of mutants were screened and identified from the T-DNA insertional library, which comprised sporulation deficient mutants, pathogenicity deficient mutants, pigment change mutants and antibiotic deficient mutant, and some of the mutants were described; the number and frequency of each type of mutant from the library were calculated, and the frequency of each type is 3.27 x 10(-3), 1.0 x 10(-4), 1.4 x 10(-4), 2.5 x 10(-4), respectively. The successful creation of the T-DNA insertional transformation library may help us to unravel the interaction between a parasite and its host at a molecular level, to clarify the differentiation and development of this fungus, and to analyze and clone functional genes from the biocontrol microorganism in tripartite associations.     

胶孢炭疽菌CgRGS2基因的克隆及生物学功能

【目的】G蛋白信号调控因子(Regulators of G-protein signaling,RGS)是G蛋白的一类负调控因子,在植物病原菌生长发育及致病过程中发挥着重要的作用,然而目前还未有关于胶孢炭疽菌RGS蛋白生物学功能的研究。本试验的目的是克隆胶孢炭疽菌的一个RGS基因CgRGS2,并分析其生物学功能。【方法】利用PCR技术扩增CgRGS2的基因并进行生物信息学分析,利用同源重组的方法获得CgRGS2基因的敲除突变体,并在突变体的基础上获得互补株,通过表型分析确定该基因的生物学功能。【结果】通过PCR扩增获得了CgRGS2的基因,其编码一个574个氨基酸的蛋白,在N末端含有一个RGS功能域。该基因敲除突变体同野生型相比,表现为营养生长缓慢,气生菌丝浓密,分生孢子产量降低且孢子呈多端萌发,对氧化压力及SDS敏感,致病性减弱等。【结论】CgRGS2蛋白参与调控胶孢炭疽菌的营养生长,分生孢子产量及萌发,氧化应激反应及细胞壁完整性,对其致病性也具有一定的影响。     

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...     

Transformation of Corynespora cassiicola by Agrobacterium tumefaciens

The fungus causing target spot disease, Corynespora cassiicola (Berk. & M. A. Curtis) C. T. Wei, poses an increasing threat to watermelon (Citrullus lanatus), muskmelon (Cucumis melo), and cucumber (Cucumis sativus); the most economically important cucurbit crops grown in China. An understanding of the molecular mechanisms underlying the pathogenicity of C. cassiicola is essential for the development of new strategies to control this disease-causing fungus. Agrobacterium tumefaciens-mediated transformation (ATMT) might be useful to obtain transformants of C. cassiicola, for the ultimate identification of genes involved in pathogenicity. In the present work, we established and optimized an ATMT protocol using A. tumefaciens strain AGL-1 carrying the vector pATMT1 for C. cassiicola. Efficiency of ATMT was 102–148 transformants per 10⁶ conidia and successive subculturing of transformants on non-selective and selective media demonstrated that the integrated transfer (T)-DNA was stably inherited in C. cassiicola transformants. The integration of the hygromycin B phosphotransferase (hph) gene into C. cassiicola was validated by PCR and Southern blot analyses, which revealed that nearly 90 % of the transformants contained single-copy T-DNA. The transformants with altered phenotypes were characterized. Three of these transformants completely lost pathogenicity and other three showed strongly impaired pathogenicity relative to the Cc-GX strain on muskmelon leaves. These results strongly suggest that ATMT may be used as a molecular tool for identifying genes relevant to pathogenicity in the fungus C. cassiicola, an emerging threat to several agronomically important plants in China.     

Transformation of the fungusPyrenopeziza brassicae,cause of light leaf spot of brassicas,and complementation of mutants using a genomic library

An efficient gene transfer system is a prerequisite for the molecular genetic analysis of pathogenicity and other genes of plant pathogens. A transformation procedure for the fungusPyrenopeziza brassicae was therefore devised. Three plasmids, encoding hygromycin resistance (pAN7-1, pAN7-2) or phleomycin resistance (pAN8-1), were used to transform conidial protoplasts ofP. brassicae in the presence of calcium chloride and polyethylene glycol. Transformation arose due to integration of transforming DNA, apparently at random sites, and multiple integration events were common. The frequency of transformation was variable but similar to that reported for other phytopathogenic fungi (up to 20 μg⁻¹ DNA) and was increased when homologous DNA was included in the vector. The pathogenicity of the transformants was unchanged by transformation and, when reisolated from inoculated host tissue, the transformants were found to have retained their antibiotic resistance. The transformation technique was used to complement adeninerequiring and extracellular enzyme-deficient, UV-induced mutants to prototrophy and extracellular protease production, respectively, with cosmids from a genomic library of the fungus.