酿酒酵母海藻糖代谢工程与抗逆性相关机制研究
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摘要
乙醇是已被大规模生产与应用的可再生生物能源之一。但乙醇作为一种新能源物质,现生产工艺的能耗与原料消耗高,能源与原料资源利用效率低,工艺废水量大质劣,环境治理面临严峻挑战,整体经济社会效益急待提高。因此,通过理论与技术创新,寻求高效清洁生产新工艺成该领域备受关注的热点。实现高浓度酒精发酵是提高乙醇生产效率效益的关键技术之一。但高浓度酒精发酵要求生产菌株酿酒酵母具有良好的抗逆性能。在酿酒酵母中,胞内海藻糖含量被认为是与其抗逆性能关系密切的一个重要指标。国内外研究显示,酵母细胞可以通过增加海藻糖的合成而抵御不良逆境的伤害。但关于菌株胞内海藻糖的积累对其高浓度酒精发酵性能影响的研究却鲜有报道。本研究用细胞与分子生物学及基因工程技术改造酿酒酵母海藻糖的代谢相关途径,增强菌株胞内海藻糖积累,构建适于工业生产的高抗逆酿酒酵母工程菌株。探讨海藻糖提高细胞抗逆性的机制。最后通过系统分析HOG (High Osmolarity Glycerol)途径的基因序列变异,阐释酵母菌渗透压耐受性差异的可能机制,为进一步深入开展提高酿酒酵母抗逆性能的研究奠定基础。主要研究结果如下：
1、海藻糖代谢途径工程菌株构建从工业菌株D308中分离得到60株单倍体,并对这些菌株进行发酵性能及胞内海藻糖含量测定,结果显示二者呈显著正相关性。构建重组质粒改造海藻糖代谢途径,得到过表达编码海藻糖-6-磷酸合成酶基因(TPSl)菌株H11ptps1、H13ptps1;敲除编码海藻糖酸性水解酶基因(ATH1)菌株H11△ath1、H13△ath1;及过表达TPS1同时敲除ATH1基因菌株H11pT△A、H13pT△A。杂交实验获得二倍体工程菌株D309ptps1、D309△ath1及D309pTΔA。
2、不同胁迫条件下工程菌株与亲株生长及发酵性能比较二倍体工程菌株D309pT△A不仅结合了优良单倍体的性状,同时增强了胞内的海藻糖积累,在高糖、高乙醇、低pH、高温等胁迫条件下其最大比生长速率比亲株D308均有显著提高,提高范围为4.92%-9.80%。在高糖、高温、低pH等条件下,工程菌株的发酵速率比亲株均有较大提高,胞内海藻糖含量也均比同一条件的亲株高。玉米糖化醪(254.906g/L葡萄糖)发酵时,工程菌株D309ptpsl、D309Aathl、D309pTΔA发酵82h的乙醇产量分别为117.171g/L、117.135g/L、117.555g/L,比亲株D308分别提高了4.71%、4.67%、5.05%；主酵期0～46h期间,以上二倍体工程菌株的乙醇发酵速率分别为2.253、2.243、2.264(g h-1L-1),分别比亲株提高了13.22%、12.20%、13.76%。
3、海藻糖工程菌株抗逆性高于亲株的机制探讨从呼吸缺陷型形成比率、细胞膜完整性分析、胞内活性氧ROS水平、抗氧化性能及SOD酶(超氧化物歧化酶)活性等五个方面进行了研究分析。在乙醇胁迫下,工程菌株D309pTAA的呼吸缺陷型比率比亲株D308显著减少。D309pTAA在高渗、高温、低pH、高乙醇等条件胁迫下,细胞膜完整性均高于亲株(如高温50℃处理1h后,其细胞存活率可达48.98%,是亲株的1.71倍)；胞内ROS积累均比亲株低(仅为亲株的0.48～0.70倍)；SOD酶活性比亲株高9.98%～19.25%,同时5mmol/L H2O2胁迫下的最大比生长速率比亲株提高7.95%。因此,海藻糖提高酵母细胞在胁迫条件下的抗逆性机制可能是通过保护其细胞膜完整性,维持活性氧代谢过程中的关键酶活性,以增强菌株的抗氧化能力,进而提高工程菌株的整体抗逆性能。
4、HOG途径基因变异与菌株高渗耐受力关系研究HOG途径基因在23株酿酒酵母(包括工业酿酒酵母H13、黄酒酵母WH1)中的基因多态性及其在10个酵母菌种间的序列突变,进化分析后发现HOG途径中的上游基因序列比下游基因序列更易发生突变,位于途径顶端的SLN1与MSB2基因碱基突变数量显著高于其它基因,且菌株H13、WH1的MSB2基因中含有一个大片段Indel区(缺失378bp)。这种现象可能是导致酵母菌株间高渗耐受力差异的分子机制之一。用通径分析等统计学方法分析发现,基因在途径中的位置、密码子偏好性、蛋白质长度、蛋白质相互作用及基因表达水平等对HOG途径基因的进化均有重要作用,为进一步从基因网络水平上阐明酵母菌在环境胁迫下耐受性差异的分子机制奠定了一定基础。
Alcohol is one kind of most important and well-established reproducible biofuel. However, there are several issues and obstacles reminded in the technology of alcohol production, for example, high biomass-consuming, low efficiency of energy production, extremely large amount of wastewater produced, and arduous environmental management. So the novel technology with high efficiency and conservation has attracted more and more attentions. The achievement of fermentation under ultrahigh alcohol concentration opens a new and important avenue to increase the efficiency of alcohol production, and the basic requirement is the developing of Saccharomyces cerevisiae with highly potential ability of stress resistance during fermentation. The one of most important factors for the ability of stress resistance in Saccharomyces cerevisiae is the intracellular trehalose content. Researchers have found that Saccharomyces cerevisiae can increase the content of trehalose inside the cells to resist the potential damage in the extreme environment, but the study on the relationship between the accumulation of trehalose in Saccharomyces cerevisiae and their capability of alcoholic fermentation was rare.
Herein, we reconstructed the correlation metabolism pathway of trehalose in yeasts to increase intracellular content of trehalose by cell and molecular biology technologies. The objective of this study is to establish one kind of Saccharomyces cerevisiae with excellent stress resistance and ability of high fermentation rate, which may have great potential in the applications of fuel industry. We also investigated the possible protection mechanism of trehalose and the evolution of HOG (High Osmolarity Glycerol) pathway in yeast to demonstrate the difference of stress resistance among yeasts. There are majorly four parts in this thesis:
1. Construction of trehalose metabolic engineering strains. We separated 60 haploids from industrial strain D308. Then we analyzed and investigated the relationship between intracellular trhalose content and capability of alcoholic fermentation, and found there is the positive correlation of them. We reconstructed the metabolism process of trehalose by recombining the plasmid and produced two over-expressing trehalose-6-phosphate synthase gene (TPS1) strains: H11ptps1, H13ptpsl, two acid hydrolase gene (ATH1) knock-out strains:H11△athl, H13△athl, and two both over-expressing TPS1 and ATH1 knock-out strains:H11pTAA, H13pTAA. We further hybridized the above strains to produce three engineered diploid strains:D309ptpsl, D309Aathl, and D309pTAA.
2. The comparison of fermentation performance between engineered strains and parental strains. The engineered diploid strain D309pTAA combined the excellent features of each haploid and increased the accumulation of trehalose inside cells. The maximal specific growth rate of D309pTAA was significantly higher than that of parental strain D308 under the same extreme stress environments such as high concentration of glucose, high concentration of alcohol, low pH value and high temperature. Moreover, the fermentation rate of D309pTAA was also higher than that of parental strain D308 under the same fermentation environments such as high concentration of glucose, high temperature, and low pH value. The intracellular trehalose content in engineered strain D309pTAA was higher than parental strain D308 over the duration of fermentation. Comparing with the parental yeast D308, the alcohol production rates from Oh to 46h of engineered strains D309ptpsl, D309Aathl, and D309pTAA were 2.253,2.243, and 2.264 (g h-1L-1), which increased 13.22%, 12.20%, and 13.76%, respectively; moreover, the alcohol yields of engineered strains D309ptpsl, D309Aathl, and D309pTAA were 117.171g/L,117.135g/L,117.555g/L, and increased 4.71%,4.67%, and 5.05%, respectively than parental strain D308 (110.905g/L) after 82 h corn mash (containing 254.906g/L glucose) fermentation.
3. The reasons about the high stress resistance of engineered strains. The existence ratio of respiratory deficient yeast, cell membrane integrality, the accumulation of ROS (reactive oxygen species), and the activity of SOD (superoxide dismutase) were investigated. Under the alcohol stress condition, we found that the existence ratio of respiratory deficient yeast in engineered yeast was dramatically lower than parental yeast. The analysis of cell membrane integrality indicated the cell viability in engineered yeast was much higher than parental yeast under the same stress conditions such as high osmolity, high temperature, low pH value, and high concentration of ethanol, especially in the case of engineered strain D309pTAA. For example, the cell viability of D309pTAA still can reach to about 48.98% after the treatment of 50℃high temperature incubation for 1 h, which was 0.7 times higher than that of parental strain. Such different stress conditions indeed induce the accumulation of ROS inside cells. The maximal specific growth rate of D309pTAA was 7.95% higher than that of parental strain D308 under the existence of H2O2.The level of reactive oxygen species in engineered yeast was significantly lower, while the SOD activity in engineered yeast was higher than parental strain under the same conditions from 9.98% to 19.25%. The data shown here indicated there are two possible reasons about the protection mechanism of trehalose in yeast under stress conditions, one is the protection of cell membrane integrality in order to insure the high cell viability, and the other one is the maintenance of ROS metabolism relative enzyme (e.g., SOD) structure to increase the oxidation resistance, so that the overall stress resistance of engineered yeast was evidently higher than that of parental strain.
4. The relationship between gene mutation of HOG pathway and osmotic tolerance of yeast. The sequence mutation of HOG signaling pathway within 23 Saccharomyces cerevisiae (including industrial yeast H13 and yellow wine yeast WH1) and among 10 different yeast species was investigated. For example, the number of mutant in upstream genes SLN1 and MSB2 was significantly higher other genes, especially the MSB2 gene in H13 and WH1 yeasts containing the segment of Indel (378bp). These variations may induce the different mechanisms to environmental osmotic stress resistance in different yeasts. Meanwhile, we further analysised the evolution of HOG pathway based on the pathway analysis and other statistical analysis methods. We found that the location of gene in the pathway, codon usage, protein length, protein interaction, and gene expression all play important roles in the evolution of HOG pathway. This could be the theoretical basis to further study on stress resistance mechanism of different yeasts.
1、海藻糖代谢途径工程菌株构建从工业菌株D308中分离得到60株单倍体,并对这些菌株进行发酵性能及胞内海藻糖含量测定,结果显示二者呈显著正相关性。构建重组质粒改造海藻糖代谢途径,得到过表达编码海藻糖-6-磷酸合成酶基因(TPSl)菌株H11ptps1、H13ptps1;敲除编码海藻糖酸性水解酶基因(ATH1)菌株H11△ath1、H13△ath1;及过表达TPS1同时敲除ATH1基因菌株H11pT△A、H13pT△A。杂交实验获得二倍体工程菌株D309ptps1、D309△ath1及D309pTΔA。
2、不同胁迫条件下工程菌株与亲株生长及发酵性能比较二倍体工程菌株D309pT△A不仅结合了优良单倍体的性状,同时增强了胞内的海藻糖积累,在高糖、高乙醇、低pH、高温等胁迫条件下其最大比生长速率比亲株D308均有显著提高,提高范围为4.92%-9.80%。在高糖、高温、低pH等条件下,工程菌株的发酵速率比亲株均有较大提高,胞内海藻糖含量也均比同一条件的亲株高。玉米糖化醪(254.906g/L葡萄糖)发酵时,工程菌株D309ptpsl、D309Aathl、D309pTΔA发酵82h的乙醇产量分别为117.171g/L、117.135g/L、117.555g/L,比亲株D308分别提高了4.71%、4.67%、5.05%；主酵期0～46h期间,以上二倍体工程菌株的乙醇发酵速率分别为2.253、2.243、2.264(g h-1L-1),分别比亲株提高了13.22%、12.20%、13.76%。
3、海藻糖工程菌株抗逆性高于亲株的机制探讨从呼吸缺陷型形成比率、细胞膜完整性分析、胞内活性氧ROS水平、抗氧化性能及SOD酶(超氧化物歧化酶)活性等五个方面进行了研究分析。在乙醇胁迫下,工程菌株D309pTAA的呼吸缺陷型比率比亲株D308显著减少。D309pTAA在高渗、高温、低pH、高乙醇等条件胁迫下,细胞膜完整性均高于亲株(如高温50℃处理1h后,其细胞存活率可达48.98%,是亲株的1.71倍)；胞内ROS积累均比亲株低(仅为亲株的0.48～0.70倍)；SOD酶活性比亲株高9.98%～19.25%,同时5mmol/L H2O2胁迫下的最大比生长速率比亲株提高7.95%。因此,海藻糖提高酵母细胞在胁迫条件下的抗逆性机制可能是通过保护其细胞膜完整性,维持活性氧代谢过程中的关键酶活性,以增强菌株的抗氧化能力,进而提高工程菌株的整体抗逆性能。
4、HOG途径基因变异与菌株高渗耐受力关系研究HOG途径基因在23株酿酒酵母(包括工业酿酒酵母H13、黄酒酵母WH1)中的基因多态性及其在10个酵母菌种间的序列突变,进化分析后发现HOG途径中的上游基因序列比下游基因序列更易发生突变,位于途径顶端的SLN1与MSB2基因碱基突变数量显著高于其它基因,且菌株H13、WH1的MSB2基因中含有一个大片段Indel区(缺失378bp)。这种现象可能是导致酵母菌株间高渗耐受力差异的分子机制之一。用通径分析等统计学方法分析发现,基因在途径中的位置、密码子偏好性、蛋白质长度、蛋白质相互作用及基因表达水平等对HOG途径基因的进化均有重要作用,为进一步从基因网络水平上阐明酵母菌在环境胁迫下耐受性差异的分子机制奠定了一定基础。
Alcohol is one kind of most important and well-established reproducible biofuel. However, there are several issues and obstacles reminded in the technology of alcohol production, for example, high biomass-consuming, low efficiency of energy production, extremely large amount of wastewater produced, and arduous environmental management. So the novel technology with high efficiency and conservation has attracted more and more attentions. The achievement of fermentation under ultrahigh alcohol concentration opens a new and important avenue to increase the efficiency of alcohol production, and the basic requirement is the developing of Saccharomyces cerevisiae with highly potential ability of stress resistance during fermentation. The one of most important factors for the ability of stress resistance in Saccharomyces cerevisiae is the intracellular trehalose content. Researchers have found that Saccharomyces cerevisiae can increase the content of trehalose inside the cells to resist the potential damage in the extreme environment, but the study on the relationship between the accumulation of trehalose in Saccharomyces cerevisiae and their capability of alcoholic fermentation was rare.
Herein, we reconstructed the correlation metabolism pathway of trehalose in yeasts to increase intracellular content of trehalose by cell and molecular biology technologies. The objective of this study is to establish one kind of Saccharomyces cerevisiae with excellent stress resistance and ability of high fermentation rate, which may have great potential in the applications of fuel industry. We also investigated the possible protection mechanism of trehalose and the evolution of HOG (High Osmolarity Glycerol) pathway in yeast to demonstrate the difference of stress resistance among yeasts. There are majorly four parts in this thesis:
1. Construction of trehalose metabolic engineering strains. We separated 60 haploids from industrial strain D308. Then we analyzed and investigated the relationship between intracellular trhalose content and capability of alcoholic fermentation, and found there is the positive correlation of them. We reconstructed the metabolism process of trehalose by recombining the plasmid and produced two over-expressing trehalose-6-phosphate synthase gene (TPS1) strains: H11ptps1, H13ptpsl, two acid hydrolase gene (ATH1) knock-out strains:H11△athl, H13△athl, and two both over-expressing TPS1 and ATH1 knock-out strains:H11pTAA, H13pTAA. We further hybridized the above strains to produce three engineered diploid strains:D309ptpsl, D309Aathl, and D309pTAA.
2. The comparison of fermentation performance between engineered strains and parental strains. The engineered diploid strain D309pTAA combined the excellent features of each haploid and increased the accumulation of trehalose inside cells. The maximal specific growth rate of D309pTAA was significantly higher than that of parental strain D308 under the same extreme stress environments such as high concentration of glucose, high concentration of alcohol, low pH value and high temperature. Moreover, the fermentation rate of D309pTAA was also higher than that of parental strain D308 under the same fermentation environments such as high concentration of glucose, high temperature, and low pH value. The intracellular trehalose content in engineered strain D309pTAA was higher than parental strain D308 over the duration of fermentation. Comparing with the parental yeast D308, the alcohol production rates from Oh to 46h of engineered strains D309ptpsl, D309Aathl, and D309pTAA were 2.253,2.243, and 2.264 (g h-1L-1), which increased 13.22%, 12.20%, and 13.76%, respectively; moreover, the alcohol yields of engineered strains D309ptpsl, D309Aathl, and D309pTAA were 117.171g/L,117.135g/L,117.555g/L, and increased 4.71%,4.67%, and 5.05%, respectively than parental strain D308 (110.905g/L) after 82 h corn mash (containing 254.906g/L glucose) fermentation.
3. The reasons about the high stress resistance of engineered strains. The existence ratio of respiratory deficient yeast, cell membrane integrality, the accumulation of ROS (reactive oxygen species), and the activity of SOD (superoxide dismutase) were investigated. Under the alcohol stress condition, we found that the existence ratio of respiratory deficient yeast in engineered yeast was dramatically lower than parental yeast. The analysis of cell membrane integrality indicated the cell viability in engineered yeast was much higher than parental yeast under the same stress conditions such as high osmolity, high temperature, low pH value, and high concentration of ethanol, especially in the case of engineered strain D309pTAA. For example, the cell viability of D309pTAA still can reach to about 48.98% after the treatment of 50℃high temperature incubation for 1 h, which was 0.7 times higher than that of parental strain. Such different stress conditions indeed induce the accumulation of ROS inside cells. The maximal specific growth rate of D309pTAA was 7.95% higher than that of parental strain D308 under the existence of H2O2.The level of reactive oxygen species in engineered yeast was significantly lower, while the SOD activity in engineered yeast was higher than parental strain under the same conditions from 9.98% to 19.25%. The data shown here indicated there are two possible reasons about the protection mechanism of trehalose in yeast under stress conditions, one is the protection of cell membrane integrality in order to insure the high cell viability, and the other one is the maintenance of ROS metabolism relative enzyme (e.g., SOD) structure to increase the oxidation resistance, so that the overall stress resistance of engineered yeast was evidently higher than that of parental strain.
4. The relationship between gene mutation of HOG pathway and osmotic tolerance of yeast. The sequence mutation of HOG signaling pathway within 23 Saccharomyces cerevisiae (including industrial yeast H13 and yellow wine yeast WH1) and among 10 different yeast species was investigated. For example, the number of mutant in upstream genes SLN1 and MSB2 was significantly higher other genes, especially the MSB2 gene in H13 and WH1 yeasts containing the segment of Indel (378bp). These variations may induce the different mechanisms to environmental osmotic stress resistance in different yeasts. Meanwhile, we further analysised the evolution of HOG pathway based on the pathway analysis and other statistical analysis methods. We found that the location of gene in the pathway, codon usage, protein length, protein interaction, and gene expression all play important roles in the evolution of HOG pathway. This could be the theoretical basis to further study on stress resistance mechanism of different yeasts.
引文
[1]Hohmann S, Osmotic stress signaling and osmoadaptation in yeasts. Microbiol Mol Biol Rev,2002. 66(2):300-372.
[2]Hersen P, et al., Signal processing by the HOG MAP kinase pathway. Proc Natl Acad Sci USA, 2008.105(20):7165-7170.
[3]Capaldi A, et al., Structure and function of a transcriptional network activated by the MAPK Hogl. Nature Genetics,2008.40(11):1300-1306.
[4]O'Rourke S, et al., Unique and redundant roles for HOG MAPK pathway components as revealed by whole-genome expression analysis. Molecular Biology of the Cell,2004.15(2):532-542.
[5]Mager W, et al., Novel insights into the osmotic stress response of yeast FEMS yeast research, 2002.2(3):251-257.
[6]Edmunds J, et al., MAP kinases as structural adaptors and enzymatic activators in transcription complexes. Journal of cell science,2004.117(17):3715.
[7]Alepuz P, et al., Glucose repression affects ion homeostasis in yeast through the regulation of the stress-activated ENA1 gene. Molecular microbiology,2003.26(01):91-98.
[8]Hirata Y, et al., Yeast glycogen synthase kinase-3 activates Msn2p-dependent transcription of stress responsive genes. Molecular biology of the cell,2003.14(1):302.
[9]Morimoto R, Cells in stress:transcriptional activation of heat shock genes. Science,1993. 259(5100):1409-1410.
[10]Heinisch J, et al, Physical and Chemical Stress Factors in Yeast. Biology of Microorganisms on Grapes, in Must and in Wine,2009:275.
[11]Glover J, et al., Hsp104,Hsp70, and Hsp40. Cell,1998.94(1):73-82.
[12]Elliott B, et al., Synergy between trehalose and Hsp104 for thermotolerance in Saccharomyces cerevisiae. Genetics,1996.144(3):923-933.
[13]Thevelein J, et al., Novel sensing mechanisms and targets for the cAMP-protein kinase A pathway in the yeast Saccharomyces cerevisiae. Molecular microbiology,2002.33(5):904-918.
[14]Estruch F, Stress-controlled transcription factors, stress-induced genes and stress tolerance in budding yeast FEMS Microbiol Rev,2000.24(4):469-486.
[15]Bose S, et al., Genetic factors that regulate the attenuation of the general stress response of yeast Genetics,2005.169(3):1215.
[16]Gross E, et al., Phosphorylation of the S. cerevisiae Cdc25 in response to glucose results in its dissociation from Ras.1992.
[17]Chen R, et al., A role for the noncatalytic N terminus in the function of Cdc25, a Saccharomyces cerevisiae Ras-guanine nucleotide exchange factor. Genetics,2000.154(4):1473.
[18]Garreau H, et al., Membrane-anchoring domains of Cdc25p, a Saccharomyces cerevisiae ras exchange factor. Biology of the Cell,1996.86(2):93-102.
[19]Zou H, et al., Candida albicans Cyrl, Capl and G-actin form a sensor/effector apparatus for activating cAMP synthesis in hyphal growthmmi_6980579..591. Molecular Microbiology,2009. 75(3):579-591.
[20]Griffioen G, et al., Molecular mechanisms controlling the localisation of protein kinase A. Current Genetics,2002.41(4):199-207.
[21]Roosen J, et al., PKA and Sch 9 control a molecular switch important for the proper adaptation to nutrient availability. Molecular Microbiology,2005.55(3):862-880.
[22]Boy-Marcotte E, et al., The transcriptional activation region of Msn2p, in Saccharomyces cerevisiae, is regulated by stress but is insensitive to the cAMP signalling pathway. Molecular Genetics and Genomics,2006.275(3):277-287.
[23]De Wever V, et al., A dual role for PP1 in shaping the Msn2-dependent transcriptional response to glucose starvation. The EMBO Journal,2005.24(23):4115.
[24]Jules M, et al., New insights into trehalose metabolism by Saccharomyces cerevisiae:NTH2 encodes a functional cytosolic trehalase, and deletion of TPS1 reveals Ath1p-dependent trehalose mobilization. Appl Environ Microbiol,2008.74(3):605-614.
[25]Eastmond P, et al., Trehalose metabolism:a regulatory role for trehalose-6-phosphate? Current opinion in plant biology,2003.6(3):231-235.
[26]Elbein A, et al., New insights on trehalose:a multifunctional molecule. Glycobiology,2003. 13(4):17-27.
[27]Satoh-Nagasawa N, et al., A trehalose metabolic enzyme controls inflorescence architecture in maize. Nature,2006.441(7090):227-230.
[28]Avonce N, et al., Insights on the evolution of trehalose biosynthesis. BMC Evol Biol,2006.6:109.
[29]Voit EO, Biochemical and genomic regulation of the trehalose cycle in yeast:review of observations and canonical model analysis. J Theor Biol,2003.223(1):55-78.
[30]Mahmud SA, et al., Effect of trehalose accumulation on response to saline stress in Saccharomyces cerevisiae. Yeast,2009.26(1):17-30.
[31]Francois J, et al., Reserve carbohydrates metabolism in the yeast Saccharomyces cerevisiae. Fems microbiology reviews,2001.25(1):125-145.
[32]Paul M, Trehalose 6-phosphate. Current opinion in plant biology,2007.10(3):303-309.
[33]Paul M, et al., Trehalose metabolism and signaling. Annu. Rev. Plant Biol.,2008:417-441.
[34]Valenzuela-Soto E, et al., Trehalose 6-phosphate synthase from Selaginella lepidophylla: purification and properties. Biochemical and Biophysical Research Communications,2004. 313(2):314-319.
[35]Richards A, et al., Trehalose:a review of properties, history of use and human tolerance, and results of multiple safety studies* 1. Food and Chemical Toxicology,2002.40(7):871-898.
[36]Aranda J, et al., Trehalose accumulation in Saccharomyces cerevisiae cells:experimental data and structured modeling. Biochemical Engineering Journal,2004.17(2):129-140.
[37]Gibson R, et al., Insights into trehalose synthesis provided by the structure of the retaining glucosyltransferase OtsA. Chemistry & biology,2002.9(12):1337-1346.
[38]Huang J, et al., The transmembrane domain of acid trehalase mediates ubiquitin-independent multivesicular body pathway sorting. Mol Biol Cell,2007.18(7):2511-2524.
[39]Garre E, et al., Acid trehalase is involved in intracellular trehalose mobilization during postdiauxic growth and severe saline stress in Saccharomyces cerevisiae. FEMS Yeast Res,2009.9(1):52-62.
[40]Hottiger T, et al., Heat-induced accumulation and futile cycling of trehalose in Saccharomyces cerevisiae. Journal of Bacteriology,1987.169(12):5518-5522.
[41]Sola-Penna M, et al., Stabilization against thermal inactivation promoted by sugars on enzyme structure and function:why is trehalose more effective than other sugars? Archives of biochemistry and biophysics,1998.360(1):10-14.
[42]Colaco C, et al., Amorphous stability and trehalose. Science,1995.268(5212):788.
[43]Singer MA, et al., Thermotolerance in Saccharomyces cerevisiae:the Yin and Yang of trehalose. Trends Biotechnol,1998.16(11):460-468.
[44]Simola M, et al., Trehalose is required for conformational repair of heat-denatured proteins in the yeast endoplasmic reticulum but not for maintenance of membrane traffic functions after severe heat stress. Mol Microbiol,2000.37(1):42-53.
[45]Mojovic L, et al., Production of bioethanol from corn meal hydrolyzates. Fuel,2006. 85(12-13):1750-1755.
[46]Burnes E, et al., Economic and policy implications of public support for ethanol production in California's San Joaquin Valley. Energy Policy,2005.33(9):1155-1167.
[47]Li L, et al., The induction of trehalose and glycerol in Saccharomyces cerevisiae in response to various stresses. Biochemical and Biophysical Research Communications,2009.387(4):778-783.
[48]朱旭芬等,酿酒酵母产孢条件及核倍性分析.科技通报,2002.18(005)：393-397.
[49]Parrou JL, et al., Acid trehalase in yeasts and filamentous fungi:localization, regulation and physiological function. FEMS Yeast Res,2005.5(6-7):503-511.
[50]Attfield P, Stress tolerance:the key to effective strains of industrial baker's yeast Nature biotechnology,1997.15(13):1351-1357.
[51]Mahmud SA, et al., Differential importance of trehalose accumulation in Saccharomyces cerevisiae in response to various environmental stresses. J Biosci Bioeng,2010.109(3):262-266.
[52]Tirosh 1, et al., A Yeast Hybrid Provides Insight into the Evolution of Gene Expression Regulatioa Science,2009.324(5927):659.
[53]Katou T, et al., Brewing characteristics of haploid strains isolated from sake yeast Kyokai No.7. Yeast,2008.25(11):799-807.
[54]Guldener U, et al., A new efficient gene disruption cassette for repeated use in budding yeast Nucleic Acids Res,1996.24(13):2519-2524.
[55]肖冬光等,酿酒酵母单倍体制备方法的优化.酿酒科技,2004(004)：21-22.
[56]诸葛健等,工业微生物试验技术手册.1994：中国轻工业出版社.
[57]Gietz R, et al., Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method Meth. Enzymol,2002.350:87-96.
[58]Streeter J, et al., Three enzymes for trehalose synthesis in Bradyrhizobium cultured bacteria and in bacteroids from soybean nodules. Applied and environmental microbiology,2006. 72(6):4250-4255.
[59]Seo H, et al., Characterization of a bifunctional enzyme fusion of trehalose-6-phosphate synthetase and trehalose-6-phosphate phosphatase of Escherichia coli. Applied and environmental microbiology,2000.66(6):2484-2490.
[60]Liang L, et al., Trehalose synthesis in Saccharomycopsis fibuligera does not respond to stress treatments. Applied microbiology and biotechnology,2007.74(5):1084-1091.
[61]Jules M, et al., Two distinct pathways for trehalose assimilation in the yeast Saccharomyces cerevisiae. Applied and environmental microbiology,2004.70(5):2771-2778.
[62]Wiemken A, Trehalose in yeast, stress protectant rather than reserve carbohydrate. Antonie Van Leeuwenhoek,1990.58(3):209-217.
[63]Soto T, et al., Accumulation of trehalose by overexpression of tps1, coding for trehalose-6-phosphate synthase, causes increased resistance to multiple stresses in the fission yeast Schizosaccharomyces pombe. Applied and environmental microbiology,1999.65(5):2020.
[64]Agosin C, Biomass Content Governs Fermentation Rate in Nitrogen-Deficient Wine Musts. Applied and environmental microbiology,2004.70(6):3392-3400.
[65]Orr-Weaver T, et al., Yeast transformation:a model system for the study of recombination. Proceedings of the National Academy of Sciences,1981.78(10):6354.
[66]Gueldener U, et al., A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic acids research,2002.30(6):e23.
[67]Huxley C, et al., Rapid assessment of S. cerevisiae mating type by PCR Trends in genetics:TIG, 1990.6(8):236.
[68]Gietz R, et al., Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method Nature Protocols,2007.2(1):35-37.
[69]Guldener U, et al., A new efficient gene disruption cassette for repeated use in budding yeast Nucleic acids research,1996.24(13):2519.
[70]Baudin A, et al., A simple and efficient methodfor direct gene deletion in Saccharomyces cerevisiae. Nucleic acids research,1993.21(14):3329-3330.
[71]马学军等,精编分子生物学实验指南.2005,北京.科学出版社：587-592.
[72]Gibson BR, et al., Yeast responses to stresses associated with industrial brewery handling. FEMS Microbiol Rev,2007.31(5):535-569.
[73]Kim J, et al., Disruption of the yeast ATH1 gene confers better survival after dehydration, freezing, and ethanol shock:potential commercial applications. Appl Environ Microbiol,1996. 62(5):1563-1569.
[74]Fujita K, et al., The genome-wide screening of yeast deletion mutants to identify the genes required for tolerance to ethanol and other alcohols. FEMS yeast research,2006.6(5):744-750.
[75]van Voorst F, et al., Genome-wide identification of genes required for growth of Saccharomyces cerevisiae under ethanol stress. Yeast,2006.23(5):131-139.
[76]Hirasawa T, et al., Comparative analysis of transcriptional responses to saline stress in the laboratory and brewing strains of Saccharomyces cerevisiae with DNA microarray. Applied Microbiology and Biotechnology,2006.70(3):346-357.
[77]He S, et al., The Saccharomyces cerevisiae vacuolar acid trehalase is targeted at the cell surface for its physiological function. FEBS J,2009.276(19):5432-5446.
[78]Nevoigt E, Progress in metabolic engineering of Saccharomyces cerevisiae. Microbiol Mol Biol Rev,2008.72(3):379-412.
[79]Singer MA, et al., Multiple effects of trehalose on protein folding in vitro and in vivo. Mol Cell, 1998. 1(5):639-648.
[80]Nwaka S, et al., Expression and function of the trehalase genes NTH1 and YBR0106 in Saccharomyces cerevisiae. Journal of Biological Chemistry,1995.270(17):10193.
[81]Alexandre H, et al., Global gene expression during short-term ethanol stress in Saccharomyces cerevisiae. FEBS letters,2001.498(1):98-103.
[82]Blomberg A, Metabolic surprises in Saccharomyces cerevisiae during adaptation to saline conditions:questions, some answers and a model. FEMS Microbiol Lett,2000.182(1):1-8.
[83]Lucero P, et al., Internal trehalose protects endocytosis from inhibition by ethanol in Saccharomyces cerevisiae. Appl Environ Microbiol,2000.66(10):4456-4461.
[84]Bandara A, et al., Trehalose promotes the survival of Saccharomyces cerevisiae during lethal ethanol stress, but does not influence growth under sublethal ethanol stress. FEMS yeast research, 2009.9:1208-1216
[85]Ding J, et al., Tolerance and stress response to ethanol in the yeast Saccharomyces cerevisiae. Applied Microbiology and Biotechnology,2009:1-11.
[86]Berlett B, et al., Protein oxidation in aging, disease, and oxidative stress. Journal of Biological Chemistry,1997.272(33):20313.
[87]Henle E, et al., Formation, prevention, and repair of DNA damage by iron/hydrogen peroxide. Journal of Biological Chemistry,1997.272(31):19095.
[88]Squier T, Oxidative stress and protein aggregation during biological aging. Experimental Gerontology,2001.36(9):1539-1550.
[89]Crowe J, et al., and Chapman D, Preservation of membranes in anhydrobiotic organisms:the role of trehalose. Science,1984.223(4637):701-704.
[90]Patist A, et al., Preservation mechanisms of trehalose in food and biosystems. Colloids and Surfaces B:Biointerfaces,2005.40(2):107-113.
[91]Benaroudj N, et al., Trehalose accumulation during cellular stress protects cells and cellular proteins from damage by oxygen radicals. Journal of Biological Chemistry,2001.276(26):242261.
[92]Bahn YS, et al., Sensing the environment: lessons from fungi. Nat Rev Microbiol,2007. 5(1):57-69.
[93]Gasch AP, Comparative genomics of the environmental stress response in ascomycete fungi. Yeast, 2007.24(11):961-976.
[94]Singh AH, et al., Modularity of stress response evolution Proc Natl Acad Sci USA,2008. 105(21):7500-7505.
[95]Nikolaou E, et al., Phylogenetic diversity of stress signalling pathways in fungi. BMC Evol Biol, 2009.9(44):1-18.
[96]Wolf MY, et al., Comparable contributions of structural-functional constraints and expression level to the rate of protein sequence evolution. Biol Direct,2008.3:40-47.
[97]Ingvarsson PK, Gene expression and protein length influence codon usage and rates of sequence evolution in Populus tremula. Mol Biol Evol,2007.24(3):836-844.
[98]Graur D, el al., Fundamentals of molecular evolution. Sinauer Associates Sunderland, MA.2000.
[99]Alvarez-Ponce D, et al., Network-level molecular evolutionary analysis of the insulin/TOR signal transduction pathway across 12 Drosophila genomes. Genome Res,2009.19(2):234-242.
[100]Chen RE, et al., Function and regulation in MAPK signaling pathways:lessons learned from the yeast Saccharomyces cerevisiae. Biochim Biophys Acta,2007.1773(8):1311-1340.
[101]Krantz M, et al., Comparative genomics of the HOG-signalling system in fungi. Curr. Genet., 2006.49(3):137-151.
[102]Pokholok DK, et al., Activated signal transduction kinases frequently occupy target genes. Science,2006.313(5786):533-536.
[103]Cherry JM, et al., SGD:Saccharomyces Genome Database. Nucleic Acids Res,1998. 26(1):73-79.
[104]Arnaud MB, et al., The Candida Genome Database (CGD), a community resource for Candida albicans gene and protein information. Nucleic Acids Res,2005.33(Database issue):D358-363.
[105]Byrne KP, et al., The Yeast Gene Order Browser:combining curated homology and syntenic context reveals gene fate in polyploid species. Genome Res,2005.15(10):1456-1461.
[106]Kellis M, et al., Sequencing and comparison of yeast species to identify genes and regulatory elements. Nature,2003.423(6937):241-254.
[107]Li YD, et al., The rapid evolution of signal peptides is mainly caused by relaxed selection on non-synonymous and synonymous sites. Gene,2009.436(1-2):8-11.
[108]Larkin MA, et al., Clustal W and Clustal X version 2.0. Bioinformatics,2007.23(21):2947-2948.
[109]Hall T. BioEdit:a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT.1999.
[110]Castresana J, Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol,2000.17(4):540-552.
[111]Rokas A, et al., Genome-scale approaches to resolving incongruence in molecular phylogenies. Nature,2003.425(6960):798-804.
[112]Ronquist F, et al., MrBayes 3:Bayesian phylogenetic inference under mixed models. Bioinformatics,2003.19(12):1572-1574.
[113]Yang Z, PAML 4:phylogenetic analysis by maximum likelihood Mol Biol Evol,2007. 24(8):1586-1591.
[114]Li YD, et al., Detecting positive selection in the budding yeast genome. J Evol Biol,2009. 22(12):2430-2437.
[115]Wong WS, et al., Accuracy and power of statistical methods for detecting adaptive evolution in protein coding sequences and for identifying positively selected sites. Genetics,2004. 168(2):1041-1051.
[116]Goldman N, et al., A codon-based model of nucleotide substitution for protein-coding DNA sequences. Mol Biol Evol,1994.11(5):725-736.
[117]Connallon T, et al., Recombination rate and protein evolution in yeast BMC Evol Biol,2007. 7:235-240.
[118]Fay JC, et al., Hypervariable noncoding sequences in Saccharomyces cerevisiae. Genetics,2005. 170(4):1575-1587.
[119]Beskow, et al., Comparative analysis of regulatory transcription factors in Schizosaccharomyces pombe and budding yeasts. Yeast,2006.23(13):929-935.
[120]Kellis M, et al., Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae. Nature,2004.428(6983):617-624.
[121]Sackton TB, et al., Dynamic evolution of the innate immune system in Drosophila. Nat Genet, 2007.39(12):1461-1468.
[122]Muzzey D, et al., A systems-level analysis of perfect adaptation in yeast osmoregulation. Cell, 2009.138(1):160-171.
[123]Rausher M, et al., Patterns of evolutionary rate variation among genes of the anthocyanin biosynthetic pathway. SMBE,1999:266-274.
[124]Jovelin R, et al., Evolutionary rates and centrality in the yeast gene regulatory network. Genome Biol,2009.10(4):R35.1-R35.10.
[125]Liti G, et al., Population genomics of domestic and wild yeasts. Nature,2009. 458(7236):337-341.
[126]Jovelin R, et al., High nucleotide divergence in developmental regulatory genes contrasts with the structural elements of olfactory pathways in Caenorhabditis. Genetics,2009.181(4):1387.
[127]Fuchs B, et al., Our paths might cross:The role of the fungal cell wall integrity pathway in stress response and cross-talk with other stress response pathways. Eukaryotic Cell,2009. 8(11):1611-1625.
[128]朱旭芬等,耐高温酿酒酵母单倍体的RAPD分析.生物工程学报,2001.17(5)：557-560.
[129]戴朝波等,酵母紫外线诱变后培养温度与酒精发酵性状变异相关性研究.科技通报,2008.24(3)：325-329.
[130]Wang PM, et al., Development and application of RAPD-SCAR markers to identify intra-species hybrids of industrial Saccharomyces cerevisiae. World J Microbiol Biotechnol,2010. in press.
[131]Zheng K, et al., Programmed cell death-involved aluminum toxicity in yeast alleviated by antiapoptotic members with decreased calcium signals. Plant physiology,2007.143:38-49.
[132]Davidson JF, et al., Oxidative stress is involved in heat-induced cell death in Saccharomyces cerevisiae. Proc Natl Acad Sci USA,1996.93:5116-5121.
[133]朱旭芬等,酿酒酵母耐热相关基因DNA片断的初探.菌物系统,2000.19(2)：248-253.
[134]吴雪昌等,酵母HOG-MAPK途径.细胞生物学杂志,2005.27：247-252.
[135]Wu XC, et al., The evolutionary rate variation among genes of HOG-signaling pathway in yeast genomes. Biology Direct,2010. in press.
[2]Hersen P, et al., Signal processing by the HOG MAP kinase pathway. Proc Natl Acad Sci USA, 2008.105(20):7165-7170.
[3]Capaldi A, et al., Structure and function of a transcriptional network activated by the MAPK Hogl. Nature Genetics,2008.40(11):1300-1306.
[4]O'Rourke S, et al., Unique and redundant roles for HOG MAPK pathway components as revealed by whole-genome expression analysis. Molecular Biology of the Cell,2004.15(2):532-542.
[5]Mager W, et al., Novel insights into the osmotic stress response of yeast FEMS yeast research, 2002.2(3):251-257.
[6]Edmunds J, et al., MAP kinases as structural adaptors and enzymatic activators in transcription complexes. Journal of cell science,2004.117(17):3715.
[7]Alepuz P, et al., Glucose repression affects ion homeostasis in yeast through the regulation of the stress-activated ENA1 gene. Molecular microbiology,2003.26(01):91-98.
[8]Hirata Y, et al., Yeast glycogen synthase kinase-3 activates Msn2p-dependent transcription of stress responsive genes. Molecular biology of the cell,2003.14(1):302.
[9]Morimoto R, Cells in stress:transcriptional activation of heat shock genes. Science,1993. 259(5100):1409-1410.
[10]Heinisch J, et al, Physical and Chemical Stress Factors in Yeast. Biology of Microorganisms on Grapes, in Must and in Wine,2009:275.
[11]Glover J, et al., Hsp104,Hsp70, and Hsp40. Cell,1998.94(1):73-82.
[12]Elliott B, et al., Synergy between trehalose and Hsp104 for thermotolerance in Saccharomyces cerevisiae. Genetics,1996.144(3):923-933.
[13]Thevelein J, et al., Novel sensing mechanisms and targets for the cAMP-protein kinase A pathway in the yeast Saccharomyces cerevisiae. Molecular microbiology,2002.33(5):904-918.
[14]Estruch F, Stress-controlled transcription factors, stress-induced genes and stress tolerance in budding yeast FEMS Microbiol Rev,2000.24(4):469-486.
[15]Bose S, et al., Genetic factors that regulate the attenuation of the general stress response of yeast Genetics,2005.169(3):1215.
[16]Gross E, et al., Phosphorylation of the S. cerevisiae Cdc25 in response to glucose results in its dissociation from Ras.1992.
[17]Chen R, et al., A role for the noncatalytic N terminus in the function of Cdc25, a Saccharomyces cerevisiae Ras-guanine nucleotide exchange factor. Genetics,2000.154(4):1473.
[18]Garreau H, et al., Membrane-anchoring domains of Cdc25p, a Saccharomyces cerevisiae ras exchange factor. Biology of the Cell,1996.86(2):93-102.
[19]Zou H, et al., Candida albicans Cyrl, Capl and G-actin form a sensor/effector apparatus for activating cAMP synthesis in hyphal growthmmi_6980579..591. Molecular Microbiology,2009. 75(3):579-591.
[20]Griffioen G, et al., Molecular mechanisms controlling the localisation of protein kinase A. Current Genetics,2002.41(4):199-207.
[21]Roosen J, et al., PKA and Sch 9 control a molecular switch important for the proper adaptation to nutrient availability. Molecular Microbiology,2005.55(3):862-880.
[22]Boy-Marcotte E, et al., The transcriptional activation region of Msn2p, in Saccharomyces cerevisiae, is regulated by stress but is insensitive to the cAMP signalling pathway. Molecular Genetics and Genomics,2006.275(3):277-287.
[23]De Wever V, et al., A dual role for PP1 in shaping the Msn2-dependent transcriptional response to glucose starvation. The EMBO Journal,2005.24(23):4115.
[24]Jules M, et al., New insights into trehalose metabolism by Saccharomyces cerevisiae:NTH2 encodes a functional cytosolic trehalase, and deletion of TPS1 reveals Ath1p-dependent trehalose mobilization. Appl Environ Microbiol,2008.74(3):605-614.
[25]Eastmond P, et al., Trehalose metabolism:a regulatory role for trehalose-6-phosphate? Current opinion in plant biology,2003.6(3):231-235.
[26]Elbein A, et al., New insights on trehalose:a multifunctional molecule. Glycobiology,2003. 13(4):17-27.
[27]Satoh-Nagasawa N, et al., A trehalose metabolic enzyme controls inflorescence architecture in maize. Nature,2006.441(7090):227-230.
[28]Avonce N, et al., Insights on the evolution of trehalose biosynthesis. BMC Evol Biol,2006.6:109.
[29]Voit EO, Biochemical and genomic regulation of the trehalose cycle in yeast:review of observations and canonical model analysis. J Theor Biol,2003.223(1):55-78.
[30]Mahmud SA, et al., Effect of trehalose accumulation on response to saline stress in Saccharomyces cerevisiae. Yeast,2009.26(1):17-30.
[31]Francois J, et al., Reserve carbohydrates metabolism in the yeast Saccharomyces cerevisiae. Fems microbiology reviews,2001.25(1):125-145.
[32]Paul M, Trehalose 6-phosphate. Current opinion in plant biology,2007.10(3):303-309.
[33]Paul M, et al., Trehalose metabolism and signaling. Annu. Rev. Plant Biol.,2008:417-441.
[34]Valenzuela-Soto E, et al., Trehalose 6-phosphate synthase from Selaginella lepidophylla: purification and properties. Biochemical and Biophysical Research Communications,2004. 313(2):314-319.
[35]Richards A, et al., Trehalose:a review of properties, history of use and human tolerance, and results of multiple safety studies* 1. Food and Chemical Toxicology,2002.40(7):871-898.
[36]Aranda J, et al., Trehalose accumulation in Saccharomyces cerevisiae cells:experimental data and structured modeling. Biochemical Engineering Journal,2004.17(2):129-140.
[37]Gibson R, et al., Insights into trehalose synthesis provided by the structure of the retaining glucosyltransferase OtsA. Chemistry & biology,2002.9(12):1337-1346.
[38]Huang J, et al., The transmembrane domain of acid trehalase mediates ubiquitin-independent multivesicular body pathway sorting. Mol Biol Cell,2007.18(7):2511-2524.
[39]Garre E, et al., Acid trehalase is involved in intracellular trehalose mobilization during postdiauxic growth and severe saline stress in Saccharomyces cerevisiae. FEMS Yeast Res,2009.9(1):52-62.
[40]Hottiger T, et al., Heat-induced accumulation and futile cycling of trehalose in Saccharomyces cerevisiae. Journal of Bacteriology,1987.169(12):5518-5522.
[41]Sola-Penna M, et al., Stabilization against thermal inactivation promoted by sugars on enzyme structure and function:why is trehalose more effective than other sugars? Archives of biochemistry and biophysics,1998.360(1):10-14.
[42]Colaco C, et al., Amorphous stability and trehalose. Science,1995.268(5212):788.
[43]Singer MA, et al., Thermotolerance in Saccharomyces cerevisiae:the Yin and Yang of trehalose. Trends Biotechnol,1998.16(11):460-468.
[44]Simola M, et al., Trehalose is required for conformational repair of heat-denatured proteins in the yeast endoplasmic reticulum but not for maintenance of membrane traffic functions after severe heat stress. Mol Microbiol,2000.37(1):42-53.
[45]Mojovic L, et al., Production of bioethanol from corn meal hydrolyzates. Fuel,2006. 85(12-13):1750-1755.
[46]Burnes E, et al., Economic and policy implications of public support for ethanol production in California's San Joaquin Valley. Energy Policy,2005.33(9):1155-1167.
[47]Li L, et al., The induction of trehalose and glycerol in Saccharomyces cerevisiae in response to various stresses. Biochemical and Biophysical Research Communications,2009.387(4):778-783.
[48]朱旭芬等,酿酒酵母产孢条件及核倍性分析.科技通报,2002.18(005)：393-397.
[49]Parrou JL, et al., Acid trehalase in yeasts and filamentous fungi:localization, regulation and physiological function. FEMS Yeast Res,2005.5(6-7):503-511.
[50]Attfield P, Stress tolerance:the key to effective strains of industrial baker's yeast Nature biotechnology,1997.15(13):1351-1357.
[51]Mahmud SA, et al., Differential importance of trehalose accumulation in Saccharomyces cerevisiae in response to various environmental stresses. J Biosci Bioeng,2010.109(3):262-266.
[52]Tirosh 1, et al., A Yeast Hybrid Provides Insight into the Evolution of Gene Expression Regulatioa Science,2009.324(5927):659.
[53]Katou T, et al., Brewing characteristics of haploid strains isolated from sake yeast Kyokai No.7. Yeast,2008.25(11):799-807.
[54]Guldener U, et al., A new efficient gene disruption cassette for repeated use in budding yeast Nucleic Acids Res,1996.24(13):2519-2524.
[55]肖冬光等,酿酒酵母单倍体制备方法的优化.酿酒科技,2004(004)：21-22.
[56]诸葛健等,工业微生物试验技术手册.1994：中国轻工业出版社.
[57]Gietz R, et al., Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method Meth. Enzymol,2002.350:87-96.
[58]Streeter J, et al., Three enzymes for trehalose synthesis in Bradyrhizobium cultured bacteria and in bacteroids from soybean nodules. Applied and environmental microbiology,2006. 72(6):4250-4255.
[59]Seo H, et al., Characterization of a bifunctional enzyme fusion of trehalose-6-phosphate synthetase and trehalose-6-phosphate phosphatase of Escherichia coli. Applied and environmental microbiology,2000.66(6):2484-2490.
[60]Liang L, et al., Trehalose synthesis in Saccharomycopsis fibuligera does not respond to stress treatments. Applied microbiology and biotechnology,2007.74(5):1084-1091.
[61]Jules M, et al., Two distinct pathways for trehalose assimilation in the yeast Saccharomyces cerevisiae. Applied and environmental microbiology,2004.70(5):2771-2778.
[62]Wiemken A, Trehalose in yeast, stress protectant rather than reserve carbohydrate. Antonie Van Leeuwenhoek,1990.58(3):209-217.
[63]Soto T, et al., Accumulation of trehalose by overexpression of tps1, coding for trehalose-6-phosphate synthase, causes increased resistance to multiple stresses in the fission yeast Schizosaccharomyces pombe. Applied and environmental microbiology,1999.65(5):2020.
[64]Agosin C, Biomass Content Governs Fermentation Rate in Nitrogen-Deficient Wine Musts. Applied and environmental microbiology,2004.70(6):3392-3400.
[65]Orr-Weaver T, et al., Yeast transformation:a model system for the study of recombination. Proceedings of the National Academy of Sciences,1981.78(10):6354.
[66]Gueldener U, et al., A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic acids research,2002.30(6):e23.
[67]Huxley C, et al., Rapid assessment of S. cerevisiae mating type by PCR Trends in genetics:TIG, 1990.6(8):236.
[68]Gietz R, et al., Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method Nature Protocols,2007.2(1):35-37.
[69]Guldener U, et al., A new efficient gene disruption cassette for repeated use in budding yeast Nucleic acids research,1996.24(13):2519.
[70]Baudin A, et al., A simple and efficient methodfor direct gene deletion in Saccharomyces cerevisiae. Nucleic acids research,1993.21(14):3329-3330.
[71]马学军等,精编分子生物学实验指南.2005,北京.科学出版社：587-592.
[72]Gibson BR, et al., Yeast responses to stresses associated with industrial brewery handling. FEMS Microbiol Rev,2007.31(5):535-569.
[73]Kim J, et al., Disruption of the yeast ATH1 gene confers better survival after dehydration, freezing, and ethanol shock:potential commercial applications. Appl Environ Microbiol,1996. 62(5):1563-1569.
[74]Fujita K, et al., The genome-wide screening of yeast deletion mutants to identify the genes required for tolerance to ethanol and other alcohols. FEMS yeast research,2006.6(5):744-750.
[75]van Voorst F, et al., Genome-wide identification of genes required for growth of Saccharomyces cerevisiae under ethanol stress. Yeast,2006.23(5):131-139.
[76]Hirasawa T, et al., Comparative analysis of transcriptional responses to saline stress in the laboratory and brewing strains of Saccharomyces cerevisiae with DNA microarray. Applied Microbiology and Biotechnology,2006.70(3):346-357.
[77]He S, et al., The Saccharomyces cerevisiae vacuolar acid trehalase is targeted at the cell surface for its physiological function. FEBS J,2009.276(19):5432-5446.
[78]Nevoigt E, Progress in metabolic engineering of Saccharomyces cerevisiae. Microbiol Mol Biol Rev,2008.72(3):379-412.
[79]Singer MA, et al., Multiple effects of trehalose on protein folding in vitro and in vivo. Mol Cell, 1998. 1(5):639-648.
[80]Nwaka S, et al., Expression and function of the trehalase genes NTH1 and YBR0106 in Saccharomyces cerevisiae. Journal of Biological Chemistry,1995.270(17):10193.
[81]Alexandre H, et al., Global gene expression during short-term ethanol stress in Saccharomyces cerevisiae. FEBS letters,2001.498(1):98-103.
[82]Blomberg A, Metabolic surprises in Saccharomyces cerevisiae during adaptation to saline conditions:questions, some answers and a model. FEMS Microbiol Lett,2000.182(1):1-8.
[83]Lucero P, et al., Internal trehalose protects endocytosis from inhibition by ethanol in Saccharomyces cerevisiae. Appl Environ Microbiol,2000.66(10):4456-4461.
[84]Bandara A, et al., Trehalose promotes the survival of Saccharomyces cerevisiae during lethal ethanol stress, but does not influence growth under sublethal ethanol stress. FEMS yeast research, 2009.9:1208-1216
[85]Ding J, et al., Tolerance and stress response to ethanol in the yeast Saccharomyces cerevisiae. Applied Microbiology and Biotechnology,2009:1-11.
[86]Berlett B, et al., Protein oxidation in aging, disease, and oxidative stress. Journal of Biological Chemistry,1997.272(33):20313.
[87]Henle E, et al., Formation, prevention, and repair of DNA damage by iron/hydrogen peroxide. Journal of Biological Chemistry,1997.272(31):19095.
[88]Squier T, Oxidative stress and protein aggregation during biological aging. Experimental Gerontology,2001.36(9):1539-1550.
[89]Crowe J, et al., and Chapman D, Preservation of membranes in anhydrobiotic organisms:the role of trehalose. Science,1984.223(4637):701-704.
[90]Patist A, et al., Preservation mechanisms of trehalose in food and biosystems. Colloids and Surfaces B:Biointerfaces,2005.40(2):107-113.
[91]Benaroudj N, et al., Trehalose accumulation during cellular stress protects cells and cellular proteins from damage by oxygen radicals. Journal of Biological Chemistry,2001.276(26):242261.
[92]Bahn YS, et al., Sensing the environment: lessons from fungi. Nat Rev Microbiol,2007. 5(1):57-69.
[93]Gasch AP, Comparative genomics of the environmental stress response in ascomycete fungi. Yeast, 2007.24(11):961-976.
[94]Singh AH, et al., Modularity of stress response evolution Proc Natl Acad Sci USA,2008. 105(21):7500-7505.
[95]Nikolaou E, et al., Phylogenetic diversity of stress signalling pathways in fungi. BMC Evol Biol, 2009.9(44):1-18.
[96]Wolf MY, et al., Comparable contributions of structural-functional constraints and expression level to the rate of protein sequence evolution. Biol Direct,2008.3:40-47.
[97]Ingvarsson PK, Gene expression and protein length influence codon usage and rates of sequence evolution in Populus tremula. Mol Biol Evol,2007.24(3):836-844.
[98]Graur D, el al., Fundamentals of molecular evolution. Sinauer Associates Sunderland, MA.2000.
[99]Alvarez-Ponce D, et al., Network-level molecular evolutionary analysis of the insulin/TOR signal transduction pathway across 12 Drosophila genomes. Genome Res,2009.19(2):234-242.
[100]Chen RE, et al., Function and regulation in MAPK signaling pathways:lessons learned from the yeast Saccharomyces cerevisiae. Biochim Biophys Acta,2007.1773(8):1311-1340.
[101]Krantz M, et al., Comparative genomics of the HOG-signalling system in fungi. Curr. Genet., 2006.49(3):137-151.
[102]Pokholok DK, et al., Activated signal transduction kinases frequently occupy target genes. Science,2006.313(5786):533-536.
[103]Cherry JM, et al., SGD:Saccharomyces Genome Database. Nucleic Acids Res,1998. 26(1):73-79.
[104]Arnaud MB, et al., The Candida Genome Database (CGD), a community resource for Candida albicans gene and protein information. Nucleic Acids Res,2005.33(Database issue):D358-363.
[105]Byrne KP, et al., The Yeast Gene Order Browser:combining curated homology and syntenic context reveals gene fate in polyploid species. Genome Res,2005.15(10):1456-1461.
[106]Kellis M, et al., Sequencing and comparison of yeast species to identify genes and regulatory elements. Nature,2003.423(6937):241-254.
[107]Li YD, et al., The rapid evolution of signal peptides is mainly caused by relaxed selection on non-synonymous and synonymous sites. Gene,2009.436(1-2):8-11.
[108]Larkin MA, et al., Clustal W and Clustal X version 2.0. Bioinformatics,2007.23(21):2947-2948.
[109]Hall T. BioEdit:a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT.1999.
[110]Castresana J, Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol,2000.17(4):540-552.
[111]Rokas A, et al., Genome-scale approaches to resolving incongruence in molecular phylogenies. Nature,2003.425(6960):798-804.
[112]Ronquist F, et al., MrBayes 3:Bayesian phylogenetic inference under mixed models. Bioinformatics,2003.19(12):1572-1574.
[113]Yang Z, PAML 4:phylogenetic analysis by maximum likelihood Mol Biol Evol,2007. 24(8):1586-1591.
[114]Li YD, et al., Detecting positive selection in the budding yeast genome. J Evol Biol,2009. 22(12):2430-2437.
[115]Wong WS, et al., Accuracy and power of statistical methods for detecting adaptive evolution in protein coding sequences and for identifying positively selected sites. Genetics,2004. 168(2):1041-1051.
[116]Goldman N, et al., A codon-based model of nucleotide substitution for protein-coding DNA sequences. Mol Biol Evol,1994.11(5):725-736.
[117]Connallon T, et al., Recombination rate and protein evolution in yeast BMC Evol Biol,2007. 7:235-240.
[118]Fay JC, et al., Hypervariable noncoding sequences in Saccharomyces cerevisiae. Genetics,2005. 170(4):1575-1587.
[119]Beskow, et al., Comparative analysis of regulatory transcription factors in Schizosaccharomyces pombe and budding yeasts. Yeast,2006.23(13):929-935.
[120]Kellis M, et al., Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae. Nature,2004.428(6983):617-624.
[121]Sackton TB, et al., Dynamic evolution of the innate immune system in Drosophila. Nat Genet, 2007.39(12):1461-1468.
[122]Muzzey D, et al., A systems-level analysis of perfect adaptation in yeast osmoregulation. Cell, 2009.138(1):160-171.
[123]Rausher M, et al., Patterns of evolutionary rate variation among genes of the anthocyanin biosynthetic pathway. SMBE,1999:266-274.
[124]Jovelin R, et al., Evolutionary rates and centrality in the yeast gene regulatory network. Genome Biol,2009.10(4):R35.1-R35.10.
[125]Liti G, et al., Population genomics of domestic and wild yeasts. Nature,2009. 458(7236):337-341.
[126]Jovelin R, et al., High nucleotide divergence in developmental regulatory genes contrasts with the structural elements of olfactory pathways in Caenorhabditis. Genetics,2009.181(4):1387.
[127]Fuchs B, et al., Our paths might cross:The role of the fungal cell wall integrity pathway in stress response and cross-talk with other stress response pathways. Eukaryotic Cell,2009. 8(11):1611-1625.
[128]朱旭芬等,耐高温酿酒酵母单倍体的RAPD分析.生物工程学报,2001.17(5)：557-560.
[129]戴朝波等,酵母紫外线诱变后培养温度与酒精发酵性状变异相关性研究.科技通报,2008.24(3)：325-329.
[130]Wang PM, et al., Development and application of RAPD-SCAR markers to identify intra-species hybrids of industrial Saccharomyces cerevisiae. World J Microbiol Biotechnol,2010. in press.
[131]Zheng K, et al., Programmed cell death-involved aluminum toxicity in yeast alleviated by antiapoptotic members with decreased calcium signals. Plant physiology,2007.143:38-49.
[132]Davidson JF, et al., Oxidative stress is involved in heat-induced cell death in Saccharomyces cerevisiae. Proc Natl Acad Sci USA,1996.93:5116-5121.
[133]朱旭芬等,酿酒酵母耐热相关基因DNA片断的初探.菌物系统,2000.19(2)：248-253.
[134]吴雪昌等,酵母HOG-MAPK途径.细胞生物学杂志,2005.27：247-252.
[135]Wu XC, et al., The evolutionary rate variation among genes of HOG-signaling pathway in yeast genomes. Biology Direct,2010. in press.