H4K5去乙酰化对镍胁迫下酿酒酵母细胞壁完整性途径的调控作用

【目的】金属镍(nickel, Ni)是人类广泛接触的重金属污染物之一,镍暴露会激活细胞内的细胞壁完整性(cell wall integrity, CWI)信号通路,也会导致细胞内组蛋白乙酰化水平降低,但CWI途径在镍胁迫时是否受组蛋白乙酰化调控尚不完全清楚。【方法】利用组蛋白定点突变型菌株H4K5R (模拟去乙酰化状态),分析镍胁迫下H4K5去乙酰化对酿酒酵母CWI途径的调控作用【结果】与野生型菌株相比,定点突变型菌株H4K5R具有较强的镍抗性:在5.0 mmol/L NiCl₂胁迫下,定点突变型菌株仍能生长良好;Western blotting与qRT-PCR结果表明,野生型菌株BY4741在5.0 mmol/L NiCl₂胁迫下细胞壁完整性途径被激活,甘露聚糖与葡聚糖调控基因Mnn9表达量显著上调3.13倍、Fks1表达量显著上调1.49倍,甘露聚糖、β-葡聚糖的含量也增加,说明此时野生型菌株激活了CWI途径,细胞壁成分含量增加;定点突变型菌株H4K5R在5.0 mmol/L NiCl₂胁迫下CWI途径激活程度较轻,虽然Mnn9、Fks1表达量上调,但甘露聚糖含量变化并不显著,而相较于野生型菌株β-葡聚糖含量增加幅度较小。【结论】在5.0 mmol/L NiCl₂胁迫下,定点突变型菌株H4K5位点的去乙酰化调控CWI途径,进而影响细胞壁组分的变化。     

酿酒酵母中SSK2基因与其他镉耐受相关基因之间的双基因缺失菌株构建

镉是一种严重的环境污染物,对人体具有致癌性,能蓄积在生物体内影响机体的生长、发育和生殖。有丝分裂原蛋白激酶(Mitogen-activated protein kinase,MAPK)在调节细胞存活、增殖和分化中是重要的信号分子,并能够被镉胁迫激活。酿酒酵母中2个MAPK信号传导途径,高渗透压甘油(High Osmolarity Glycerol,HOG)途径和细胞壁完整性(Cell Wall Integrity,CWI)途径都参与Cd2+胁迫下的细胞应答。为了进一步研究这两条途径在调控Cd2+胁迫方面的相互作用,以HOG途径的蛋白激酶SSK2基因为例,通过合成遗传阵列(Synthetic Genetic Array,SGA)方法,成功构建了SSK2基因与其他52个Cd2+耐受相关基因之间的双基因缺失菌株。为大规模研究Cd2+耐受基因之间在调控镉胁迫方面的遗传学相互作用奠定了基础,也为酿酒酵母的相关研究提供了一个新的遗传学手段。     

植物调控盐胁迫下细胞壁完整性的分子机制

植物细胞壁不仅起着支撑和保护细胞的作用,还被认为是植物抵抗逆境胁迫环境的第一道屏障。作为限制农业生产的一个主要非生物胁迫因子,盐胁迫能造成植物细胞壁的组分和结构发生改变,而植物可以通过细胞壁完整性感受器如CrRLK1Ls、LRXs和WAKs等蛋白来感知这些变化并启动下游盐胁迫响应。在细胞内,植物通过盐胁迫诱导的Ca²⁺内流、植物激素等信号促进细胞壁多聚糖合成和修饰相关基因的表达,从而有助于维持细胞壁的完整性,增强植物盐胁迫适应性。本文概述了植物初生细胞壁多聚糖的主要组分和各组分之间的相互结合关系,并且阐述了盐胁迫对细胞壁各组分的影响,以及盐胁迫下植物感知和维持细胞壁完整性的分子机制,最后讨论了盐胁迫下细胞壁完整性感知和调控研究领域还需要解决的科学问题。     

镉胁迫下水稻OsPT1的表达及功能分析

OsPT1编码的水稻磷酸盐（Pi）转运蛋白在水稻生长发育、非生物胁迫应答等方面发挥重要的调控作用。前期研究表明OsPT1为镉（Cd）响应基因,但其在Cd胁迫下的功能及作用机制仍然未知。阐明OsPT1在Cd胁迫下的作用,并为低Cd水稻品种的选育奠定基础。通过生物信息学方法对该基因的序列特征、结构和功能进行分析和预测,利用实时荧光定量PCR（RT-qPCR）方法检测Cd胁迫下水稻不同组织、不同时间点OsPT1的相对表达量。此外,利用PCR的方法克隆OsPT1的编码序列,构建pGADT7-OsPT1重组质粒载体,并将其转入Δycf1 BY4741酵母菌株（Cd敏感酵母菌株）用以验证OsPT1对酵母Cd耐受性的影响。结果表明,OsPT1编码序列全长为1 584 bp,编码分子量为57.46 kD,由527个氨基酸构成的蛋白。在水稻基因组中该基因上游启动子区含有与光、厌氧、茉莉酸甲酯等环境和激素响应相关的调控元件。系统进化分析表明,水稻OsPT1与高粱SbPT1亲缘关系最近。基因的镉响应表达分析结果表明,与对照相比,经100 μmol/L Cd处理的水稻在1、6和12 h后,地上部分OsPT1的转录水平分别上调1.31、1.34和2.46倍;水稻根部OsPT1在处理1和6 h后分别上调1.28和1.14倍,但在Cd处理12 h后,其表达水平下调至处理前的0.62倍。转基因酵母Cd耐受性结果表明,与对照（0 μmol/L Cd）相比,经25 μmol/L Cd处理后,转OsPT1的酵母对Cd的耐受性有一定的下降。OsPT1可能在水稻应对Cd胁迫过程中发挥一定的作用。     

外源油菜素内酯对镉胁迫下菊芋幼苗光合作用及镉富集的调控效应

以两种菊芋(Helianthus tuberosus L.)品系南芋2号(NY2)和南芋5号(NY5)为材料,研究了外源24-表油菜素内酯(24-EBL)对镉胁迫下菊芋幼苗干重、根冠比(R/S)、光合色素含量、叶片气体交换参数和水分利用效率(WUE)的调节效应,还测定了其不同器官的镉(Cd)含量.结果表明:在镉胁迫下,2种菊芋幼苗的干重、R/S、光合色素含量、净光合速率(Pn)、气孔导度(Gs)、蒸腾速率(Tr)、WUE均呈下降趋势,而胞间二氧化碳浓度(Ci)升高.(2)与镉胁迫相比,胁迫下外源喷施10-10、10-9、10-8、10-7mol/L 24-EBL作用下,两品系植株干重和R/S值均不同程度的上升,NY2、NY5的植株干重分别在10-9 mol/L 24-EBL(EBL2)和10-8mol/L 24-EBL(EBL3)处理下达到最大值,分别增加50％和64％.镉胁迫下,外源24-EBL处理均提高菊芋的叶绿素(Chl)和类胡萝卜素(Car)含量,Pn、Gs、Tr也由此得到不同程度的上升,而Ci均下降,NY5的Ci下降更显著.镉胁迫下,外源EBL2和EBL3作用下均不同程度地提高其WUE,NY5的WUE增幅远大于NY2.镉胁迫下NY5的新完全展开叶Cd含量的积累明显高于NY2;而EBL2处理下可降低NY2的新完全展开叶Cd含量,但EBL3却显著增加NY5的叶片Cd含量.镉胁迫下,喷施EBL2的NY2的不同器官、NY5根的Cd含量均显著下降,而NY5其他器官的Cd含量变化不显著.NY5不同器官的Cd含量均明显高于NY2.上述表明,24-EBL可明显提高菊芋的耐镉水平,主要是因为外源喷施24-EBL能显著促进其光合和提高水分利用效率,从而改善Cd胁迫下菊芋幼苗的生长;而24-EBL对菊芋NY5非气孔限制的更显著改善是其促进其光合、水分利用的重要原因,也是其对NY5的生长调控效果优于NY2的重要原因之一.结果还显示,菊芋NY5植株生物量大,从环境中提取Cd的能力较好,因此可作为重金属污染土壤的植物修复的材料来利用.     

壳聚糖对镉胁迫下玉米幼苗叶片AsA-GSH循环的调控效应

以玉米（Zea mays L.）品种‘郑单958’为实验材料,分析外施壳聚糖对镉胁迫下玉米幼苗生物量、叶片镉含量、叶片超氧阴离子（O₂^·-）产生速率和过氧化氢（H₂O₂）的含量,以及抗坏血酸-谷胱甘肽（AsA-GSH）循环中抗氧化酶的活性及抗氧化物含量的影响。结果显示,随着镉胁迫时间的延长,玉米幼苗发生氧化胁迫,叶片抗氧化酶（APX、GR、DHAR、MDHAR）活性和抗氧化物（AsA、GSH）的含量降低,镉积累过量会抑制玉米幼苗的生长。施加壳聚糖可以降低镉胁迫下玉米幼苗叶片O₂^·-的产生速率和H₂O₂含量,提高上述抗氧化酶活性和抗氧化物的含量,促进AsA和GSH的再生,维持细胞的氧化还原状态,促进玉米幼苗的生长。研究结果表明壳聚糖处理后玉米幼苗保持了较高的AsA-GSH循环运作效率,提高了抗氧化能力,可有效缓解镉胁迫对玉米幼苗生长的抑制。     

外源脯氨酸对镉胁迫下小麦幼苗生长的影响

以高蛋白小麦品种“北农9549”为试材,研究喷施不同浓度脯氨酸(0、1.0、5.0和10.0 mmol·L^-1)对镉胁迫下小麦幼苗生长和重金属吸收的影响.结果表明： 以不施镉为对照,1.0 mmol·L^-1CdCl₂胁迫下,小麦幼苗的根长、株高和干质量分别显著下降24.0%、15.0%和27.5%,叶绿素a、b和类胡萝卜素含量分别显著下降23.3%、6.7%和30.8%,超氧化物歧化酶(SOD)活性降低了18.4%,内源脯氨酸、抗坏血酸和丙二醛(MDA)含量分别显著上升78.6%、31.5%和17.9%,细胞膜相对透性显著升高24.8%,过氧化物酶(POD)活性为对照的2.4倍,并且促进对铜的吸收,抑制锌的吸收.随外源脯氨酸浓度的增加,小麦幼苗的根长、株高、干质量、叶绿素和类胡萝卜素含量均逐渐恢复到对照水平,抗坏血酸、内源游离脯氨酸含量和SOD活性均上升,可溶性蛋白含量先上升后下降,POD活性、MDA含量和细胞膜相对透性下降,而锌积累量升高,镉、铜积累量下降.叶面喷施外源脯氨酸可缓解镉对小麦幼苗生长的胁迫,以喷施5.0～10.0 mmol·L^-1外源脯氨酸效果最佳.     

外源脯氨酸对镉胁迫下小麦幼苗生长的影响

以高蛋白小麦品种“北农9549”为试材,研究喷施不同浓度脯氨酸(0、1.0、5.0和10.0 mmol·L-1)对镉胁迫下小麦幼苗生长和重金属吸收的影响.结果表明:以不施镉为对照,1.0 mmol·L-1CdCl2胁迫下,小麦幼苗的根长、株高和干质量分别显著下降24.0％、15.0％和27.5％,叶绿素a、b和类胡萝卜素含量分别显著下降23.3％、6.7％和30.8％,超氧化物歧化酶(SOD)活性降低了18.4％,内源脯氨酸、抗坏血酸和丙二醛(MDA)含量分别显著上升78.6％、31.5％和17.9％,细胞膜相对透性显著升高24.8％,过氧化物酶(POD)活性为对照的2.4倍,并且促进对铜的吸收,抑制锌的吸收.随外源脯氨酸浓度的增加,小麦幼苗的根长、株高、干质量、叶绿素和类胡萝卜素含量均逐渐恢复到对照水平,抗坏血酸、内源游离脯氨酸含量和SOD活性均上升,可溶性蛋白含量先上升后下降,POD活性、MDA含量和细胞膜相对透性下降,而锌积累量升高,镉、铜积累量下降.叶面喷施外源脯氨酸可缓解镉对小麦幼苗生长的胁迫,以喷施5.0～10.0 mmol·L-1外源脯氨酸效果最佳.     

NO对镉胁迫下小麦根系生长发育的生理影响

为了探究外源物一氧化氮（NO）供体硝普钠（sodium nitroprusside,SNP）对Cd²⁺胁迫下小麦根系生长发育和活性氧代谢的影响,以小麦（Triticum aestivum L.）为材料,研究10 mmol/L CdCl₂胁迫下,30 μmol/L硝普钠（含一氧化氮NO）对小麦根系生长发育和活性氧代谢的影响。结果显示,外施SNP后,Cd²⁺胁迫下的小麦根长度、鲜重与干重较单独镉胁迫处理分别上升了48.0%、107.7%和87.3%,根系超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）、抗坏血酸过氧化物酶（APX）的活性分别上升了28.5%、7.4%、19.2%和9.8%,根中超氧自由基（O₂^.-）和过氧化氢（H₂O₂）的含量分别降低了80.5％和47.0％;同时外施SNP,使镉胁迫下小麦根中的可溶性糖含量和脯氨酸含量分别上升了24.7％和22.1%;使根中丙二醛（MDA）含量降低了30.2％;使根系活力上升了15.3%。因此,外源NO在一定程度上可以显著提高小麦根的抗氧化能力,增强小麦的抗逆性,缓解镉对小麦根系的毒害,进而促进小麦幼苗根系的生长发育。     

过量表达蛋白激酶YPK1使酵母细胞对高盐胁迫的高度敏感依赖于雷帕霉素靶蛋白

摘要：YPK1是酵母中和哺乳动物蛋白激酶SGK同源的一种丝氨酸∕苏氨酸蛋白激酶,在酿酒酵母（Saccharomyces cerevisiae）生理调节中有重要的作用,和酵母细胞壁的完整性、细胞骨架中肌动蛋白极性、细胞内吞作用、细胞在氮源缺乏和营养条件调节下细胞内部的翻译情况密切相关。【目的】为了深入研究YPK1蛋白激酶的细胞功能以及在细胞信号传导中的作用,【方法】我们构建了过量表达YPK1的高拷贝质粒,研究了过量表达YPK1的酵母细胞在盐胁迫条件下的生长情况,【结果】发现过量表达YPK1会导致酵母细胞对盐胁迫高度敏感,并且这种敏感性依赖于TOR1的存在。【结论】我们的研究结果首次初步揭示YPK1与细胞盐胁迫应答的关系,并初步证明YPK1的功能充分发挥需要TOR1的参与。     

Wide-Ranging Effects of the Yeast Ptc1 Protein Phosphatase Acting Through the MAPK Kinase Mkk1

The Saccharomyces cerevisiae type 2C protein phosphatase Ptc1 is required for a wide variety of cellular functions, although only a few cellular targets have been identified. A genetic screen in search of mutations in protein kinase–encoding genes able to suppress multiple phenotypic traits caused by the ptc1 deletion yielded a single gene, MKK1, coding for a MAPK kinase (MAPKK) known to activate the cell-wall integrity (CWI) Slt2 MAPK. In contrast, mutation of the MKK1 paralog, MKK2, had a less significant effect. Deletion of MKK1 abolished the increased phosphorylation of Slt2 induced by the absence of Ptc1 both under basal and CWI pathway stimulatory conditions. We demonstrate that Ptc1 acts at the level of the MAPKKs of the CWI pathway, but only the Mkk1 kinase activity is essential for ptc1 mutants to display high Slt2 activation. We also show that Ptc1 is able to dephosphorylate Mkk1 in vitro. Our results reveal the preeminent role of Mkk1 in signaling through the CWI pathway and strongly suggest that hyperactivation of Slt2 caused by upregulation of Mkk1 is at the basis of most of the phenotypic defects associated with lack of Ptc1 function.     

Phenotype analysis of Saccharomyces cerevisiae mutants with deletions in Pir cell wall glycoproteins

Proteins with internal repeats (Pir) belong to a minor group of covalently linked yeast cell wall proteins. They are not essential for viability but important for cell wall strength, reduced permeability against plant antifungal enzymes and maintenance of osmotic stability. Here we show the importance of Pir proteins of Saccharomyces cerevisiae for growth at low pH and in presence of various inhibitors. Cell wall analysis of Deltapir1,2,3,4 deletion strain revealed slightly increased chitin content and changes in relative proportion of alkali-soluble and insoluble glucan and chitin fractions. Activation of the cell wall integrity pathway was indicated by increased levels of double phosphorylated Mpk1p/Slt2p in the pir deletants.     

Thermosensitivity of the <Emphasis Type="Italic">Saccharomyces cerevisiae gpp1gpp2</Emphasis> double deletion strain can be reduced by overexpression of genes involved in cell wall maintenance

A Saccharomyces cerevisiae strain in which the GPP1 and GPP2 genes, both encoding glycerol-3-phosphate phosphatase isoforms, are deleted, displays both osmo- and thermosensitive (ts) phenotypes. We isolated genes involved in cell wall maintenance as multicopy suppressors of the gpp1gpp2 ts phenotype. We found that the gpp1gpp2 strain is hypersensitive to cell wall stress such as treatment with β-1,3-glucanase containing cocktail Zymolyase and chitin-binding dye Calcofluor-white (CFW). Sensitivity to Zymolyase was rescued by overexpression of SSD1, while CFW sensitivity was rescued by SSD1, FLO8 and WSC3—genes isolated as multicopy suppressors of the gpp1gpp2 ts phenotype. Some of the isolated suppressor genes (SSD1, FLO8) also rescued the lytic phenotype of slt2 deletion strain. Additionally, the sensitivity to CFW was reduced when the cells were supplied with glycerol. Both growth on glycerol-based medium and overexpression of SSD1, FLO8 or WSC3 had additive suppressing effect on CFW sensitivity of the gpp1gpp2 mutant strain. We also confirmed that the internal glycerol level changed in cells exposed to cell wall perturbation.     

代谢工程改造酿酒酵母提高法尼醇产量

[目的]法尼醇(FOH,C15H26O)是一种具有芳香气味的非环状倍半萜醇,被广泛应用于化妆品和医学药物的工业化生产,也可作为航空燃料的理想替代品.具有食品级安全性的酿酒酵母细胞能够合成内源性法尼醇,但其产量很低,无法满足工业生产的需要.因此,需要采用代谢工程手段,改造法尼醇合成途径,以有效提高法尼醇在酿酒酵母中的产量...     

Effect of low pH on organization of the actin cytoskeleton in Saccharomyces cerevisiae

Cell growth in the yeast Saccharomyces cerevisiae depends on polarization of the actin cytoskeleton. In this study, we investigated how the cell regulates the distribution of actin in response to low pH conditions, focusing on the role of mitogen-activated protein kinases, Hog1 and Slt2. Changing the extracellular pH from 6.0 to 3.0 caused a transient depolarization of the actin cytoskeleton. Actin cables were no longer visible, and actin patches appeared randomly distributed after 30 min at pH 3.0. The deletion strain hog1Δ did not show this low-pH phenotype, suggesting that Hog1 is involved in depolarization of the actin cytoskeleton in response to low-pH stress. Yeast cells incubated at pH 3.0 also showed markedly increased endocytosis compared with the control at neutral pH, as indicated by the uptake of Lucifer Yellow (LY). Both the hog1Δ and slt2Δ mutants took up LY into the vacuole to a similar extent as the wild-type strain. In addition, cells grown at pH 3.0 showed a 2-fold increase in phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) levels, as did the hog1Δ or slt2Δ cells. Efficient uptake of LY and actin repolarization at pH 3.0 might therefore require activation of PI(4,5)P2 synthesis.     

Comparative analysis of trehalose production by Debaryomyces hansenii and Saccharomyces cerevisiae under saline stress

The comparative analysis of growth, intracellular content of Na⁺ and K⁺, and the production of trehalose in the halophilic Debaryomyces hansenii and Saccharomyces cerevisiae were determined under saline stress. The yeast species were studied based on their ability to grow in the absence or presence of 0.6 or 1.0 M NaCl and KCl. D. hansenii strains grew better and accumulated more Na⁺ than S. cerevisiae under saline stress (0.6 and 1.0 M of NaCl), compared to S. cerevisiae strains under similar conditions. By two methods, we found that D. hansenii showed a higher production of trehalose, compared to S. cerevisiae; S. cerevisiae active dry yeast contained more trehalose than a regular commercial strain (S. cerevisiae La Azteca) under all conditions, except when the cells were grown in the presence of 1.0 M NaCl. In our experiments, it was found that D. hansenii accumulates more glycerol than trehalose under saline stress (2.0 and 3.0 M salts). However, under moderate NaCl stress, the cells accumulated more trehalose than glycerol. We suggest that the elevated production of trehalose in D. hansenii plays a role as reserve carbohydrate, as reported for other microorganisms.     

The Pisum sativum MAP kinase homologue (PsMAPK) rescues the Saccharomyces cerevisiae hog1 deletion mutant under conditions of high osmotic stress

Previous analysis of the MAP kinase homologue from Pisum sativum (PsMAPK) revealed a potential MAP kinase motif homologous to that found in eukaryotic cdc2 kinases. Sequence comparison showed a 47% identity on amino acid sequence basis to the Saccharomyces cerevisiae Hog 1p MAP kinase involved in the osmoregulatory pathway. Under conditions of salt-stress aberrant morphology of a hog1 deletion mutant was completely restored and growth was partially restored by expression of the PsMAPK. This shows that PsMAPK is functionally active as a MAP kinase in S. cerevisiae. Comparison of PsMAPK with other kinases involved in osmosensitivity, showed a high degree of homology and implicates a possible role for PsMAPK in a P. sativum osmosensing signal transduction pathway.