新生隐球菌转录共激活因子MBF1基因的鉴定与敲除

目的构建新生隐球菌转录共激活因子MBF1基因缺陷菌株。方法采用套叠PCR方法 ,构建含有抗性基因NEO以及靶基因上下游同源DNA片段的基因敲除框,通过基因枪将重组片段转化入新生隐球菌,应用PCR筛选和DNA序列测序方法对基因突变株进行鉴定与验证。结果成功构建了新生隐球菌基因突变株mbf1裣。结论通过基因突变株mbf1裣的构建,为深入研究新生隐球菌转录辅助因子Mbf1的功能机制奠定基础。     

STE12α基因对新生隐球菌形态学影响的初步研究

目的研究STE12α基因对新生隐球菌形态学的影响。方法分别敲除血清A型和血清B型新生隐球菌菌株的STE12α基因,建立缺陷株,再将STE12α基因重新导入建立重建株。观察并比较野生株、STE12α基因缺陷株及重建株在体内、外孵育后菌落和菌落的形态学差异。结果 STE12α基因缺陷株组形成的菌落明显偏少,菌株直径偏小,荚膜发育不良,而重构株组这些方面的改变都得到了恢复。结论 STE12α基因对新生隐球菌的形态学改变有着重要的影响,可能直接影响其毒力。     

条件启动子pCTR4在隐球菌基因表达调控中的应用

目的通过条件启动子pCTR4的质粒构建以及其在新生隐球菌中的同源置换,研究其在隐球菌基因表达调控中的应用。方法应用套叠PCR,构建含报告基因NEO的铜离子抑制性启动子质粒pNEO/CTR4和启动子同源重组框,并利用基因枪将其转化入新生隐球菌感受态细胞,常规及实时定量PCR检测条件启动子对目的基因的转录调控效应。结果成功构建了质粒pNEO/CTR和隐球菌条件启动子重建菌株,条件启动子pCTR4对目的基因具有预期的转录诱导和抑制效果。结论新建铜离子抑制性启动子质粒pNEO/CTR4可以应用于对隐球菌目的基因表达水平的调控;隐球菌泛素编码基因UBI 1并非致死性关键基因。我们的研究为今后新生隐球菌泛素系统的分子致病机制研究奠定了基础。     

新生隐球菌MIS1基因的siRNA表达载体的构建及鉴定

目的 构建靶向新生隐球菌MIS1基因的siRNA重组表达载体质粒,并进行鉴定.方法 根据GenBank的MIS1基因序列,按照载体要求设计单链引物,克隆到空载体psilencer4.I-CMV neo中,经过LiAc化学法将重组质粒转染到新生隐球菌细胞中并用G418筛选,利用real time PCR鉴定阳性细胞的MIS1基因水平.结果 重组表达质粒Psilencer4,1-CMV-si-MIS1经PCR、双酶切及测序鉴定,结果证明重组表达载体构建成功,其能在mRNA水平显著抑制MIS1的表达.结论 已成功构建新生隐球菌MIS1基因的siRNA表达载体,为深入研究MIsl在隐球菌相关疾病的发生及发展中的作用提供了技术手段.     

中国首株格特隐球菌VGII基因型分离株XH91的分子和表型特征鉴定

【目的】研究我国首次临床分离的一株格特隐球菌VGII基因型菌株(XH91)的分子和表型特征。【方法】对受试株XH91进行血清型的分子鉴定;选取核基因组和线粒体基因组中共16个基因片段进行多位点序列分型;对受试株进行单倍体繁育、同性交配及异性交配能力评价;观察受试株的黑色素生成、荚膜厚度及37℃生长等表型特征。【结果】我国首株格特隐球菌VGII基因型菌株XH91为血清B型;该菌株在多位点序列分型上与温哥华岛致病基因型VGIIb一致;XH91能与a交配型发生交配,产生担孢子,而不能与α交配型发生交配且不具备单倍体繁育能力;XH91的黑色素生成、荚膜厚度及37℃生长等表型特征与参考株无明显差异。【结论】我国首株格特隐球菌VGII基因型菌株XH91在基因型、表型特征上均与温哥华岛VGIIb基因亚型一致,该结果将为我国格特隐球菌VGII基因型的分子流行病学和疾病监控提供重要资料。     

新生隐球菌STE12α基因的克隆及表达载体的构建

目的从新生隐球菌的基因组中扩增出STE12α基因,并构建相应的表达载体,以进一步研究STE12α基因对隐球菌的生长特性及致病性的影响。方法采用PCR方法以及基因重组方法扩增并克隆新生隐球菌基因组中的STE12α基因,建立具有表达野生型STE12α基因的表达载体。结果从新生隐球菌的基因组获得STE12α全基因,建立重组子pUCm—STE12α／NovaBlue以及重组表达载体质粒pGAPZ—STE12α,实现了STE12α基因的转化并获得表达。结论成功地克隆了新生隐球菌STE12α基因并构建了可表达野生型STE12α基因的表达载体,为进一步研究STE12α基因功能打下了良好的基础。     

格特隐球菌交配型的多重PCR鉴定

【目的】建立一种基于格特隐球菌α交配型位点内SXI1α基因和a交配型位点内SXI2a基因的多重PCR分析,用于快速鉴定格特隐球菌的交配型。【方法】设计针对格特隐球菌α交配型位点内SXI1α基因部分片段和格特隐球菌a交配型位点内SXI2a基因部分片段的特异性引物,用于多重PCR鉴定格特隐球菌的交配型;并与交配试验以及已报道的扩增α交配型位点的引物MFα、STE12α,及扩增a交配型位点的引物STE20a、STE3a进行扩增效果的比较。【结果】基于SXI1α基因和SXI2a基因的多重PCR分析,准确鉴定所有受试格特隐球菌(包括VGI、VGII、VGIII和VGIV基因型)的交配型,引物STE12α、STE20a和STE3a在常规PCR鉴定中不能鉴定部分菌株的交配型;66.7%的受试菌株不能发生交配,交配试验无法鉴定其交配型。【结论】建立的多重PCR方法明显优于常规PCR或交配试验鉴定。     

中国广西地区格特隐球菌菌种复合体的基因型分析

目的探讨中国广西地区格特隐球菌菌种复合体的基因型特点、种群结构特征和全球菌株的进化关系。方法收集2014—2018年间分离自临床确诊为隐球菌病患者的隐球菌临床株,利用CGB培养基初步筛选格特隐球菌菌种复合体。采用多位点序列分型方法(MLST)确定基因型。通过MEGA7软件构建系统发育树,利用R语言进行主成分分析。使用微量肉汤稀释法M27-A3方案行体外抗真菌药物敏感性检验。结果120株临床隐球菌中,分离出11株格特隐球菌菌种复合体,6株属于C.deuterogattii(AFLP6/VGII),5株属于C.gattii sensus stricto(AFLP4/VGI)。分离自广西的AFLP6/VGII呈遗传多样性,主要起源进化自南美巴西格特隐球菌菌种复合体。11株分离菌株均对常用抗真菌药物敏感。结论中国广西可能出现高致病性AFLP6/VGII,对格特隐球菌菌种复合体进行有效的全国性监测是必要的。     

白念珠菌SPE1基因高表达菌株构建及鉴定

目的构建白念珠菌SPEl基因高表达菌株。方法将白念珠菌．SPEl基因的ORF置于高表达质粒载体pCaE—xP的MErF3启动子后面,构建pCaEXP—SPEJ的高表达质粒,然后采用醋酸锂转染法将高表达质粒转染白念珠菌RMl000中,在SD—ura’met—cys-选择性固体培养基上筛选阳性克隆,抽取基因组进行PCR验证,将验证为阳性转染子的菌落采用RealTimeRT．PCR方法进行SPE1基因转录水平的表达验证。结果通过酶切鉴定pCaEXP—SPEl高表达质粒构建正确;通过PCR验证表明SPEl基因整合到亲本菌中的RPl0位点;通过RealTimeRT—PCR方法筛选出sPEJ基因在转录水平高表达的菌株。结论利用高表达质粒载体pCaEXP通过基因同源重组等方法正确构建SPEl基因高表达的白念珠菌。     

绿色荧光蛋白GFP基因与新生隐球菌荚膜基因的融合表达系统

目的构建新生隐球菌荚膜基因与绿色荧光蛋白的融合表达系统。方法PCR法扩增CAP60基因片段,测序验证其准确性。将其与多个必需基因共同连人穿梭质粒。结果获得6150bps大小的质粒,该质粒含有荚膜基因启动子、终止子及荧光蛋白的基因。结论将新生隐球菌荚膜基因与荧光蛋白基因融合表达,将会有利于对荚膜的生化合成途径作进一步研究。     

Osmolarity hypersensitivity of hog1 deleted mutants is suppressed by mutation in KSS1 in budding yeast Saccharomyces cerevisiae

An osmosensing mechanism of Saccharomyces cerevisiae involves a mitogen-activated protein kinase (MAPK) cascade (HOG pathway). This study aimed to investigate the response of the yeast to osmotic stress. A mutant strain, in which the HOG1 gene was disrupted by TRP1, was constructed. A spontaneous mutant, named YJY45, which suppresses the osmosensitive growth phenotype of the hog1 deletion mutant, was selected and showed a secondary phenotype of temperature sensitivity on YPD containing 0.5 M NaCl at 37 degrees C. Our data indicate that the spontaneous mutation in YJY45 mutant was mapped in KSS1, which is one of the MAPK family. The mutation in KSS1 suppresses the osmolarity-hypersensitive phenotype of the hog1 deletion mutation and restores GPD1 induction.     

Diversity among strains of Cryptococcus neoformans var. gattii as revealed by a sequence analysis of multiple genes and a chemotype analysis of capsular polysaccharide.

We demonstrated the diversity of Cryptococcus neoformans var. gattii strains by a sequence analysis of multiple genes: (i) the intergenic spacer (IGS) 1 and 2 regions of the rRNA gene; (ii) the internal transcribed spacer (ITS) region, including 5.8S of the rRNA gene; (iii) TOP1 (topoisomerase); and (iv) CAP59. In these studies, we compared C. neoformans var. gattii with varieties grubii, and neoformans of C. neoformans. Phylogenetic analysis indicated that both C. neoformans var. grubii and C neoformans var. neoformans are monophyletic, but C. neoformans var. gattii showed polyphyletic. C. neoformans var. gattii can be divided into three phylogenetic groups, I, II, and III, with high bootstrap support. Phylogenetic group I contains serotype B and C strains, and groups II and III include serotype B strains. Because the serotype B strains of C. neoformans var. gattii exhibited more genetic divergence, the serological characteristics and chemotypes of their capsular polysaccharide were further investigated. No remarkable difference among the serotype B strains was found in the reactivities to factor serum 5, which is specific for serotype B. The NMR spectra of the capsular polysaccharide from serotype B strains could be divided into three characteristic patterns, but the chemical shifts were very similar. These results suggested that the serotype B strain of C. neoformans var. gattii has more genetic diversity than the serotype C strain of C. neoformans var. gattii or the varieties grubii and neoformans of C. neoformans, but there was no correlation between genotype and chemotype.     

Cdc37p is involved in osmoadaptation and controls high osmolarity-induced cross-talk via the MAP kinase Kss1p

Cdc37p, the p50 homolog of Saccharomyces cerevisiae, is an Hsp90 cochaperone involved in the targeting of protein kinases to Hsp90. Here we report a role for Cdc37p in osmoadaptive signalling in this yeast. The osmosensitive phenotype that is displayed by the cdc37-34 mutant strain appears not to be the consequence of deficient signalling through the high osmolarity glycerol (HOG) MAP kinase pathway. Rather, Cdc37p appears to play a role in the filamentous growth (FG) pathway, which mediates adaptation to high osmolarity parallel to the HOG pathway. The osmosensitive phenotype of the cdc37-34 mutant strain is aggravated upon the deletion of the HOG gene. We report that the hyper-osmosensitive phenotype of the cdc37-34, hog1 mutant correlates to a reduced of activity of the FG pathway. We utilized this phenotype to isolate suppressor genes such as KSS1 that encodes a MAP kinase that functions in the FG pathway. We report that Kss1p interacts physically with Cdc37p. Like Kss1p, the second suppressor that we isolated, Dse1p, is involved in cell wall biogenesis or maintenance, suggesting that Cdc37p controls osmoadapation by regulating mitogen-activated protein kinase signalling aimed at adaptive changes in cell wall organization.     

Identification of salt-tolerant gene HOG1 in Torulopsis versatilis

To understand the osmo-adaptation mechanism in Torulopsis versatilis (T), we investigated the salt-tolerant gene HOG1 from the wild-type and a salt-tolerant mutant strain (T5) constructed using genome shuffling. The HOG1 genes from T and T5 were sequenced and revealed several mutations had occurred. The expression level of T5HOG1 was stronger than that of THOG1, indicating a reason for the increase of salt-tolerance in T. versatilis. Moreover, overexpression of T5HOG1 and THOG1 improved the tolerance of salt in Saccharomyces cerevisiae. Identification and overexpression of THOG1 and T5HOG1 from the wild-type T. versatilis and the mutant T. versatilis, respectively, play an important role for the osmo-adaption mechanism of the T. versatilis used in soy-sauce fermentation.     

ASR1, a stress-induced tomato protein, protects yeast from osmotic stress

Asr1 , a tomato gene induced by abiotic stress, belongs to a family, composed by at least three members, involved in adaptation to dry climates. To understand the mechanism by which proteins of this family seem to protect cells from water loss in plants, we expressed Asr1 in the heterologous expression system Saccharomyces cerevisiae under the control of a galactose-inducible promoter. In a mutant yeast strain deficient in one component of the stress-responsive high-osmolarity glycerol (HOG) pathway, namely the MAP kinase Hog 1, the synthesis of ASR1 protein restores growth under osmotic stress conditions such as 0.5  M NaCl and 1.2  M sorbitol. In contrast, the rescuing of this phenotype was less evident using a wild-type strain or the upstream MAP kinase kinase (Pbs2)-deficient strain. In both knock-out strains impaired in glycerol synthesis because of a dysfunctional HOG pathway, but not in wild-type, ASR1 led to the accumulation of endogenous glycerol in an osmotic stress-independent and unrestrained manner. These data suggest that ASR1 complements yeast HOG-deficient phenotypes by inducing downstream components of the HOG pathway. The results are discussed in terms of the function of ASR proteins in planta at the molecular and cellular level.     

Isolation and functional analysis of a gene,tcsB, encoding a transmembrane hybrid-type histidine kinase from Aspergillus nidulans

We cloned and characterized a novel Aspergillus nidulans histidine kinase gene, tcsB, encoding a membrane-type two-component signaling protein homologous to the yeast osmosensor synthetic lethal N-end rule protein 1 (SLN1), which transmits signals through the high-osmolarity glycerol response 1 (HOG1) mitogen-activated protein kinase (MAPK) cascade in yeast cells in response to environmental osmotic stimuli. From an A. nidulans cDNA library, we isolated a positive clone containing a 3,210-bp open reading frame that encoded a putative protein consisting of 1,070 amino acids. The predicted tcsB protein (TcsB) has two probable transmembrane regions in its N-terminal half and has a high degree of structural similarity to yeast Sln1p, a transmembrane hybrid-type histidine kinase. Overexpression of the tcsB cDNA suppressed the lethality of a temperature-sensitive osmosensing-defective sln1-ts yeast mutant. However, tcsB cDNAs in which the conserved phosphorylation site His(552) residue or the phosphorelay site Asp(989) residue had been replaced failed to complement the sln1-ts mutant. In addition, introduction of the tcsB cDNA into an sln1delta sho1delta yeast double mutant, which lacked two osmosensors, suppressed lethality in high-salinity media and activated the HOG1 MAPK. These results imply that TcsB functions as an osmosensor histidine kinase. We constructed an A. nidulans strain lacking the tcsB gene (tcsBdelta) and examined its phenotype. However, unexpectedly, the tcsBdelta strain did not exhibit a detectable phenotype for either hyphal development or morphology on standard or stress media. Our results suggest that A. nidulans has more complex and robust osmoregulatory systems than the yeast SLN1-HOG1 MAPK cascade.     

HOG1基因对白念珠菌超微结构的影响

目的探讨HOG1基因对白念珠菌超微结构的影响。方法设置实验组、HOG21组(hog1/hog1双等位基因缺陷株);对照组、WT组(标准株),分别在扫描电镜及透射电镜下观察两组菌株细胞的超微结构。结果扫描电镜下观察两组细胞均呈圆形或椭圆形,呈多边出芽繁殖的生长方式。但HOG1基因缺陷株细胞表面粗糙、凹凸不平,出芽数目比标准株少;标准株细胞表面光滑,出芽数量较多,可见"花瓣样"结构的芽痕;透射电镜下HOG1基因缺陷株细胞壁结构不完整,电子透明层厚薄不一,部分细胞可见棉絮状电子致密外层局灶性缺失、细胞膜外凸、不连续以及细胞膜周围见囊泡聚集等现象。标准株细胞壁各层结构完整。结论HOG1基因对白念珠菌细胞壁结构具有一定影响。     

The mitogen-activated protein kinase homolog HOG1 gene controls glycerol accumulation in the pathogenic fungus Candida albicans.

The Candida albicans HOG1 gene (HOG1CA) was cloned by functional complementation of the osmosensitive phenotype associated with Saccharomyces cerevisiae hog1 delta mutants. HOG1CA codes for a 377-amino-acid protein, 78% identical to S. cerevisiae Hog1p. A C. albicans hog1 null mutant was found to be sensitive to osmotic stress and failed to accumulate glycerol on high-osmolarity media.