香梨果萼黑斑病菌Alternaria alternata遗传转化体系的建立及GFP标记菌株的获得

 为建立香梨果萼黑斑病菌链格孢(Alternaria alternata)的原生质体遗传转化体系,本实验以香梨果萼黑斑病菌强致病性菌株LI1为供试材料,研究菌龄、酶系统、酶解时间等对链格孢菌原生质体制备的影响。链格孢菌菌丝在CM液体培养基中培养20 h,以0.7 mol·L^-1 NaCl为稳渗剂,1%裂解酶+1%崩溃酶+1%蜗牛酶的酶液组合下,28 ℃酶解4 h,原生质体制备效率最高。通过PEG/CaCl₂介导法将含有潮霉素B抗性基因和绿色荧光蛋白基因的质粒转入链格孢菌LI1,转化子生长表型及外源基因的PCR鉴定结果表明抗性基因已成功整合到香梨果萼黑斑病菌中。成功建立了香梨果萼黑斑病菌链格孢菌的原生质体遗传转化体系,并成功获得GFP标记菌株,为病原菌侵染定殖过程及致病机制研究奠定了基础。     

根癌农杆菌介导的胶孢炭疽菌遗传转化体系的建立

本研究基于农杆菌介导的遗传转化方法,建立了芒果胶孢炭疽菌高效的遗传转化体系,获得一批炭疽菌的T-DNA插入突变体,其目的是为炭疽菌的功能基因组学研究和致病相关基因的克隆奠定基础。结果如下:通过摸索并优化了体系的各项因子,在潮霉素筛选浓度为200μg/mL,菌液浓度为OD₆₆₀=0.15条件下,AS为200μmol/L,选择pH5.5的IM共培养基中转化效果最好;进一步通过对转化子的继代稳定性和PCR检测,结果发现潮霉素抗性稳定遗传和假阳性率低;通过菌落形态观察和产孢能力的测定获得3个菌落形态异常突变体,6个产孢能力下降突变体。     

一种优化的辣椒尖孢炭疽菌遗传转化体系

由辣椒尖孢炭疽菌(Colletotrichum acutatum)侵染引起的炭疽病是辣椒生产中最具有破坏性的真菌病害,严重影响辣椒的品质和产量。本研究以辣椒尖孢炭疽病菌HHDL02为对象,采用1 mol·L^-1 NH₄Cl 2%的酶裂解液裂解分生孢子萌发2 h的芽管,裂解2～3 h可以高效制备原生质体,结合PEG介导转化法成功将GFP基因导入,其转化菌株菌落形态、菌丝生长速率、分生孢子形态、孢子萌发、附着胞形成、产孢量和致病性与野生型菌株无明显差异,且后代荧光信号遗传稳定。PEG介导的原生质体转化适合辣椒尖孢炭疽菌遗传操作,有助于其致病机理的研究。     

莲腐败病菌遗传转化体系的构建

由共享镰刀菌Fusarium commune引起的莲腐败病是我国莲生产上的主要病害之一。本研究以强致病力菌株FCN23为供试菌株,通过PEG介导的遗传转化法研究了菌龄、酶解时间和渗透压稳定剂等对原生质体制备的影响。结果表明,分生孢子在YPD液体培养基培养24 h获得新鲜菌丝,在酶解时间为2.5 h,渗透压稳定剂为0.7 mol/L NaCl溶液时原生质体制备效率最高。进而将遗传霉素的抗性基因和绿色荧光蛋白基因转入莲腐败病菌原生质体中,获得了稳定表达的转化子,能够稳定遗传gfp基因,说明莲腐败病菌的遗传转化体系构建成功。该体系的建立为莲腐败病菌的侵染过程及基因功能研究奠定了基础。     

转DsRed荧光蛋白的新月弯孢Curvularia-lunata菌株构建

本研究通过农杆菌介导转化方法(ATMT),利用DsRed荧光蛋白基因对玉米弯孢叶斑病致病菌新月弯孢进行遗传转化。通过转化子的荧光蛋白基因和潮霉素B抗性基因的PCR检测,菌丝体和分生孢子的荧光观察,hyg基因的Southern杂交验证,以及荧光蛋白基因插入位点的TAIL-PCR分析,确定了DsRed荧光蛋白基因插入与表达对新月弯孢转化子的影响。结果表明:测定的4株转化子基因组中均成功整合了DsRed荧光蛋白目的基因片段;转化子在生长发育和致病性方面与野生型菌株存在一定差异,分别有2株在产孢量方面略高于野生型菌株,4株转化子在纤维素酶活性和粗毒素致病力方面均低于野生型菌株,有3株转化子在果胶酶活性上较野生型菌株有提高,1株转化子的致病力显著低于野生型菌株;获得其中3个转化子插入位点的侧翼序列。     

臂形草内生真菌HND5的GFP标记及其抑菌作用

臂形草Brachiaria brizantha内生真菌HND5及其产生的挥发性气体对多种植物病原真菌均有拮抗作用。本研究通过原生质体转化将绿色荧光蛋白（GFP）表达载体质粒pPKNTG转入HND5菌株中,获得了带绿色荧光且遗传稳定的转化子;PCR鉴定结果表明绿色荧光与外源质粒的插入相关。随机选取7个荧光转化子进行拮抗活性评价,发现其中5个转化子及其产生的挥发性气体对多主棒孢Corynespora cassiicola的拮抗活性与野生型菌株HND5相当。该结果为深入研究HND5菌株内生性及生防作用机理奠定了基础。     

茎基腐亚洲镰孢菌侵染抗感小麦品种的组织病理学观察

小麦茎基腐是由多种镰孢菌侵染的世界性土传病害,亚洲镰孢菌(Fusarium asiaticum)是我国冬小麦主产区茎基腐镰孢菌的优势种群,对小麦生产造成巨大损失。本研究利用绿色荧光蛋白报告基因标记亚洲镰孢菌,研究其侵染抗感小麦的病理组织学过程,建立了茎基腐病菌与寄主互作的直观性的研究体系,对病害防治及抗病育种具有重要意义。基于PEG-CaCl_2介导原生质体转化法将gfp导入亚洲镰孢菌株CF0915,对转化子进行荧光表达、PCR验证、遗传稳定性、生长特性及致病力分析,选取与野生型表现相近的转化子进行侵染分析。结果表明,绿色荧光蛋白基因(gfp)与潮霉素基因(hyg)PCR扩增表明gfp已整合入真菌基因组中,转化子菌丝与分生孢子表现强烈绿色荧光信号,gfp能够在转化子中稳定遗传,菌落形态、生长速度及致病力与野生型菌株无显著差异;将gfp标记病菌分生孢子接种感病品种1 d后,大量孢子附着于根毛及根表皮细胞开始萌发,接种2 d后观察到抗性品种分生孢子萌发;感病品种接种3 d后,菌丝直接侵入表皮细胞或沿表皮细胞间层定殖生长,扩展至皮层组织,8 d后菌丝从根部迅速扩展至茎基部,至第10 d大量菌丝充塞根皮层细胞,叶鞘维管束也被菌丝侵染,并产生大量大型分生孢子,植株表现褐色病斑,14 d后根部及茎维管束被大量菌丝体填充,而后产生大量厚垣孢子,至25 d大部分感病品种幼苗萎蔫死亡;与感病品种相比,抗性品种在整个侵染过程中表现时间滞后。本研究对引起茎基腐病的亚洲镰孢菌侵染小麦的组织学过程观察,为病菌致病机理的阐释及抗病资源的利用提供了重要理论依据。     

梨黑斑病菌遗传操作体系的建立与RFP标记转化子的致病性分析

由链格孢(Alternaria alternata)引起的梨黑斑病是我国梨生产上的主要病害之一,导致梨叶与果实产生黑斑症状及早期落叶,对我国梨产业的健康发展构成严重威胁。本实验在前期获得强致病性梨黑斑病菌菌株HN-5基础上,建立该病菌的遗传转化体系。通过优化原生质体制备过程以及转化体系发现梨黑斑病菌HN-5原生质体制备的最优条件为:菌丝在M R液体培养基中培养36 h;以0.7 M NaCl+0.8 M Mannitol为稳渗剂,配置复合细胞壁裂解酶(1%崩溃酶+1%裂解酶+1%纤维素酶+1%蜗牛酶),酶解菌丝3 h,原生质体的产量可达0.5×10~7个·mL~(-1)。通过PEG介导的原生质体遗传转化体系,将含有RFP基因的质粒p KD7转入链格孢菌HN-5中,转化效率为6个·μg~(-1)DNA。PCR检测和转化子荧光观察均表明RFP基因整合到了HN-5基因组中并成功表达。致病性分析发现RFP (Red Fluorescent Protein)标记转化子致病性未发生变化。本研究成功建立了PEG (Polyethylene glycol)介导的梨黑斑病菌遗传转化体系,构建了RFP标记菌株,为研究该病菌的致病机理奠定基础。     

甘蓝枯萎病菌REMI转化体系的建立

建立高效、稳定的甘蓝枯萎病菌REMI转化体系,为进一步获得特定表型突变体及基因功能研究建立技术储备.利用REMI(restriction enzyme mediate intergration)转化方法,将线性化的含有潮霉素抗性基因的pUCAT-PH质粒转化甘蓝枯萎病菌A6菌株的原生质体,摸索获得转化子最适的潮霉素筛选浓度以及不同限制性内切酶和酶量对转化效率的影响;利用PCR(polymerase chain reaction)技术对潮霉素抗性转化子进行验证.结果表明转化子的最适潮霉素筛选浓度为50 μg/mL;转化效率较高的限制性内切酶为HindⅢ,并且转化效率最高时的酶量为20 U.利用该转化体系构建了含1 050个转化子的甘蓝枯萎病菌转化子库,对转化子进行Southern验证,证明该转化体系是可行的.     

辣椒炭疽病生防菌株的筛选、鉴定及其抑菌机理

为有效防控由胶孢炭疽菌Colletorichum gloeosporioides引起的辣椒炭疽病,自辣椒上分离得到内生细菌,通过平板拮抗和辣椒离体生防试验筛选对胶孢炭疽菌有抑制作用的拮抗菌株,通过形态学特征、生理生化特征以及分子生物学技术对其进行鉴定,并于室内测定其对胶孢炭疽菌菌丝生长的影响、对辣椒炭疽病的防效及接种后辣椒内抗病活性物质含量以及防御酶活性。结果显示,从辣椒上共分离纯化获得46株细菌,其中菌株SQ-6对胶孢炭疽菌有明显的抑制作用,抑制率为61.11%,显著高于其他45株。结合菌株SQ-6的形态学特征、生理生化特征以及分子生物学特征,将该菌株鉴定为解淀粉芽胞杆菌Bacillus amyloliquefaciens。SQ-6菌株的50%无细胞滤液可引起胶孢炭疽菌菌丝畸形、断裂等,对其抑制率为57.87%。SQ-6菌株的10%、50%发酵液和10%、50%无细胞滤液均能显著降低由胶孢炭疽菌引起的辣椒炭疽病的发病率和病情指数,其中50%无细胞滤液的防效最好。SQ-6菌株能够提高辣椒内Vc、酚类和黄酮类物质含量,诱导辣椒内过氧化物酶（peroxidase,POD）、丙氨酸解氨酶（p...     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation as a tool for random mutagenesis of <Emphasis Type="Italic">Colletotrichum trifolii</Emphasis>

We transformed Colletotrichum trifolii, the causal agent of alfalfa anthracnose, using Agrobacterium tumefaciens as a new tool for random insertional mutagenesis. Fungal spores of C. trifolii were transformed with T-DNA including the hygromycin phosphotransferase gene (hph). Southern analysis showed that every randomly selected transformant had a unique hybridization pattern of T-DNA, suggesting that the T-DNA was randomly integrated into the fungal genome. More significantly, about 75% of transformants had a single copy of the T-DNA. The results demonstrate that insertional mutagenesis via A. tumefaciens is a useful tool for studying the function of C. trifolii genes.     

哈茨木霉T2-16的GFP标记及其生防特性

优化高效拮抗生防菌哈茨木霉T2-16的转化条件，筛选出与野生型菌株具有相似生防特性的阳性转化子，为生防木霉菌T2-16的定殖动态、分布规律等研究打下基础。通过PCR和分子克隆技术构建具有G418抗性基因的绿色荧光（GFP）表达载体pKN-sGFP，利用PEG-CaCl₂介导的原生质体转化法，获得强荧光表达的哈茨木霉T2-16转化子，并将其与野生型菌株的生物特性进行比较，筛选出与野生型菌株具有相似生防特性的阳性转化子。试验结果显示，哈茨木霉T2-16在20℃培养条件下对1000 μg/mL G418敏感，在上述优化条件下，转化获得稳定遗传的阳性转化子TG2-10；进一步比较其与野生型菌株的生物特性发现，两者之间无明显差异，可用于下一步哈茨木霉T2-16生防机理的研究。     

Transformation of Sclerotinia sclerotiorum with the green fluorescent protein gene and fluorescence of hyphae in four inoculated hosts

To obtain a genetic marker to observe and study the interaction of Sclerotinia sclerotiorum with its hosts, isolates ND30 and ND21 were transformed using pCT74 and gGFP constructs, both containing genes for the green fluorescent protein (GFP) and hygromycin B phosphotransferase. Putative transformants were obtained using polyethylene glycol-mediated transformation of protoplasts. Seven stable gfp transformants were identified and evaluated for fluorescence in vitro and in planta , pathogenicity and colonization of host tissues. Real-time quantitative polymerase chain reaction detected a single copy of the gfp gene in transformants. Fluorescence was quantified directly from mycelium and protein extracted from hyphae. The seven transformants (four from ND30 and three from ND21) were pathogenic on dry bean, canola, soybean and sunflower. However, depending on the host, three transformants differed significantly ( P  = 0·05) in the length of lesions formed compared to the wild-type. Hyphae fluoresced in plant tissue and could clearly be distinguished from plant cells. Infection and colonization of tissues were clearly visible with a fluorescent microscope. Transformants differed in the intensity of GFP expression both in vitro and in planta .     

绿色荧光蛋白基因标记棉花黄萎病菌

以携带有潮霉素抗性筛选标记的pCTHyg载体为骨架,构建了含有增强型绿色荧光蛋白基因sGFP的载体pCH-sGFP,并通过农杆菌介导的遗传转化法导入引起棉花黄萎病的高致病力大丽轮枝菌Vd991,获得了sGFP整合到大丽轮枝菌基因组的转化株。通过转化子荧光信号、生长表型和致病力筛选鉴定,获得了1株与Vd991生长和致病力无显著差异且荧光信号强烈的转化株Vd gfp77。侵染棉花根部试验表明,Vd-gfp77侵染棉花后快速扩展繁殖,子代仍然能发出强烈的荧光信号。本试验绿色荧光蛋白标记大丽轮枝菌的成功构建,为后续大丽轮枝菌侵染棉花过程的组织学和致病机理研究提供了良好的研究材料。     

增强型绿色荧光蛋白基因转化大豆疫霉菌及其表达

为了探讨大豆疫霉菌Phytophthora sojae的侵染过程及其在土壤中的生态学,采用CaCl2-PEG介导的原生质体转化方法,将外源增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因转化到大豆疫霉菌中,观察EGFP基因在大豆疫霉菌不同生育时期的表达,对转化子中的EG-FP基因进行PCR检测,并对转化子的生长、发育及致病性进行测定。结果显示,EGFP基因能够在大豆疫霉菌菌丝、游动孢子囊和卵孢子中稳定表达,并在475 nm蓝光激发下发出绿色荧光;转化子的菌丝生长速率与野生型无显著差异,个别转化子在无性繁殖和有性生殖方面与野生型有显著或极显著差异,其中一个转化子的致病型发生了改变。研究表明获得的EGFP基因转化子可以作为研究大豆疫霉菌侵染及定殖的材料。     

Role of secondary metabolites in the biocontrol activity of <Emphasis Type="Italic">Pseudomonas corrugata</Emphasis> and <Emphasis Type="Italic">Pseudomonas mediterranea</Emphasis>

Colletotrichum gloeosporioides is the causal agent of Camellia oleifera anthracnose, mainly infecting fruits and leaves. The fungus secretes degrading enzymes to destroy the cuticle of aerial plant parts and help infect the host successfully. To validate whether a cutinase gene (CglCUT1) was required for cutinase activity and pathogenicity of C. gloeosporioides, the CglCUT1 gene was cloned and analyzed. The characterization of CglCUT1 predicted protein suggests that the cloned DNA encoded a cutinase in C. gloeosporioides affecting C. oleifera. The CglCUT1 showed a high homology to those from C. gloeosporioides causing papaya anthracnose and C. capsici causing pepper anthracnose, as well as those of other ascomycetes. The whole CglCUT1 gene was knocked-out and the knockout mutant (?CglCUT39) was subsequently complemented using Agrobacterium tumefaciens mediated transformation. The knockout transformants exhibited significant decreases in cutinase activity and virulence compared with the wild-type strain. The complemented transformants of the disrupted transformant ?CglCUT39 showed a significant increase in cutinase activity and virulence compared with the disrupted transformant ?CglCUT39. This study suggests that the CglCUT1 gene has a positive effect on fungal virulence of the hemibiotrophic C. gloeosporioides on C. oleifera.     

农杆菌介导的哈茨木霉T-DNA转化系统优化及拮抗能力突变子的性质分析

采用单因子试验方法研究了农杆菌EHA105介导的哈茨木霉Th-33转化过程中,各主要因素对转化效率的影响,建立了高效的转化系统,使农杆菌转化哈茨木霉的效率达到60～150个转化子/10^6个木霉孢子,利用该转化系统构建了含有8000多个转化子的T-DNA插入突变库。通过转化子与立枯丝核菌的对峙试验,从1260株转化子中筛选到23株拮抗能力发生变化的突变子。随机挑选5株突变子对其遗传稳定性进行分析,表明5株突变子都具有稳定性,聚合酶链式反应（PCR）表明上述突变子均有T-DNA片段的插入。     

Colonization of Vitis spp. wood by sGFP-transformed Phaeomoniella chlamydospora, a tracheomycotic fungus involved in Esca disease

To evaluate wood colonization and interactions with Vitis spp. of Phaeomoniella chlamydospora, a fungal agent involved in Esca disease, isolate CBS 229.95 was transformed using a pCT74 construct which contained the genetic markers for synthetic green fluorescent protein (sGFP) and hygromycin B phosphotransferase. Nine stable P. chlamydospora fungal transformants (Pch-sGFP lines) were obtained using polyethylene-glycol-mediated transformation of protoplasts. These were characterized for sgfp and hygromycin B phosphotransferase (hph) genome insertions and for sGFP fluorescence emission, using quantitative polymerase chain reaction and fluorimetric systems, respectively. No correlation was observed between sgfp copy number genome insertion and sGFP fluorescence expression. Cuttings of Vitis vinifera 'Montepulciano', 'Verdicchio', 'Sangiovese', 'Biancame', and 'Cabernet Sauvignon'; and the grapevine rootstocks 'Kober 5BB', 'SO4', '420A', '1103P', and V. rupestris were inoculated by immersion in a conidial suspension of the selected fungal Pch-sGFP71 line and incubated at 4 ± 1 and 25 ± 1°C. Wood colonization was estimated through epifluorescence microscopy and was affected by incubation temperature. After 6 months at 4 ± 1°C, the fungal growth was completely inhibited. At 25 ± 1°C, the highest extent of wood colonization was recorded in Montepulciano and Verdicchio, with the lowest in the rootstocks SO4 and V. rupestris. The expression of the Pch-sGFP71 transformed line was localized in the xylem area, primarily around the vessels. The use of sGFP-transformed P. chlamydospora helped to clarify different aspects associated with the location of this pathogen in grapevine tissue, before disease symptom expression.