根癌农杆菌介导的赭曲霉遗传转化体系的建立

赭曲霉在工业上用于甾体C-11α羟基化。为了对现有赭曲霉生产菌株进行遗传改造,以农杆菌LBA4404作为侵染菌株,以赭曲霉(TCCC41060)作为受体菌株,以潮霉素B基因作为筛选标记,成功建立了根癌农杆菌介导的赭曲霉遗传转化体系并对其转化效率的主要影响因素,如农杆菌浓度、孢子浓度、共培养时间和温度等进行了分析。在最佳条件下,转化效率可以达到57个转化子/108个分生孢子。随机挑选的8个阳性转化子10代连续培养结果表明所获得的转化子能稳定遗传。该遗传转化体系的建立为赭曲霉菌株的定向遗传改造提供了重要的操作平台。     

不同诱导方法对农杆菌介导的木霉菌遗传转化效率的影响

[目的]比较不同诱导方法对根癌农杆菌EHA105介导的哈茨木霉T88遗传转化体系转化效率的影响,以确定最佳的诱导方法.[方法]通过对复铺培养基法、转膜培养法和液体共培养法转化效率的比较来确定3种方法的优劣.[结果]复铺培养基法转化效率约为10个转化子/107个木霉孢子,转膜培养法转化效率约为20个转化子/107个木霉孢子,液体共培养法转化效率达到100个转化子/107个木霉孢子.[结论]在农杆菌介导的木霉菌遗传转化体系中,液体共培养法的转化效率最高,是常用的复铺培养基法及转膜培养法转化效率的5-10倍.     

根癌农杆菌介导的日本曲霉转化体系的建立

【目的】通过根癌农杆菌介导的方法构建日本曲霉转化子库,从而筛选出高产甘没氧化酶的日本曲霉突变菌株。【方法】本文通过三亲杂交的方法将双元载体pBI-hphII转移至根癌农杆菌EHA105中并作为侵染菌株,以日本曲霉As5999为受体菌株,建立了农杆菌介导的日本曲霉转化体系,构建了突变体库,并对影响转化效率的根癌农杆菌浓度,乙酰丁香酮(As)加入与否,共培养时间,共培养温度等因素进行了分析。【结果】对转化子的PCR检测和Southern杂交分析表明,T-DNA已整合进日本曲霉基因组中,随机挑选的9个转化子连续转接10代后均能稳定遗传。【结论】该转化体系的建立为筛选出高产甘油氧化酶的日本曲霉突变菌株奠定了基础。     

根癌农杆菌介导里氏木霉转化体系的建立和条件优化

目的:采用根癌农杆菌介导的转化方法实现丝状真菌里氏木霉的遗传转化,并优化转化条件.方法:构建含潮霉素抗性基因(hph)的双元载体pCAM-hph后,转化根癌农杆菌LBA4404获得转化菌株.将根癌农杆菌的转化菌株和里氏木霉的分生孢子共培养后在含100μg/mL潮霉素的抗性平板上筛选里氏木霉转化子,并采用PCR扩增和序列测定对转化子中的插入片段进行了分析.结果:使用根癌农杆菌介导的转化方法转化里氏木霉,每106个分生孢子可获得25.8个转化子.最佳的转化条件为:农杆菌初始浓度为OD660约为0.8,孢子数为106个,共培养时间为48h,pH为5.0～5.5,培养温度为28℃.结论:建立了根癌农杆菌介导的里氏木霉转化方法,并获得了最佳的转化条件.     

根癌农杆菌介导的金边狗牙根遗传转化条件的优化

为建立根癌农杆菌(Agrobacterium tumefaciens,菌(L.)Pers.]最佳遗传转化体系,以葡萄糖醛酸糖苷酶(GUS)基因的瞬间表达率为指标,从愈伤组织继代时间、根癌农杆菌侵染时间、负压条件及光照时间等方面进行了筛选.结果表明,根癌农杆菌介导的金边狗牙根最佳的转化体系是:以继代培养2周的愈伤组织为起始材料,根癌农杆菌介导感染10 min,经负压处理(抽拉20次)并在全光条件下共培养转化.     

根癌农杆菌介导转化红曲菌及转化子发酵性能的研究

利用根癌农杆菌介导转化技术成功将潮霉素抗性基因转入发白红曲菌中,优化了抗生素浓度,发白红曲菌孢子浓度,根癌农杆菌浓度,共培养温度及时间,以及乙酰丁香酮浓度等转化条件,最终转化效率可达52个转化子/105个红曲孢子.将转化子在含有潮霉素B的培养基继代培养5代,得到了多株稳定的转化子,对部分转化子进行PCR鉴定,结果进一步...     

链格孢ATMT转化体系的构建及弱毒突变株验证

本文利用来自质粒pUCATPH的构巢曲霉色氨酸合成基因trpC的启动子(PtrpC)、终止子(Ttrpc)和潮霉素磷酸转移酶抗性基因(hph)以及植物表达载体pROK Ⅱ成功构建了适用于丝状真菌的根癌农杆菌介导转化的双元载体pROKIIHPH,并转化根癌农杆菌Agrobacterium tumefaciens LBA4404,建立了根癌农杆菌LBA4404介导的链格孢Alternaria alternata分生孢子转化体系;再从乙酰丁香酮(AS)浓度、不同的共培养时间、受体菌分生孢子浓度和农杆菌菌液浓度对A.alternata转化效率的影响对体系进行优化.结果确定了根癌农杆菌菌液体积为200 mL(OD_(600)=0.15),A.alternata分生孢子浓度为10~6个/mL,在A.tumefaciens的预培养时期以及与A.alternata共培养时期分别加入200 μmol/LAS,共培养时间48 h,转化率达120～200个/10~5分生孢子.在所筛选的约800个转化子中获得了1株毒性明显低于野生菌sd1的弱毒突变株t108,通过PCR验证推测其毒力降低可能由于T-DNA插入阻断了基因的表达.该实验结果为与突变相关基因的研究以及弱毒株t108的进一步研究和利用提供了实验材料,也为深入研究链格孢sd1菌株的基因功能奠定了基础.     

农杆菌介导的马尔尼菲青霉基因转化技术的建立及优化

目的建立农杆菌介导的马尔尼菲青霉(PM)基因转化技术,并对该技术条件进行优化。方法以二元质粒p DHt/SK为载体,通过农杆菌介导将pyr G基因插入马尔尼菲青霉尿嘧啶缺陷株SPM4(pyr G-,nia D-)中,在不含尿嘧啶的培养基中筛选阳性转化子。运用PCR验证重组子。进一步对影响转化效率的农杆菌类型、共培养浓度、转化媒介、共培养温度、共培养时间、乙酰丁香酮(AS)等六个条件进行优化。结果 PCR验证pyr G基因成功的插入SPM4中,所得到转化子可稳定传代,通过条件优化,得到转化子约300个/106个细胞。选用AGL-1,以农杆菌共培养浓度为OD600=0.8,AS浓度为200μmol/L,无膜IM固体共培养基为介质,25℃共培养48 h为最适转化条件。结论成功建立了农杆菌介导PM基因转化技术,简化并优化了转化条件,该方法可用于PM基因功能研究。     

农杆菌介导转化莱茵衣藻体系的优化

为建立根癌农杆菌介导的莱茵衣藻快速简便高效的遗传转化体系,本研究以模式生物莱茵衣藻为受体材料,从转化方法和转化子快速鉴定两个方面进行了优化。[方法]比较了固体培养基共培养转化方法和液体培养基共培养转化方法对根癌农杆菌LBA 4404介导的莱茵衣藻CC425转化效率的影响;研究并比较了(1)首先经过TE裂解再进行PCR(两步法)和(2)不经TE裂解直接进行PCR(一步法)的两种转化子鉴定方法的最佳反应条件和扩增效率。[结果]农杆菌LBA 4404和莱茵衣藻CC425液体培养基共培养5 d后的转化效果最好,转化率达43.33±1.67个转化子/10⁶个藻细胞。PCR最佳反应条件为:使用高保真DNA聚合酶Taq 1进行扩增;参加PCR反应的细胞密度为5×10³-5×10⁶个/mL;TE裂解缓冲液沸水浴20 min(两步直接PCR方法),或者预变性15 min(一步直接PCR方法)。两步法直接PCR的扩增效率优于一步法,但后者反应步骤更简洁。[结论]本研究建立并优化了农杆菌液体介导莱茵衣藻遗传转化体系,该体系可快速获得遗传转化子,减少转化工作量。     

农杆菌介导的粘帚菌遗传转化北大核心CSCD

【目的】建立分离自西藏林芝的能够产生一系列具有抗肿瘤活性的二酮哌嗪类化合物(epipolythiodioxopiperazine,ETP)的一株冬虫夏草定殖真菌——粘帚菌(Gliocladium sp.)6.102的遗传转化体系。【方法】利用农杆菌介导的方式建立了粘帚菌的遗传转化体系;用正交试验研究了影响转化效率的因素,包括共培养所用农杆菌与真菌孢子的比例、共培养的时间、pH值和乙酰丁香酮的浓度。【结果】成功建立了农杆菌介导的粘帚菌的遗传转化体系,得到了转化的最佳条件,其转化效率为50-100个转化子/106真菌孢子。通过农杆菌介导的方式分别将潮霉素B磷酸转移酶基因(hph)和绿色荧光蛋白基因(egfp)转入粘帚菌中,实现了表达并且可以稳定存在。【结论】首次建立了农杆菌介导的粘帚菌遗传转化体系,为研究ETP化合物的生物合成及其调控机制奠定了基础。     

农杆菌介导生防棘孢木霉菌转化体系的优化

目的:实现棘孢木霉菌T4的遗传转化并优化其转化体系.方法:以潮霉素抗性为选择标记,利用农杆菌转化法介导转化棘孢木霉菌.结果:潮霉素基因成功整合到受体菌基因组中,转化子抗性基因可稳定遗传.结论:最优的转化体系和条件为:IM和CM培养基中AS浓度为200 μg/mL,棘孢木霉T4孢子浓度为106/mL,农杆菌浓度为200 μL( OD600约0.8),共培养时间为48 h,转化效率约为50个转化子/106个孢子.     

Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus, Magnaporthe grisea.

An effective way to study the infection mechanisms of fungal pathogens is to disrupt their genes via transformation in both targeted and random manners. This isolates the mutants that exhibit altered virulence. In this paper, we report the successful transformation of Magnaporthe grisea, the causal agent for rice blast, that is mediated by Agrobacterium tumefaciens. Employing the binary vector pBHt2, which carries the bacterial hygromycin B phosphotransferase gene under the control of the Aspergillus nidulans trpC promoter as a selectable marker, led to the production of 500 to > 1,000 hygromycin B-resistant transformants per 1 x 10(6) conidia of M. grisea. The transformation efficiency is correlated with the number of A. tumefaciens cells used, pre-treating bacterial cells with acetosyringone prior to co-cultivation with fungal spores, and the duration of co-cultivation. All of the transformants tested remained mitotically stable, maintaining their hygromycin B resistance after several generations of growth in the absence of hygromycin B. A genomic Southern blot analysis showed that over 60% of the transformants contained a single T-DNA insert on their genome. Considering the efficiency and flexibility of A. tumefaciens-mediated transformation (ATMT), this technique offers highly efficient means for characterizing the genes that are important for the pathogenicity of M. grisea.     

Agrobacterium tumefaciens-mediated transformation in Colletotrichum falcatum and C. acutatum

Agrobacterium tumefaciens-mediated transformation (ATMT) is becoming an effective system as an insertional mutagenesis tool in filamentous fungi. We developed and optimized ATMT for two Colletotrichum species, C. falcatum and C. acutatum, which are the causal agents of sugarcane red rot and pepper anthracnose, respectively. A. tumefaciens strain SK1044, carrying a hygromycin phosphotransferase gene (hph) and a green fluorescent protein (GFP) gene, was used to transform the conidia of these two Colletotrichum species. Transformation efficiency was correlated with cocultivation time and bacterial cell concentration and was higher in C. falcatum than in C. acutatum. Southern blot analysis indicated that about 65% of the transformants had a single copy of the T-DNA in both C. falcatum and C. acutatum and that T-DNA integrated randomly in both fungal genomes. T-DNA insertions were identified in transformants through thermal asymmetrical interlaced PCR (TAIL-PCR) followed by sequencing. Our results suggested that ATMT can be used as a molecular tool to identify and characterize pathogenicity-related genes in these two economically important Colletotrichum species.     

Agrobactrium tumefaciens-mediated transformation of Monascus ruber

Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied to Monascus ruber. The optimum cocultivation time was 84 h with an efficiency of 900 to 1,000 transformants when 1x106 spores were used with the same volume of bacteria. The stability of transformants was over 98% after five generations. When M. ruber was transformed with A. tumefaciens YL-63 containing the green fluorescent protein gene (egfp), the green fluorescent signal was observed throughout hyphae, confirming expression of the gene. This efficient transformation and expression system of M. ruber by ATMT will facilitate the study of this fungus at a molecular genetic level.     

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。     

根癌农杆菌介导丝状真菌遗传转化的研究进展

根癌农杆菌介导的丝状真菌遗传转化是近年建立起来的一种新方法,该方法和以往的真菌转化体系相比具有转化方法简单、材料易得、效率高以及转化子中T-DNA单拷贝插入比例高等特点。就根癌农杆菌转化的丝状真菌种类、转化的具体过程以及影响转化效率的因素等方面进行了综述,并展望了该方法的应用前景。     

黑曲霉电转化条件的研究

研究避免了繁琐的原生体制备过程,直接使用萌发的黑曲霉孢子进行电转化,以潮霉素B作为筛选标记,从孢子萌发时间、电场强度及质粒浓度等方面考察了电转化效率的影响因素。研究表明,针对A.nigerMGG029-ΔaamA,其理想的电转化条件:孢子龄为4d,孢子萌发时间为2h,电场强度为5kV/cm。在上述条件下分别使用1μg环状或线状pBC-Hygro质粒DNA进行转化,平均可以得到34个和51个转化子,而在同样条件下使用质粒pRS303H平均可以获得163个和258个转化子。     

A highly efficient Agrobacterium mediated transformation system for chickpea wilt pathogen Fusarium oxysporum f. sp. ciceri using DsRed-Express to follow root colonisation

The soil-borne fungus Fusarium oxysporum f. sp. ciceri (Foc) causes vascular wilt of chickpea (Cicer arietinum L.), resulting in substantial yield losses worldwide. Agrobacterium tumefaciens mediated transformation (ATMT) has served as a resourceful tool for plant-pathogen interaction studies and offers a number of advantages over conventional transformation systems. Here, we developed a highly efficient A. tumefaciens mediated transformation system for Foc. In addition, a binary vector for constitutive expression of red fluorescent protein (DsRed-Express) was used to study developmental stages and host-pathogen interactions. Southern hybridisation was performed to confirm the transformation event and the presence of T-DNA in selected hygromycin resistant transformants. Most of the transformants showed single copy integrations at random positions. Microscopic studies revealed significant levels of fluorescent protein, both in conidia and mycelia. Confocal microscopy of chickpea roots infected with the transformed Foc showed rapid colonisation. These studies will allow us to develop strategies to determine the mechanisms of Foc-chickpea interaction in greater detail and to apply functional genomics for the characterisation of involved genes at the molecular level either by insertional mutagenesis or gene knock-out.