在钙离子敏感性方面与RCH1遗传互作的酿酒酵母基因的筛选

酿酒酵母ScRCH1是白念珠菌CaRCH1的同功基因,作为人体溶质转运蛋白SLC10A7的同源蛋白,两者都是细胞质膜上钙离子内流的抑制因子。为了研究酿酒酵母RCH1与基因组中其他基因之间的遗传互作,利用合成遗传阵列(Synthetic Genetic Array,SGA)方法构建了RCH1分别与其他非必需基因之间的双基因缺失株文库。钙离子表型筛选表明RCH1与17个基因之间存在遗传互作,其中4个基因BUD9、THR1、RAS2和CPR7在钙离子敏感性方面的功能以前没有报道过。这些结果为深入研究Rch1对钙离子稳态的调控提供了参考。     

酿酒酵母中与钙离子稳态调控因子ScRCH1细胞质膜定位相关的转录因子基因的筛选检测

酿酒酵母细胞中ymr034c基因与白念珠菌的Carch1基因同源,CaRCH1与白念珠菌对钙离子、锂离子和硝唑类药物的耐受性相关。因此,把ymr034c命名为Scrch1。前期研究结果显示,在胞外高钙离子胁迫条件下,ScRCH1定位于细胞质膜上。为了研究Scrch1基因表达的调控机理,通过荧光显微镜技术对酵母细胞基因组中编码转录因子的223个单基因缺失菌株进行了筛选,分别检测了ScRCH1-GFP融合蛋白在它们中的亚细胞定位情况。结果发现,钙离子处理后,ngg1、hal9、crz1、ada2和swi6五个转录因子基因的缺失造成ScRCH1-GFP没有细胞质膜定位,而ino2基因的缺失导致ScRCH1-GFP在不经钙离子处理的条件下即定位到细胞质膜上。     

白念珠菌CaSPO75基因的敲除与功能研究

酿酒酵母中ScCsc1是一个最近鉴定的钙离子通透性压力门控阳离子通道蛋白,参与调控胞内离子稳态。ScSpo75是ScCsc1的一个同源蛋白,可能参与孢子壁的装配过程。在白念珠菌中存在一个与ScSPO75同源的基因CaSPO75,其功能尚不清楚,因此采用SAT1-flipper方法敲除CaSPO75的两个等位基因以研究其功能。通过表型筛选,发现该基因的缺失导致白念珠菌对特比萘芬(Teb)、荧光增白剂(CFW)、氯化锰和氯化锌耐受,对酮康唑(KCZ)敏感。因此,CaSPO75基因可能参与白念珠菌耐药性和细胞壁压力反应的调控,以及胞内锰离子和锌离子稳态的调控。     

鼠伤寒沙门氏菌群体感应复苏oxyR基因缺失引起的VBNC状态

探讨鼠伤寒沙门氏菌oxyR基因缺失株引起的VBNC状态及其与群体感应的关系。运用同源重组的方法构建oxyR基因无痕缺失的鼠伤寒沙门氏菌并检测该菌株对H_2O_2的敏感性;将oxyR基因缺失株和亲本株(WT)涂布或滴于LB固体培养基,观察其是否生长及其浓度依赖性生长情况;用swimming和swarming平板检测oxyR基因缺失株和WT的运动能力;检测固体培养基和液体培养基中沙门氏菌分解H_2O_2的能力。成功构建了oxyR无痕缺失菌株;oxyR基因缺失株在0.1 mmol/L H_2O_2的LB平板上形成的菌苔发生了变形,在1 mmol/L H_2O_2的LB平板上不能生长,而WT均能生长;6×10~6和6×10~5 cfu/mL的WT涂布于LB平板上能长满菌苔,而等量的oxyR缺失株不能生长菌落;不同浓度的WT滴于LB平板均能形成菌苔,而oxyR缺失株仅在OD_(600)≥10~(-1)浓度时才能形成菌苔;oxyR缺失株泳动距离无显著性变化,而群集运动距离显著性大于WT;固体培养的沙门氏菌比液体培养的沙门氏菌有更强的分解H_2O_2的能力。鼠伤寒沙门氏菌的群体感应系统通过调控其群集运动和H_2O_2分解能力来复苏由oxyR基因缺失引起的VBNC状态。     

酿酒酵母细胞基因组中与转录因子Rim101亚细胞定位相关的磷酸酶和激酶基因的筛选

Rim101是一个具有锌指结构的转录因子,在调控酿酒酵母细胞耐受碱性和高盐环境、钙离子稳态、细胞分裂以及硒毒性方面起作用。前人研究结果显示,细胞周期依赖性激酶基因PHO85的缺失,导致Rim101蛋白在细胞核内积累。为了探索Rim101亚细胞定位的新调节因子,通过荧光显微镜技术对酿酒酵母细胞基因组中编码磷酸酶的73个非必需基因缺失株和编码激酶的139个非必需基因缺失株进行了筛选,发现编码磷脂酰肌醇磷酸(Ptd Ins P)的磷酸酶Sac1调控Rim101的亚细胞定位。     

CCH1或MID1基因缺失对白念珠菌药物耐受性及致病性的影响

CCH1和MID1基因编码的钙闸门是外源钙进入细胞内的重要通道,Ca2+作为细胞内重要的第2信使分子,其浓度的升高可激活相应的途径参与各种细胞反应过程。该研究将利用钙通路CCH1和MID1基因的单缺失菌株,并构建其相应的回补菌株,研究CCH1或MID1基因缺失后对白念珠菌药物耐受性和致病性的影响作用。通过药物平板敏感性试验和微量液基稀释法比较不同菌株对唑类药物敏感性的变化,进一步添加钙通道阻滞剂和钙离子螯合剂来分析钙离子浓度变化对药物作用的影响,结果发现CCH1或MID1基因的缺失明显对氟康唑和伊曲康唑表现出敏感性,且药物作用受到钙离子浓度变化的调节。最后建立小鼠感染模型分析不同菌株的毒力变化差异,确定CCH1或MID1基因的缺失显著减弱了白念珠菌的致病性。     

白念珠菌SIM1基因敲除与功能研究

目的构建白念珠菌SIM1基因缺失菌,初步考察SIM1基因的功能。方法采用同源重组的方法构建SIM1基因双臂缺失菌,通过测定生长曲线、菌丝诱导、黏附上皮细胞等实验考察SIM1基因缺失菌表型。结果成功构建SIM1基因缺失菌,SIM1基因缺失后没有显著影响白念珠菌生长繁殖、菌丝及被膜形成,但白念珠菌对Caco-2细胞和KB细胞的黏附能力显著下降,对部分药物的敏感性增加。结论白念珠菌SIM1基因缺失导致细胞壁成分改变,并影响白念珠菌对宿主的黏附。     

酵母细胞中碱性应答蛋白Rim101与ESCRT复合物的相互调控作用

本研究采用同源重组、倍比稀释及荧光定位等方法研究了ESCRT基因对Rim101蛋白定位的影响及Rim101其对钙稳态相关基因VNX1和SPF1的调控作用。结果显示,7个ESCRT基因(SNF7, SNF8, VPS20,STP22, VPS25, VPS28和VPS36)的缺失导致GFP-Rim101不能进入细胞核,VNX1基因的缺失不影响7个ESCRT缺失株对钙离子的敏感性,而SPF1基因的缺失导致7个ESCRT缺失株对钙离子更加敏感。研究表明ESCRT基因不仅调控Rim101的功能也影响其定位,VNX1和SPF1基因是Rim101组成型活性蛋白Rim101-531抑制ESCRT缺失株对钙离子敏感性所必需的,这些研究结果为进一步研究ESCRT的功能提供了基础。     

粗糙脉孢菌转录因子LAH-3参与渗透压调控

【目的】通过构建转录因子lah-3基因缺失突变体,研究lah-3基因缺失突变体菌株的渗透压表型,进而探究lah-3基因在渗透压调控中的作用。【方法】采用同源基因重组敲除技术构建lah-3基因缺失突变体。用4%NaCl和1 mol/L Sorbitol进行渗透压处理。利用Northern blot检测渗透压应答基因的表达。利用Westhern blot检测LAH-3蛋白磷酸化修饰水平,OS-2蛋白的表达水平及其磷酸化修饰水平。【结果】在转录因子lah-3基因缺失突变体中,渗透压应答基因gcy-1、stl-1以及pck-1的表达水平都明显降低,而且在渗透压刺激下,LAH-3蛋白磷酸化修饰水平升高。LAH-3的磷酸化修饰不受OS-2调控。lah-3基因的缺失既不影响OS-2蛋白的表达水平,也不影响其在渗透压刺激后的磷酸化修饰。【结论】粗糙脉孢菌中转录因子LAH-3参与调控渗透压应答基因的转录,但其响应过程不依赖于OS-2 MAPK信号通路。     

菜心SOD基因的克隆及其在低温条件下的表达分析

采用同源克隆法从菜心中获得3个SOD基因,并进行生物信息学分析,采用qRT-PCR分析3个基因在不同组织器官和低温胁迫下的表达模式。结果表明:(1)获得Cu/Zn-SOD、Fe-SOD、Mn-SOD基因的ORF,分别命名为BclCZSD、BclFSD、BclMSD,序列长分别为459、639、696bp,分别编码152、212、231个氨基酸。(2)生物信息学分析显示,3种蛋白均为稳定的亲水性蛋白,均不存在跨膜结构和信号肽,BclCZSD的二级结构以无规则卷曲为主,含有2个Cu/Zn-SOD结构域,BclFSD和BclMSD的二级结构以α-螺旋为主,含有一个相同的Mn/Fe-SOD结构域;进化分析显示BclCZSD和BclMSD与油菜最先聚在一个分支,BclFSD与甘蓝、萝卜、油菜、芜菁聚在一个分支。(3)qRT-PCR结果显示,BclCZSD、BclFSD和BclMSD基因在菜心根、茎、叶和叶柄中均有表达,且在根、茎、叶和叶柄中的表达模式不完全相同;低温条件下,3个基因的表达量随着胁迫时间的延长均呈先升高后降低的变化趋势。研究结果为进一步探讨菜心SOD基因在低温胁迫下的响应机制奠定了基础。     

酿酒酵母细胞中内质网应激与未折叠蛋白反应的研究进展

内质网应激激活的未折叠蛋白反应(Unfolded protein response,UPR)途径在酿酒酵母和哺乳动物细胞中是非常保守的。内质网(Endoplasmic reticulum,ER)是蛋白质合成、折叠和修饰的细胞器,也是贮存钙的主要场所之一。酵母细胞内质网钙平衡与UPR的作用是相互的;两个MAPK途径——HOG途径和CWI途径都是细胞应答内质网应激压力时生存所必需的;重金属镉离子能够激活UPR途径,它通过激活钙离子通道Cch1/Mid1进入细胞影响钙离子的功能。本文结合最新研究进展对酿酒酵母细胞中的两个MAPK途径、镉离子和钙离子稳态与内质网应激激活的UPR途径之间相互关系进行综述。     

A Drosophila gene encoding a DEAD box RNA helicase can suppress loss of wee1/mik1 function in Schizosaccharomyces pombe

We describe a screen to isolate cDNAs encoding Drosophila mitosis inhibitors capable of suppressing the mitotic catastrophe phenotype resulting in Schizosaccharomyces pombe from the combination of the weel-50 mutation with either a deletion allele of mil1, or with overexpression of cdc25 ⁺. One plasmid was isolated which could suppress the temperature sensitive lethality of both these strains. The cDNA in this plasmid encodes a protein highly homologous to the DEAD-box family of ATP-dependent RNA helicases, rather than to protein kinases as might be expected. It is possible that the RNA helicase described here may regulate entry into mitosis by down regulating the expression of other genes whose activity may be rate-limiting for entry into mitosis.     

Relationship of vector insert size to homologous integration during transformation of Neurospora crassa with the cloned am (GDH) gene

Summary We used lambda and plasmid vectors containing the am ⁺ gene in an insert of from 2.7 to 9.1 kb, to transform am point mutant and deletion strains. A total of 199 transformants were examined with the potential to yield am ^– transformants by homologous recombination. When we used vectors that had 9.1 kb of homology with the chromosomal DNA, 30% of the transformants obtained were the result of homologous recombination regardless of whether the vector was a lambda molecule, a circular plasmid, or a plasmid that had been linearized prior to transformation. When vectors with up to 5.1 kb of homology were used, very few transformants (1 of 89 tested) resulted from homologous recombination. Of a sample of 29 ectopic integration events obtained by transformation with the 9.1 kb fragment cloned in a vector, 18 included a major part (usually almost all) of both arms of lambda with the entire Neurospora 9.1 kb insert between them. Four included only long arm sequence together with an adjacent segment of the insert containing the am gene. The remaining seven were the result of multiple integrations. There was no evidence of circularization of the vector prior to integration. All transformants that had multiple copies of the am gene appeared to be subject to the RIP process, which causes multiple mutations in duplicated sequences during the sexual cycle.     

Recombination between repeats in Escherichia coli by a recA-independent, proximity-sensitive mechanism

We have examined the influence of proximity on the efficiency of recombination between repeated DNA sequences in Escherichia coli. Our experiments have employed a plasmid-based assay to detect deletions between direct repeats of 100 bp. The rate of deletion of the juxtaposed direct repeats was reasonably high at 6 × 10^–5 per cell. A comparison of recA+ and recA mutant strains showed that these deletion events are primarily the result of recA-independent recombination at these homologous sequences. Random restriction fragments of yeast or E. coli genomic DNA were used to separate the two repeats. Deletion rates decreased over two orders of magnitude with increasing separation of up to 7 kb. There was a surprisingly strong effect of even short sequence separations, with insertions of a few hundred base pairs exhibiting 10-fold reductions of deletion rates. No effect of recA on the efficiency of deletion was observed at any distance between repeats.     

Candida amazonensis FLP/FRT基因敲除系统的构建及初步验证

【目的】构建一个适用于Candida amazonensis抗性标记可重复使用的FLP/FRT基因敲除系统,并通过敲除C.amazonensis的丙酮酸脱羧酶基因(Pyruvate decarboxylase,PDC)对该系统进行初步验证。【方法】以gfpm(绿色荧光蛋白基因)为报告基因,通过添加相应诱导剂评估Spathaspora passalidarum来源启动子(SpXYLp、SpMAL6p、SpMAL1p、SpGAL1p)和Saccharomyces cerevisiae来源Sc GAL1p启动子在C.amazonensis中的诱导调控性能。选择严格诱导型启动子调控FLP重组酶的表达,并在FLP表达盒和潮霉素(Hygromycin B)抗性标记基因(hphm)两端添加同向重复的FRT位点,以PDC基因作为靶基因构建敲除盒PRFg HRP,转化宿主菌C.amazonensis CBS 12363,筛选得到阳性转化子后,通过添加诱导剂,表达FLP重组酶,实现FRT位点间片段切除。【结果】诱导调控实验表明启动子SpGAL1p(受半乳糖诱导)和SpMAL1p(受麦芽糖诱导)是适用于C.amazonensis的严格诱导型启动子。以SpGAL1p调控FLP基因表达,构建的敲除盒PRFg HRP成功转化宿主菌,获得阳性转化子C.amazonensis PDC01,通过添加半乳糖诱导,成功切除基因组中FLP表达盒和抗性标记盒,获得突变株C.amazonensis PDC02。【结论】首次建立了一个适用于C.amazonensis抗性标记可重复使用的FLP/FRT基因敲除系统,并利用该系统成功敲除了C.amazonensis内的PDC基因,为进一步利用代谢工程改造C.amazonensis酵母奠定了良好基础。     

Molecular cloning of thentrA gene of the broad host-rangeRhizobium sp. NGR234, and phenotypes of a site-directed mutant

Summary The clonedntrA (rpoN) gene andntrA mutants ofRhizobium meliloti were used to isolate the homologous gene from the broad-host rangeRhizobium sp. NGR234 by hybridization and interspecies complementation. The NGR234 locus was analyzed by deletion and insertional mutagenesis. A site-directedntrA mutant, NGR234rn1, was made with an interposon, GmI, and its phenotype was examined ex planta and in symbiosis. NGR234rn1 formed Fix⁻ nodules on six genera tested from among its legume hosts, including both indeterminate and determinate nodule-type plants. Formation of nodules onMacroptilium was delayed, and expression of anR. meliloti nodABC-lacZ fusion was reduced by the mutant allele.     

Identification of nifE-, nifN- and nifS-like genes in Bradyrhizobium japonicum

Summary The 17 kb region between the Bradyrhizobium japonicum nitrogenase genes (nifDK and nifH) was investigated for the presence of further nif or fix genes by site-directed insertion or deletion/replacement mutagenesis and interspecies hybridization. Mutant strains were tested for their ability to reduce acetylene in free-living, microaerobic culture (Nif phenotype) and in soybean root nodules (Fix phenotype). The presence of a gene, previously identified by hybridization with the Klebsiella pneumoniae nifB gene, was proved by isolation of a nifB insertion mutant which was completely Nif^- and Fix^-. Three other regions were found to be homologous to the K. pneumoniae genes nifE, nifN, and nifS, NifE and nifN insertion mutants were completely Nif^-/Fix^- whereas nifS mutants were leaky with 30% residual Fix activity. Taken together, the data show that the B. japonicum genome harbours a cluster of closely adjacent genes which are directly concerned with nitrogenase function.