【作者】 龙朝钦；

【导师】 郝飞；

【作者基本信息】 第三军医大学 ， 皮肤病与性病学， 2008， 博士

【摘要】 一、研究背景和目的烟曲霉是引起过敏性支气管肺曲霉病、曲霉球和侵袭性曲霉病的空气播散性的条件致病真菌。在免疫抑制患者中,导致致死性感染的重要原因是由于吸入烟曲霉分生孢子进而发展为侵袭性曲霉病。由于癌症、器官移植和其它原因造成免疫抑制患者逐渐增多,侵袭性曲霉病的发病率也逐年增加。尽管进行了积极的抗真菌治疗,侵袭性曲霉病的病死率仍维持在一个较高的水平。如何预防和治疗这一类疾病是个突出而且亟待解决的问题。很多分子和基因与烟曲霉的毒力相关。但是,对于侵袭性曲霉病具体是哪一个毒力因子起主导作用目前仍然不是很清楚。几丁质是烟曲霉细胞壁上第三大类多糖化合物并且有多种几丁质合成酶被检测到。烟曲霉chs基因家族至少包括7种不同的基因,分别为chsA(组Ⅰ)、chsB(组Ⅱ)、chsC(组Ⅲ)、chsD(组Ⅵ)、chsE(组Ⅴ)、chsF(组Ⅳ)和chsG(组Ⅲ)。这些基因对烟曲霉的表型和毒力的改变起着重要作用。chsE-突变株能够减少菌丝体和所有菌丝上几丁质的水平,也能减少分生孢子的受精作用;chsG-突变株显示几丁质合成酶活性的降低,减少菌群的生长半径和高端菌丝生长,揭示这种酶在菌丝顶端发挥作用;chsE-/chsG-双重突变对烟曲霉并不是致命的,它降低了几丁质合成酶的活性,其克隆的生长速度也明显减慢,此双重突变体能够产生更多的分枝菌丝。但是烟曲霉几丁质合成酶chsC在其生长方式、形态及对抗真菌药物的敏感性方面的作用仍然不是十分清楚。目前,烟曲霉全基因组测序已经完成,通过反向或正向遗传转化构建突变体是一个有效的研究烟曲霉未知基因功能的方法。用原生质体通过同源重组来打断基因的方法曾经被广泛运用,但此方法费时、费力,而且其同源重组的频率也比较低。电穿孔和其它一些生物学的方法也经常被应用到烟曲霉的转化中,但是这些方法都存在多拷贝插入和低同源重组等缺点。作为一种替代方法,根癌农杆菌介导转化(ATMT)已经在多种曲霉的转化中得到应用。本研究将把根癌农杆菌作为工程菌应用于插入突变烟曲霉的转化中,构建出烟曲霉几丁质合成酶基因缺失突变体,从而对烟曲霉几丁质合成酶基因在烟曲霉的生活方式、表型以及抗真菌药物敏感性方面的作用作一探讨。同时作为对ATMT这一新方法的探讨,我们观察了ATMT转化的优化条件。二、方法与结果1.根癌农杆菌介导转化的二元载体的构建及鉴定高保真扩增几丁质合成酶chsC基因两端约500bp的片段,分别插入质粒pPK2中潮霉素抗性序列的两侧,将构建好的的质粒命名为pPK2/ chsC-::hph。用限制性内切酶HindIII/SacI进行双酶切,把切下来约5.5kb的片段与用限制性内切酶HindIII/SacI进行双酶切的pDHt/SK大片段相连接,构建出的二元载体pDHt/chsC-::hph用于ATMT。二元载体用HindIII/SacI进行双酶切鉴定,载体切出两条条带,一条为5.5kb包含潮霉素抗性基因、构巢曲霉启动子以及两侧的约500bp的chsC基因片段,另一条为约7.45kb的pDHt/SK载体片段,通过测序比对进一步验证片段插入的正确性。2.根癌农杆菌介导转化对烟曲霉chsC基因的定点突变二元载体pDHt/chsC-::hph用氯化钙法转化至根癌农杆菌EHA105中,转化后的根癌农杆菌命名为EHA105/chsC-。ATMT中我们用不同的共培养温度(23、24、25、26、27、28℃),不同的转化媒介(尼龙膜、硝化纤维素膜、液态静置培养),不同的共培养时间(12、24、36、48h),预培养加或不加AS,共培养时培养基中加入不同浓度的AS(100、200、300、400μg/ml),共培养时用不同的分生孢子与根癌农杆菌的比例(1:1、1:10、1:100、1:1000),以此来探讨影响ATMT转化效率的因素。用潮霉素筛选阳性转化子,转化子接种到含高浓度潮霉素( 200、400μg/ml)的LB培养基中初步鉴定转化子,进一步鉴定用RT-PCR的方法。二元载体pDHt/chsC-::hph可用氯化钙法转化进入根癌农杆菌EHA105中。用尼龙膜和液态静置培养法在ATMT中具有相似的转化效率,但硝化纤维素膜的转化效率明显低于尼龙膜和液态静置培养;共培养温度为25℃时ATMT转化效率最高,高于或低于该温度转化效率均有下降;随着共培养时间的延长,转化效率也随之增加,但共培养时间过长如72h,由于转化子太多而不容易挑取单克隆;当分生孢子与农杆菌的比例在1:1到1:100时,随着比例的增加ATMT转化效率相应提高,但当其比例达到1:1000时,转化效率不再增加;预培养加入AS与否对转化效率无明显影响,共培养时随着加入AS浓度的增加其转化效率也相应提高。阳性转化子在含有高浓度的潮霉素环境中能正常生长。RT-PCR法检测转化子发现ATMT能成功转化T-DNA到烟曲霉染色体基因组中,定点插入并打断失活chsC基因。3.烟曲霉chsC基因缺失突变体基本特征的研究等量突变体分生孢子和野生型烟曲霉分生孢子接种到察氏培养基中,37℃孵育3d观察二者的生长速度。钢圈法进行小培养以观察烟曲霉chsC-突变体和野生型烟曲霉MIDA1在形态学上的差异,用微量培养法观察二者对抗真菌药物敏感性的差异。应用美国临床实验室标准化委员会制定的M38-A方案探讨了烟曲霉chsC-突变体和野生型烟曲霉MIDA1对两性霉素B(AmB)、特比奈芬(TBF)、伊曲康唑(ICZ)、氟胞嘧啶(5-Fc)和氟康唑(FCZ)的敏感性。在生长速度方面,烟曲霉chsC-突变体生长速度明显比野生型烟曲霉MIDA1慢,但大体菌落形态和颜色并无显著变化。光镜下野生型烟曲霉MIDA1,分生孢子链较短,分生孢子头圆而饱满,菌丝均匀饱满。烟曲霉chsC-分生孢子链长,菌丝不均匀,部分可见膨大。电镜高真空下野生型烟曲霉MIDA1,菌丝脱水塌陷,分生孢子较圆而饱满;烟曲霉chsC-,菌丝脱水塌陷,分生孢子也脱水塌陷如瘪乒乓球样。电镜低真空下野生型烟曲霉MIDA1,分生孢子及菌丝都较饱满,分生孢子链较短;烟曲霉chsC-,菌丝易脱水塌陷,分生孢子链较长。野生型烟曲霉MIDA1与烟曲霉MIDA1(chsC-)对5-Fc、AmB、FCZ、ICZ的MIC值上无明显差异,但相对于TBF烟曲霉MIDA1(chsC-)的MIC值明显低于野生型烟曲霉MIDA1。三、结论1.用烟曲霉EHA105和质粒pDHt/SK组成的转化系统能够成功转化T-DNA到烟曲霉染色体基因组中,当T-DNA两侧含有与烟曲霉基因组同源序列时,T-DNA可以定点插入从而打断和失活目标基因;2.转化媒介、共培养温度、共培养时间、分生孢子与根癌农杆菌的比例、共培养时加入AS的浓度均对ATMT的转化效率产生较大的影响,而预培养时加入AS与否对其转化效率影响不显著。3.烟曲霉几丁质合成酶基因chsC在烟曲霉的生长、形态和抗真菌药物敏感性方面起着重要的作用。更多还原

【Abstract】 Backgrounds and ObjectivesAspergillus fumigatus, an airborne fungal pathogen, is the major cause of allergic bronchopulmonary aspergillosis, aspergilloma and invasive aspergillosis. Of particular importance is the invasive aspergillosis that starts with inhalation of conidia and progresses to life-threatening infection in immunocompromised patients. Due to the rising prevalence of cancer, organ transplantation, and other causes of immunosuppression, the number of patients at risk of invasive aspergillosis is on the rise. Despite aggressive antifungal therapy, the overall mortality of invasive aspergillosis remains high, and new strategies to prevent and treat this disease are urgently needed. Many molecular and genes involved the virulence of Aspergillus fumigatus. But, at present, it is not known what molecular or gene play a predominant role in invasive aspergillosis (IA). Chitin is the third polysaccharide component of the wall,several chitin synthases (chs) have been detected in Aspergillus fumigatus. The family of chs genes in Aspergillus fumigatus include at least seven different genes, called chsA (class I), chsB (class II), chsC (class III), chsD (class VI), chsE (class V), chsF (class IV) and chsG (clase III). Disruption of these genes may leads to an altered phenotype in Aspergillus fumigatus. The chsE- mutant has reduced levels of chitin in the mycelium, swellings throughout the length of the hypha and conidiation. The strains with chsG- mutations showed a reduced activity of the chitin synthetase enzyme, reduced radial growth and highly branched hyphae, suggesting that the function of the enzyme is in the apex of the hypha. The chsE-/chsG- double mutantion is not lethal for this fungus, it has reduced chitin synthase activity and colony radial growth rate, and produced highly branched hyphae. But whether or not chitin synthase gene chsC plays a role in lifestyle, appearance and sensitivity for antifungus drugs in Aspergillus fumigatus is unknown.As genomic sequencing of Aspergillus fumigatus has been succeeded, investigation of the functions of such genes can be accomplished via mutational analysis either by reverse or forward genetic approaches. To disrupt a gene by homologous integration in Aspergillus fumigatus, transformation via the spheroplast method has been most widely used; this is a laborious and time-consuming method, and the homologous recombination frequency can be relatively low. Electroporation and biolistic methods were also used to transform Aspergillus fumigatus. these methods, however, resulted in a high frequency of multiple integrations and a low frequency of homologous recombination. As an alternative method, Agrobacterium tumefaciens-mediated transformation (ATMT) has been used to transform several Aspergillus species. Here we report transformation of Aspergillus fumigatus mediated by Agrobacterium tumefaciens as a tool for insertional mutagenesis to construct chsC- mutant of Aspergillus fumigatus, and investgate the function of chsC gene in lifestyle, appearance and sensitivity for antifungus drugs in Aspergillus fumigatus. For ATMT is a new method of transformation of Aspergillus fumigatus, we investgate the optimize condition of ATMT as well.Methods and Results1. The construction and identification of pDHt/chsC-::hph in ATMT. To construct the plasmid used for ectopic mutagenesis, , we amplificate two approximately 500 bp fragments templeted two side of chsC gene. Following, insert the two fragments into pPK2, and made the hygromycin resistance gene (hph) in pPK2 flanked by two chsC gene fragment sequence, this plasmid named as pPK2/ chsC-::hph. The pDHt/chsC-::hph was constructed by insertion of a 5.5-kb HindIII/SacI fragment from pPK2/ chsC-::hph containing the hygromycin resistance gene flanked by two chsC gene fragment sequence into the HindIII/SacI restricted pDHt/SK plasmid. The plasmid used for ectopic mutagenesis named as pDHt/chsC-::hph. To confirm the pDHt/chsC-::hph been constructed successfully, we digested the plasmid by HindIII/SacI restriction enzyme and found the plasmid be digested into two fragment, 5.5-kb fragment and 7.45-kb fragment. All constructs were sequenced and blasted for the further confirmation.2. Tageted disruption of chsC of Aspergillus fumigatus by ATMT. Agrobacterium tumefaciens strain EHA105 was transformed by Calcium chloride with pDHt/chsC-::hph plasmids. Strain of Agrobacterium tumefaciens harboring pDHt/chsC-::hph was named EHA105/chsC-. To investget the tansformation efficience, the Agrobacterium tumefaciens-mediated transformation was conducted at different co-cultivation temperature (23、24、25、26、27、28℃),different transformation media(nylon、nitrocellulose filters、liquid phase cultivation),different co-cultivation period(12、24、36、48h),AS in preincubation or not,different concentration of AS in co-cultivation(100、200、300、400μg/ml),co-cultivated at different ratios of conidia to bacteria(1:1、1:10、1:100、1:1000),hygromycin resistance cassette was used to select the positive transformants. Tansformants were inoculated to LB broth with high concentration hygromycin( 200、400μg/ml) to identify the positive transformant, reverse transcription PCR (RT-PCR) was carried out for further identification.Agrobacterium tumefaciens strain EHA105 could be transformed by Calcium chloride with pDHt/chsC-::hph plasmids. Nylon and liquid phase cultivation have the same tansformation efficience. Compared with the two method, nitrocellulose filters had lower tansformation efficience. Co-cultivation at 25℃during ATMT could gain the most transformants, Co-cultivation at lower or higher than this temperature during ATMT usually got lower tansformation efficience. With the elongation of co-cultivationt period, we ould gain more transformants. Co-cultivated for 72 hours, it is difficult to pick the monoclone for too many transformants. From 1:1 to 1:100, with the increasing of co-cultivationt ratios, the transformants were increasing as well. But if the ratio of conidia to bacteria reached to 1:1000, the transformants do not increase any more. The tansformation efficience of ATMT is not changed significantly while the media contain AS or not. Higher concentration of AS in inducing medium usually got higher tansformation efficience. The positive transformant growed well in the media with high concentration hygromycin. RT-PCR showed that ATMT could transfor T-DNA to Aspergillus fumigatus and made the chsC gene tageted disruption.3. Investegation of basical character of chsC- mutant. Conidia were inoculated into Czapek medium and grown at 37℃for 3 days to observe the growth velocity. Aspergillus fumigatus chsC- mutant and wild type Aspergillus fumigatus were microcultured to observe the difference of morphous and the sensitivity to antifungus drugs of the chsC- mutant. NCCLS M38-A protocol was employed to investigate the activities of amphotericin B (AmB), itraconazole (ICZ), terbinafine (TBF), fluconazole (FCZ) and 5-flucytosine (5-FC) against Aspergillus fumigatus.On growth velocity chsC- mutant is obviously slower than wild type Aspergillus fumigatus, but there have no difference on figure and color in following observation. Under light microscope , the conidium chains of wild type Aspergillus fumigatus MIDA1 are short, and the hypha are uniformity, conidial head are round and full. The conidium chains of chsC- mutant are long, and the hypha are not uniformity, intumescence can be seen in some section. Under high vacuum of electron microscope,the hypha of wild type Aspergillus fumigatus MIDA1 are collapse because of anhydration, but the conidium are round and full. Under high vacuum of electron microscope,The hypha of chsC- mutant are collapse because of anhydration, and the conidium are collapse as well. Under black vacuum of electron microscope,the hypha and conidium of wild type Aspergillus fumigatus MIDA1 are full, the conidium chains are short. The hypha of chsC- mutant are collapse because of anhydration, the conidium chains are long. ChsC- mutant and wild type Aspergillus fumigatus have the same sensitivity to 5-Fc、AmB、FCZ、ICZ,but to TBF the value of MIC chsC- mutant obviously lower than wild type Aspergillus fumigatusConclusions1. The transformat system composed of Agrobacterium tumefaciens species EHA105 and pDHt/SK plasmid Agrobacterium tumefaciens-mediated transformation can transformat T-DNA to Aspergillus fumigatus and targeted disrupt the destinating gene.2. During ATMT,co-cultivation temperature, transformat media, co-cultivation period, concentration of AS in co-cultivation,co-cultivated ratios of conidia to bacteria play important roles in the transformation efficience,but AS in preincubation or not does not affect the tansformation efficience seriously.3. ChsC gene of Aspergillus fumigatus play an important role in its lifestyle, appearance and sensitivity for antifungus drugs.更多还原