巴斯德毕赤酵母甲醇诱导表达磷脂酶A2的转录组学分析

【目的】基于转录组学技术研究表达磷脂酶A_2的毕赤酵母重组菌在甲醇诱导表达外源蛋白时的基因表达差异,从而解析外源蛋白高效诱导表达机制,为进一步工程菌株的改造提供理论支撑。【方法】以一株产磷脂酶(PLA_2)的毕赤酵母为出发菌株,采用RNA-Seq二代测序方法,研究在甘油培养和甲醇诱导两种条件下,重组毕赤酵母转录组基因表达差异情况。【结果】重组毕赤酵母中共鉴定到5225个转录本。甘油培养与甲醇诱导相比,共有857个基因发生显著变化。依据代谢途径分类,差异基因集中在核糖体成分、甲醇代谢、磷酸戊糖途径、糖酵解途径、柠檬酸循环、乙醛酸循环以及蛋白质加工过程。【结论】通过分析甲醇诱导前后的差异表达基因,结果表明碳源改变对胞内代谢会产生全局影响。本研究结果为进一步研究毕赤酵母表达外源蛋白的机制提供了基础。     

乙醇胁迫抑制毕赤酵母表达外源蛋白的转录组学分析

在5 L发酵罐中进行毕赤酵母发酵表达猪?干扰素的实验,发现甘油培养末期乙醇的积累会抑制外源蛋白的表达。从转录组学角度系统分析不同浓度乙醇胁迫条件下,毕赤酵母甘油培养期和甲醇诱导期细胞的生理状态变化。研究结果表明,在甘油培养期,乙醇胁迫使得毕赤酵母细胞中的545个基因发生了显著差异表达(265个基因表达上调,280个基因表达下调),这些差异表达基因的功能主要涉及蛋白质合成、能量代谢、细胞周期和过氧化物酶代谢。乙醇胁迫增加了蛋白质错误折叠的情况,降低了核糖体和线粒体的结构完整性,使得甘油培养末期无法得到大量具有健全功能的酵母细胞。在甲醇诱导期,与甲醇代谢、蛋白质加工合成、氨基酸代谢等途径相关的294个基因发生了显著差异表达(171个基因表达上调,123个基因表达下调),导致内质网胁迫不能被及时解除,破坏了细胞内的氨基酸正常代谢。     

产酱香地衣芽孢杆菌CGMCC 3963耐受特征及基于转录组学的耐受机制分析

【目的】地衣芽孢杆菌是茅台酒高温大曲中能产酱香风味物质的主要微生物,对酱香型白酒的酿造具有重要价值。而酱香型白酒的酿造环境具有高渗、高温、酸性、高乙醇胁迫等特征,研究产酱香地衣芽孢杆菌在环境胁迫下的耐受特征有利于认识酱香型白酒的酿造特征。【方法】以一株产酱香地衣芽孢杆菌(Bacillus licheniformis CGMCC 3963)为研究对象,测定其耐渗、耐酸、耐乙醇特征,并从比较转录组学角度系统分析B.licheniformis CGMCC 3963的耐受机制。【结果】B.licheniformis CGMCC 3963在15%的KCl、15%的Na Cl、p H 4.0的酸性环境或6%乙醇浓度下的生长情况明显优于不产酱香的模式菌株B.licheniformis ATCC 14580。转录组比较分析显示B.licheniformis CGMCC 3963中一系列与耐受相关的基因表达有差异。【结论】来源于酿造环境的B.licheniformis CGMCC 3963耐受能力强于B.licheniformis ATCC 14580,一系列与耐受相关的基因表达有差异。编码脯氨酸和甜菜碱等溶质转运、离子外排、钾离子通道蛋白等基因的差异表达,使得高渗胁迫下B.licheniformis CGMCC 3963生长明显优于B.licheniformis ATCC 14580;编码II类热休克蛋白、乙醇脱氢酶、氧化应激、p H动态平衡等相关基因的差异表达,在提高菌株耐受酸性环境能力上起了重要作用;II类及III类热休克基因的高表达对B.licheniformis CGMCC 3963耐乙醇能力起了重要作用。     

地果的转录组学分析

为探究地果(Ficus tikoua)的遗传变异特征,利用Illumina HiSeq-2500平台获取地果的基因表达谱。结果表明,共获得了197 362个转录本,总长度和平均长度分别为114 072 125和577 bp。使用BlastX和BlastN共注释了139 992个转录本(占总数的70.93%)。从3个品种叶片和茎的6对样品中,分别鉴定了12 397、12 340、10 373、94 431、71 830和44 465个差异表达基因,以及注释了126、129、125、134、138和137条代谢途径。这将有助于理解地果的遗传特征,以及不同组织中代谢途径的变化。     

吡唑啉酮铜配合物P-FAH-Cu-phen对金黄色葡萄球菌的转录组影响分析

【背景】金黄色葡萄球菌是目前食品和临床引起感染的重要病原菌,迫切需要开发新型抗菌药物。【目的】分析吡唑啉酮铜配合物P-FAH-Cu-phen对金黄色葡萄球菌的转录组影响和主要代谢信号通路。【方法】采用液体稀释法测定P-FAH-Cu-phen作用金黄色葡萄球菌的最低抑菌浓度(minimum inhibitory concentration, MIC)和最低杀菌浓度(minimum bactericidal concentration, MBC)。将终浓度2 μg/mL的配合物分别作用于对数生长期的金黄色葡萄球菌30 min和2 h,进行转录组测序及分析。【结果】 P-FAH-Cu-phen作用金黄色葡萄球菌的MIC和MBC分别为2 μg/mL和4 μg/mL。与空白对照相比,配合物处理细菌30 min后,其差异基因共有356个,其中上调表达180个、下调表达176个;配合物处理细菌2 h后,其差异基因共有23个,其中上调表达3个、下调表达20个。差异基因功能主要富集于膜的组成部分、细胞质、质膜、ATP结合、发病机制、金属离子结合、组氨酸生物合成过程、DNA结合、水解酶活性、跨膜转运蛋白活性、硝酸盐同化、硝酸盐代谢过程、硝酸还原酶复合物、硝酸还原酶活性等。差异基因涉及的信号通路主要有双组分系统、群体感应、氮代谢、三羧酸循环、氨基酸代谢等。【结论】影响细菌质膜组成、毒素生成、生物膜形成、细胞壁合成、能量代谢等可能是吡唑啉酮铜配合物P-FAH-Cu-phen对金黄色葡萄球菌的主要抑菌作用。研究为揭示吡唑啉酮铜配合物抑制金黄色葡萄球菌分子机制提供了理论依据。     

转录组学技术在急性肾损伤中的应用进展

急性肾损伤(acute kidney injury,AKI)是一种常见的临床危重症,发病机制复杂,缺乏有效的防治策略.近年来,转录组测序(RNA sequencing,RNA-seq)技术已广泛应用于AKI分子机制的研究,加深了对AKI亚型和病理生理过程的认知.与常规的全转录组测序(bulk RNA sequencin...     

基于RNA-Seq转录组测序技术揭示肉兔生长发育调控的分子机制

为研究肉兔肌肉生长发育调控的分子机制,本试验以84日龄齐卡巨型白兔和齐兴肉兔为研究对象,屠宰后取背最长肌组织并提取总RNA,反转录建立c DNA文库,利用Illumina HiSeq 2500测序平台进行测序,并进行序列分析和注释。筛选与肉兔肌肉生长发育相关的信号通路及差异表达基因,最后进行荧光定量PCR验证。结果表明:齐卡巨型白兔和齐兴肉兔背最长肌中显著差异表达基因总数为833个,其中齐卡巨型白兔中显著上调基因325个,显著下调基因508个。KEGG功能注释发现差异基因显著富集到PI3K-AKT通路、癌症通路和黏着斑通路,最终筛选出4个可能与生长发育显著相关的差异表达基因,为进一步对肉兔进行遗传改良提供了理论基础。     

基于转录组学的梅花鹿茸皮组织修复机制研究

为研究不同生长时期梅花鹿东北亚种Cervus nippon hortulorum鹿茸茸皮差异基因表达变化,以期为鹿茸生长机制及组织修复的临床治疗提供理论依据,本研究采用Illumina测序技术和生物信息学方法对快速生长期和骨化期鹿茸生长中心茸皮进行转录组测序和差异基因表达分析。从快速生长期和骨化期茸皮中分别获得45422254条和44530430条clean reads,测序质量值分别为98.50和98.39;通过Trinity组装,分别获得44352条和43194条组装序列,平均长度分别为977nt和1135nt。通过数据库比对和差异基因表达分析筛选出生长因子7种、转录因子25种和胶原分子6种。在快速生长期鉴定出多种参与组织修复的重要生长因子和转录因子,包括Pdgfa、Tgfβ3、Egfl7、Vegfb、Sox4、Sox12、Scx、E4f1、Tcf4和Elf4等。     

琥珀酸对酵母细胞转录组的影响

寻找抗衰老活性小分子并研究其作用机制是衰老药物学研究的重点和热点。本文报道了一种新的抗衰老活性小分子琥珀酸,发现琥珀酸可以显著延缓芽殖酵母细胞的衰老并增强细胞的压力抗性。随后,利用DNA Microarray技术及生物信息学手段较系统分析了琥珀酸处理对基因表达谱、基因本体聚类及相关信号通路的影响。结果显示,琥珀酸处理对细胞转录组产生了显著影响,共导致3 485个基因的差异表达(P 0.05),其中1 335个基因显著上调,2 150个基因显著下调。进一步对基因本体聚类及信号通路分析显示,线粒体及核糖体生物合成相关的分子功能、细胞组分、生物学过程和信号通路可能是琥珀酸作用的主要靶点,其他可能的作用靶点还包括蛋白酶体、细胞内吞、过氧化物酶体代谢及细胞自噬等。本研究为进一步阐明琥珀酸介导的寿命及压力调控机制提供了理论参考和研究线索。     

转录组和代谢组分析瘤菌根菌对铁皮石斛的促生机制

为筛选出促进铁皮石斛(Dendrobium officinale)生长的差异表达基因和差异代谢物,对瘤菌根菌与无菌盆栽铁皮石斛苗共生后形成的侧根根系进行转录组、代谢组和双组学联合分析。结果表明,转录组分析共找到262条差异表达基因富集到了35条通路中,其中内质网蛋白质加工通路途径的差异基因最多,其次为氨基糖和核苷酸糖代谢通路。代谢组分析共检测出194个差异代谢物富集到33个KEGG通路中,其中代谢途径的差异代谢物最多有133个,其次为不同环境的微生物代谢途径的差异代谢物有70个。通过联合分析,有9个差异基因的差异表达导致丝氨酸、谷氨酸、D-甘露糖和激素等代谢物的积累量发生变化,这可能是瘤菌根菌促进铁皮石斛生长的重要原因。因此,推测瘤菌根菌促进铁皮石斛生长与氨基酸、糖、植物激素的积累及相关基因的表达变化有关。     

基于转录组学的Vip3Aa杀美洲大蠊的作用机制研究

美洲大蠊Periplaneta americana是常见的家居害虫,前期研究发现营养期杀虫蛋白3Aa(Vegetative insecticidal proteins 3Aa, Vip3Aa)蛋白具备杀美洲大蠊作用,但其机制尚不明确。本研究对美洲大蠊饲喂Vip3Aa蛋白后,进行中肠转录组分析,所得raw data过滤获得44.60 Gb Clean Data。通过生物信息学分析,共注释了23 470条基因,发掘新基因5 757个。与对照组比较,共获得2 302个差异表达基因,其中1 539个上调、763个下调。KEGG分析表明,差异基因主要涉及调控溶酶体、其他多糖降解、淀粉和蔗糖代谢、糖胺聚糖降解、昆虫激素合成、鞘糖脂生物合成、药物代谢和凋亡等信号通路,其中溶酶体相关差异表达基因50个,凋亡相关差异表达基因21个。透射电镜和Hoechst 33342染色显示,Vip3Aa给药后,中肠上皮细胞出现凋亡典型的形态改变,线粒体肿胀、空泡、破裂,出现大量次级溶酶体,可见部分细胞的细胞核凝集、固缩、荧光强度增加。结果表明Vip3Aa蛋白可能通过上皮细胞溶酶体-线粒体损伤介导凋亡通路的激活,发挥杀...     

Induction of flocculation of brewer's yeast strains of Saccharomyces cerevisiae by changing the calcium concentration and pH of culture medium

Strains of Saccharomyces cerevisiae (NCYC 1190, 1214 and 1364) behaved as nonflocculent in defined culture medium, as well as the strain NCYC 1214 in rich medium. Flocculation was induced by Ca²⁺ addition and/or by correcting the pH to a suitable value. Since the free Ca²⁺ concentration is pH-dependent, these two factors (total Ca²⁺ concentration and pH) cannot be dissociated and are the critical parameters governing flocculation, in culture medium, of the brewing strains studied.     

Crystallization and preliminary structural analysis of catalase A from Saccharomyces cerevisiae.

Yeast peroxisomal catalase A, obtained at high yields by over expression of the C-terminally modified gene from a 2 mu-plasmid, has been crystallized in a form suitable for high resolution X-ray diffraction studies. Brownish crystals with bipyrimidal morphology and reaching ca. 0.8 mm in size were produced by the hanging drop method using ammonium sulphate as precipitant. These crystals diffract better than 2.0 A resolution and belong to the hexagonal space group P6(1)22 with unit cell parameters a = b = 184.3 A and c = 305.5 A. An X-ray data set with 76% completeness at 3.2 A resolution was collected in a rotating anode generator using mirrors to improve the collimation of the beam. An initial solution was obtained by molecular replacement only when using a beef liver catalase tetramer model in which fragments with no sequence homology had been omitted, about 150 residues per subunit. In the structure found a single molecule of catalase A (a tetramer with accurate 222 molecular symmetry) is located in the asymmetric unit of the crystal with an estimated solvent content of about 61%. The preliminary analysis of the structure confirms the absence of a carboxy terminal domain as the one found in the catalase from Penicillium vitalae, the only other fungal catalase structure available. The NADPH binding site appears to be involved in crystal contacts, suggesting that heterogeneity in the occupancy of the nucleotide can be a major difficulty during crystallization.     

Morphometric analysis of autophagy-related structures in Saccharomyces cerevisiae

When macroautophagy (autophagy) is induced by nutrient starvation or rapamycin treatment, Atg (autophagy-related) proteins are assembled at a restricted region close to the vacuole. Subsequently, the phagophore expands to form a closed autophagosome. In Saccharomyces cerevisiae cells overexpressing precursor Ape1 (prApe1), a specific autophagosome cargo protein, the phagophore can be visualized as a cup-shaped structure labeled with green fluorescent protein (GFP)-tagged Atg8. Previously, our group has shown that the maximum length of GFP-Atg8-labeled structures reflects the magnitude of bulk autophagy. In that study, the morphological parameters of the autophagy-related structures were extracted manually, requiring a great deal of time. Moreover, only well-expanded phagophores were subjected to further analysis. Here we report Qautas (Quantitative autophagy-related structure analysis system), a high-throughput and comprehensive system for morphological analysis of autophagy-related structures using a combination of image processing and machine learning. We describe both the manual method and Qautas in detail.     

Comprehensive phenotypic analysis for identification of genes affecting growth under ethanol stress in Saccharomyces cerevisiae

We quantified the growth behavior of all available single gene deletion strains of budding yeast under ethanol stress. Genome-wide analyses enabled the extraction of the genes and determination of the functional categories required for growth under this condition. Statistical analyses revealed that the growth of 446 deletion strains under stress induced by 8% ethanol was defective. We classified these deleted genes into known functional categories, and found that many were important for growth under ethanol stress including several categories that have not been characterized, such as peroxisome. We also performed genome-wide screening under osmotic stress and identified 329 osmotic-sensitive strains. We excluded these strains from the 446 ethanol-sensitive strains to extract the genes whose deletion caused sensitivity to ethanol-specific (359 genes), osmotic-specific (242 genes), and both stresses (87 genes). We also extracted the functional categories that are specifically important for growth under ethanol stress. The genes and functional categories identified in the analysis might provide clues to improving ethanol stress tolerance among yeast cells.     

Proteomic analysis of a distilling strain of Saccharomyces cerevisiae during industrial grain fermentation

The fermentation performance of industrial yeast strains is influenced, among other things, by their genetic composition and the nature of the fermentable sugar, availability of nitrogen, and temperature. Therefore, to manipulate the fermentation process, it is important to understand, at a molecular level, the changes occurring in the yeast cell throughout industrial fermentation processes. With this aim in mind, using two-dimensional gel electrophoresis and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS), we have examined the proteome of distillers yeast in an industrial context. Using yeast sampled from a local grain whisky distillery, we have prepared a detailed reference map of the proteome of distillers yeast and have examined in some detail the alterations in protein levels that occur throughout fermentation. In particular, as fermentation progresses, there is a significant increase in the levels of a variety of proteins involved in protecting against stress and nitrogen limitation. These results therefore give an insight into the stresses that yeast are exposed to in industrial fermentations and reveal some of the proteins and enzymes that are either necessary or important for efficient fermentation.     

Functional genomic analysis of a commercial wine strain of Saccharomyces cerevisiae under differing nitrogen conditions

DNA microarray analysis was used to profile gene expression in a commercial isolate of Saccharomyces cerevisiae grown in a synthetic grape juice medium under conditions mimicking a natural environment for yeast: High-sugar and variable nitrogen conditions. The high nitrogen condition displayed elevated levels of expression of genes involved in biosynthesis of macromolecular precursors across the time course as compared to low-nitrogen. In contrast, expression of genes involved in translation and oxidative carbon metabolism were increased in the low-nitrogen condition, suggesting that respiration is more nitrogen-conserving than fermentation. Several genes under glucose repression control were induced in low-nitrogen in spite of very high (17%) external glucose concentrations, but there was no general relief of glucose repression. Expression of many stress response genes was elevated in stationary phase. Some of these genes were expressed regardless of the nitrogen concentration while others were found at higher levels only under high nitrogen conditions. A few genes, FSP2, RGS2, AQY1, YFL030W, were expressed more strongly with nitrogen limitation as compared to other conditions.     

Survival of inoculated Saccharomyces cerevisiae strain on wine grapes during two vintages

AIMS: To investigate the influence of a specific ecological niche, the wine grape, on the survival and development of Saccharomyces cerevisiae. METHODS AND RESULTS: A strain with a rare phenotype was sprayed onto the grape surfaces and monitored through two vintages using a specific indicative medium and analysing the internal transcribed spacer regions in the 5.8S rDNA. During the ripening process, there was a progressive colonization of the surface of the undamaged and damaged grapes by epiphytic yeasts, up to the time of harvest. The damaged wine grapes showed a much greater epiphytic yeast population. However, the inoculated S. cerevisiae strain showed a scarce persistence on both undamaged and damaged wine grapes, and the damaged grapes did not appear to improve the grape surface colonization of this strain. CONCLUSIONS: Results indicated that wine grape is not a favourable ecological niche for the development and colonization of S. cerevisiae species. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this work are further evidence that S. cerevisiae is not specifically associated with natural environments such as damaged and undamaged wine grapes.