环链棒束孢hsp90基因的克隆及冷热胁迫下的表达特征分析

通过本地Blast筛选转录组数据库方法,首次克隆了环链棒束孢热休克蛋白90基因全长cDNA序列,命名为Ichsp90（GenBank登录号KT944289）。克隆结果表明,该序列含有2 284个碱基,包括一个含2 097个碱基的开放阅读框,编码699个氨基酸,推测蛋白的分子量为79.23kDa,等电点（pI）为4.86,且含有5个Hsp90家族特征基序和胞质特征序列MEEVD,推导的氨基酸序列与其他丝状真菌相似性在92%-96%之间。用qRT-PCR方法分析了冷热胁迫下,该基因在环链棒束孢中的相对表达情况,结果表明：在4℃冷胁迫下15min检测到Ichsp90表达量下降到最低点,为对照的-1.8倍;随后表达量开始上升,至120min表达量是对照的1.07倍。在39℃高温胁迫下,60min Ichsp90表达量达到最高峰,为对照样品的5.02倍;随后表达量开始下降,至110min为对照样品的2.46倍。因此推测,Ichsp90基因在环链棒束孢抵抗外界温度胁迫中发挥重要的作用。     

盐胁迫对昆虫病原真菌环链棒束孢生长及基因表达的影响

环链棒束孢Isaria cateniannulata是一种重要的昆虫病原真菌,广泛应用于茶园害虫的防治。环境胁迫是影响该菌株生长、扩散和菌株毒力的不利外界因素,其中盐胁迫对环链棒束孢的生长和基因转录表达的影响尚不清楚。本研究采用0（对照）、0.6和1.0mol/L的NaCl处理菌株,观测不同浓度NaCl对菌株表型的生长抑制作用,并对3组处理做了转录组分析。结果表明,3组处理共拼接到37 833个转录本,得到10 441个unigenes。与对照组相比,0.6和1.0mol/L NaCl处理组共鉴定出1 074个和2 412个差异表达基因,其中697个和1 201个表达上升,377个和1 211个表达下降。这些差异表达基因分别参与了碳水化合物和氨基酸的代谢、核糖体的生物合成、脂肪酸的合成与降解。为了验证转录组结果的准确性,本研究进一步通过qRT-PCR技术验证了12个差异表达基因的表达谱,研究结果为进一步了解环链棒束孢抵抗盐胁迫的分子机理奠定了理论基础。     

盐胁迫对昆虫病原真菌环链棒束孢生长及基因表达的影响（英文）

环链棒束孢Isaria cateniannulata是一种重要的昆虫病原真菌,广泛应用于茶园害虫的防治。环境胁迫是影响该菌株生长、扩散和菌株毒力的不利外界因素,其中盐胁迫对环链棒束孢的生长和基因转录表达的影响尚不清楚。本研究采用0 (对照)、0.6和1.0mol/L的NaCl处理菌株,观测不同浓度NaCl对菌株表型的生长抑制作用,并对3组处理做了转录组分析。结果表明,3组处理共拼接到37 833个转录本,得到10 441个unigenes。与对照组相比,0.6和1.0mol/L NaCl处理组共鉴定出1 074个和2 412个差异表达基因,其中697个和1 201个表达上升,377个和1 211个表达下降。这些差异表达基因分别参与了碳水化合物和氨基酸的代谢、核糖体的生物合成、脂肪酸的合成与降解。为了验证转录组结果的准确性,本研究进一步通过qRT-PCR技术验证了 12个差异表达基因的表达谱,研究结果为进一步了解环链棒束孢抵抗盐胁迫的分子机理奠定了理论基础。     

烟蚜热激蛋白基因MpHsp70的克隆及在UV-B胁迫下的表达分析

【目的】为探讨烟蚜Myzus persicae适应UV-B胁迫的分子机制。【方法】采用RT-PCR和RACE技术克隆了烟蚜热激蛋白基因Hsp70的全长,利用生物信息学方法分析了其特征;采用实时荧光定量PCR检测了不同时长(0,15,30,60,90和120 min) UV-B胁迫下烟蚜成虫中该基因的相对表达量。【结果】克隆获得烟蚜Hsp70基因并命名为MpHsp70(GenBank登录号:MF509827),该基因全长为2 221 bp,开放阅读框(ORF) 1 965 bp,编码654个氨基酸,蛋白相对分子量为71. 41kD,等电点(p I)为5. 34,末端高度保守序列EEVD显示该蛋白属于胞质热激蛋白。系统进化关系分析表明,MpHsp70与多种昆虫的Hsp70的同源性较高,表现出Hsp70基因的高度保守性。实时荧光定量PCR分析表明,随着UV-B照射时间的延长,烟蚜成虫体内MpHsp70基因的表达量先上升后下降,当照射时间为30 min时表达量最大。【结论】烟蚜MpHsp70基因可以响应UV-B的胁迫,它可能在烟蚜适应UV-B胁迫过程中起到重要作用。     

柑橘全爪螨热激蛋白基因PcHsp90的克隆及表达模式分析

【目的】研究热激蛋白基因Hsp90在柑橘全爪螨Panonychus citri生长发育及响应高温和低温胁迫方面的作用。【方法】采用RT-PCR和RACE技术克隆柑橘全爪螨Hsp90基因cDNA全长序列;利用生物信息学软件分析该基因的序列特性;运用荧光Real-time PCR技术,分析Hsp90基因mRNA在该螨各发育阶段、高温及低温胁迫条件下的表达模式。【结果】克隆鉴定出柑橘全爪螨一条Hsp90基因的c DNA全长序列,命名为PcHsp90(Gen Bank登录号:GQ495086),全长为2 763 bp,包含2 193 bp的开放阅读框,编码730个氨基酸,编码蛋白质的理论分子量和等电点分别为83.85kDa和4.99,氨基酸序列包括Hsp90家族的5个特征基序及细胞质型Hsp90的特征序列"MEEVD"。系统进化分析表明,PcHsp90与朱砂叶螨Tetranychus cinnabarinus的Hsp90首先聚为一支,然后再与肩突硬蜱Ixodes scapularis的Hsp90聚合,说明它们较近的亲缘关系。PcHsp90在柑橘全爪螨的各发育阶段均有所表达,其中在幼螨期表达量较低,且显著低于若螨和成螨期的表达水平(P=0.015)。0~10℃低温胁迫下,Pc Hsp90的mRNA相对表达量无显著变化(P=0.492);但在35~41℃高温胁迫下,Pc Hsp90的mRNA相对表达量随胁迫温度的升高而上调,尤其是当温度升高到41℃时,mRNA相对表达量达到对照(25℃)的6.75倍,且差异达显著水平(P=0.007)。【结论】柑橘全爪螨PcHsp90不仅对该螨的生长发育具有重要作用,而且是其响应高温胁迫的重要机制之一。     

玫烟色棒束孢(Isaria fumosorosea)侵染小菜蛾的表达谱分析

【背景】昆虫病原真菌对寄主的侵染是一个十分复杂的过程,是多基因共同作用的结果。玫烟色棒束孢(Isaria fumosorosea) IFCF01菌株对小菜蛾具有很高的致病力,然而有关玫烟色棒束孢对小菜蛾致病的相关基因少见报道。【目的】筛选玫烟色棒束孢侵染小菜蛾相关基因,为更好地利用玫烟色棒束孢防治小菜蛾提供基因靶点。【方法】采用第二代高通量测序技术RNA-Seq,对玫烟色棒束孢侵染小菜蛾2–3龄幼虫4、8、12、16、24、30、36 h的虫菌混合样品(处理组)及纯培养玫烟色棒束孢(对照组)进行测序分析并筛选差异表达基因,结合生物信息学方法分析差异基因涉及的功能模块和信号通路。【结果】玫烟色棒束孢侵染小菜蛾混合样品与纯培养玫烟色棒束孢对照组对比分析共获得28 384个差异基因,其中显著差异表达基因274个,上调表达118个,下调表达156个。筛选获得的显著差异表达基因,特别是上调表达基因可能与玫烟色棒束孢对小菜蛾的侵染有关。GO二级分类显示,差异表达基因能够注释到36个GO条目中,包含18个生物学过程、9个细胞组分和9个分子功能。KEGG通路分析显示共有171个差异表达基因(differentially expressed gene,DEG)注释到132个通路中,其中有66个DEG显著富集在14个通路中。这些显著差异表达基因中大部分为玫烟色棒束孢侵染过程中潜在致病毒力相关基因。【结论】本研究为筛选玫烟色棒束孢侵染小菜蛾致病相关基因提供重要数据库,也为阐明玫烟色棒束孢对小菜蛾的侵染机制提供基础。     

蠋蝽热激蛋白基因AcHsp83a和AcHsp83b的克隆、表达谱及对高低温和UV-B胁迫的响应

【目的】探索天敌昆虫蠋蝽Arma chinensis响应高低温和UV-B胁迫的分子机制。【方法】利用RT-PCR克隆蠋蝽热激蛋白基因Hsp83a和Hsp83b,并利用生物信息学分析其序列特征；利用RT-qPCR检测Hsp83a和Hsp83b在蠋蝽不同发育阶段(卵、1-5龄若虫、雌成虫和雄成虫)、成虫不同组织(头、胸、腹、翅、触角、脂肪体、足、马氏管、口器、中肠、卵巢和精巢)及38℃高温、4℃低温0(CK), 6和24 h时和UV-B胁迫0(CK), 6和12 h时雌成虫和雄成虫中的表达量。【结果】克隆获得蠋蝽2个Hsp90基因，分别命名为AcHsp83a(GenBank登录号：OP791883)和AcHsp83b(GenBank登录号：OP791884),开放阅读框(ORF)分别长2 172和2 163 bp,分别编码723和720个氨基酸，编码蛋白相对分子量分别为83.12和82.90 kD,等电点(pI)分别为4.94和4.97,C末端序列都含保守基序EEVD,均为胞质型热激蛋白。AcHsp83a和AcHsp83b高度保守。AcHsp83a在卵中表达量最高，AcHsp83b在成虫中...     

割手密SnRK2基因家族全基因组鉴定及干旱胁迫表达分析

【目的】探究割手密SnRK2基因家族成员在干旱胁迫中的调控机制,为抗旱性甘蔗品种的选育提供侯选基因。【方法】以全基因组数据为基础从割手密中鉴定SnRK2基因,并对其进行生物信息学分析和干旱胁迫下的表达分析。【结果】在割手密基因组中共鉴定出11个SnRK2基因家族成员,命名为SsSnRK2.1-SsSnRK2.11,且这些基因不均匀地分布于8条染色体上。SnRK2蛋白的氨基酸残基数为227～580,分子质量为25 683.53～64 695.8 kD,等电点为4.62～8.94,且均为亲水性蛋白。系统发育树可将其分为3个亚组,且同亚组中的保守基序基本相似,外显子数量以7～9个为主。SsSnRK2基因家族成员的启动子中含有多种激素类和逆境胁迫响应类的作用元件。割手密SsSnRK2基因家族成员的表达具有组织特异性。所有的SsSnRK2基因均能不同程度地响应干旱胁迫。【结论】割手密SnRK2基因家族在响应干旱胁迫过程中发挥重要作用,可为割手密的抗逆性研究提供一定的理论依据。     

番茄LeHsp110/ClpB基因的分子克隆及其对植物耐热性的影响

HSP100ClpB是Clp蛋白家族的一员,具有分子伴侣功能,与细胞“获得耐热性(acquiredthermotolerance)”相关。从番茄cDNA文库中筛选到长度达3144bp的cDNA,依据最长的开放读码框推导出的多肽含980个氨基酸残基,分子进化分析结果表明该蛋白属于HSP100ClpB家族,因其计算分子量为110kD,所以命名为LeHSP110ClpB。实验证明,LeHsp110ClpB在番茄叶片中没有组成型表达,为热诱导型基因,其编码蛋白定位于叶绿体基质。利用农杆菌介导法,将CaMV35S驱动的反义LeHsp110ClpBcDNA片段导入番茄,高温下转反义基因的番茄株系中LeHsp110ClpBmRNA水平明显低于对照,转基因株系的PSⅡ对高温胁迫更加敏感,说明HSP110ClpB在植物耐热性方面起重要作用。     

温度胁迫下棉花粉蚧热激蛋白基因的表达分析

【目的】为了研究热激蛋白 Hsp70, Hsp70-4和Hsp90在棉花粉蚧Phenacoccus solenopsis抵抗逆温中的作用。【方法】在测序棉花粉蚧转录组的基础上,分析了该虫热激蛋白Hsp70基因家族的2个序列［Pshsp70（GenBank登录号为KJ909505）和Pshsp70-4（GenBank登录号为KJ909506）］和Hsp90基因家族的1个序列,［Pshsp90(GenBank登录号为KJ909507)］,采用实时荧光定量 PCR（RT-qPCR）检测了在不同温度（18和32℃恒温, 37, 39, 41, 43和45℃热激1 h 后26℃恢复1 h)下棉花粉蚧不同发育阶段（2龄若虫、3龄若虫、雌成虫）3种热激蛋白基因的表达量。【结果】Pshsp70 cDNA序列包含1 923 bp的开放阅读框,编码641个氨基酸,理论分子量和等电点分别为70.9 kDa和5.65; Pshsp70-4 cDNA序列包含1 962 bp的开放阅读框,编码654个氨基酸,理论分子量和等电点分别为71.8 kDa和5.38;Pshsp90 cDNA序列包含2 172 bp的开放阅读框,编码724个氨基酸,理论分子量和等电点分别为83.5 kDa和4.93。Pshsp70 和Pshsp70-4均含有Hsp70基因家族高度保守的基序,Pshsp70编码的氨基酸序列与烟粉虱Bemisia tabaci和家蚕Bombyx mori等昆虫的Hsp70 的氨基酸序列一致性为 85%;Pshsp70-4编码的氨基酸序列与白蜡蚧Ericerus pela和点蜂缘蝽Riptortus pedestris等昆虫的Hsp70的氨基酸序列一致性高达95%;Pshsp90也含有Hsp90基因家族高度保守的基序,Pshsp90编码的氨基酸序列与赤拟谷盗Tribolium castaneum和东亚小花蝽Orius sauteri等昆虫的Hsp90 的氨基酸序列一致性为 87%。热激蛋白基因表达量分析结果表明,在18℃恒温条件下,粉蚧2龄若虫的3个PsHsps基因的mRNA相对表达量均比对照(26℃)低,在32℃恒温条件下,各龄期的Hsp70基因的相对表达量均显著高于对照。在37~45℃下热激1 h并在26℃下恢复1 h,棉花粉蚧3个龄期的3个热激蛋白PsHsps基因的相对表达量随温度的升高总体呈增加趋势,相关性分析表明,除Pshsp70-4在雌成虫中的表达量与热胁迫温度的相关系数为0.225外,各龄期中3个基因的表达量与温度的相关系数均大于0.6,显著相关;43℃和45℃胁迫下,各龄期的3个热激蛋白基因相对表达量均显著高于对照组（P<0.05）。【结论】棉花粉蚧热激蛋白基因的表达与温度呈正相关,在该虫应对高温中起着重要作用。     

Plant Hsp100/ClpB-like proteins: poorly-analyzed cousins of yeast ClpB machine

ClpB/Hsp100 proteins act as chaperones, mediating disaggregation of denatured proteins. Recent work shows that apart from cytoplasm, these proteins are localized to nuclei, chloroplasts, mitochondria and plasma membrane. While ClpB/Hsp100 genes are essentially stress-induced (mainly heat stress) in vegetative organs of the plant body, expression of ClpB/Hsp100 proteins is noted to be constitutive in plant reproductive structures like pollen grains, developing embryos, seeds etc. With global warming looming large on the horizon, ways to genetically engineer plants against high temperature stress are urgently needed. Yeast mutants unable to synthesize active ClpB/Hsp100 protein show a clear thermosensitive phenotype. ClpB/Hsp100 proteins are implicated in high temperature stress tolerance in plants. We herein highlight the selected important facets of this protein family in plants.     

Prokaryotic chaperones support yeast prions and thermotolerance and define disaggregation machinery interactions

Saccharomyces cerevisiae Hsp104 and Escherichia coli ClpB are Hsp100 family AAA+ chaperones that provide stress tolerance by cooperating with Hsp70 and Hsp40 to solubilize aggregated protein. Hsp104 also remodels amyloid in vitro and promotes propagation of amyloid prions in yeast, but ClpB does neither, leading to a view that Hsp104 evolved these activities. Although biochemical analyses identified disaggregation machinery components required for resolubilizing proteins, interactions among these components required for in vivo functions are not clearly defined. We express prokaryotic chaperones in yeast to address these issues and find ClpB supports both prion propagation and thermotolerance in yeast if it is modified to interact with yeast Hsp70 or if E. coli Hsp70 and its cognate nucleotide exchange factor (NEF) are present. Our findings show prion propagation and thermotolerance in yeast minimally require cooperation of species-specific Hsp100, Hsp70, and NEF with yeast Hsp40. The functions of this machinery in prion propagation were directed primarily by Hsp40 Sis1p, while thermotolerance relied mainly on Hsp40 Ydj1p. Our results define cooperative interactions among these components that are specific or interchangeable across life kingdoms and imply Hsp100 family disaggregases possess intrinsic amyloid remodeling activity.     

The ClpB/Hsp104 molecular chaperone-a protein disaggregating machine

ClpB and Hsp104 (ClpB/Hsp104) are essential proteins of the heat-shock response and belong to the class 1 family of Clp/Hsp100 AAA+ ATPases. Members of this family form large ring structures and contain two AAA+ modules, which consist of a RecA-like nucleotide-binding domain (NBD) and an alpha-helical domain. Furthermore, ClpB/Hsp104 has a longer middle region, the ClpB/Hsp104-linker, which is essential for chaperone activity. Unlike other Clp/Hsp100 proteins, however, ClpB/Hsp104 neither associates with a cellular protease nor directs the degradation of its substrate proteins. Rather, ClpB/Hsp104 is a bona fide molecular chaperone, which has the remarkable ability to rescue proteins from an aggregated state. The full recovery of these proteins requires the assistance of the cognate DnaK/Hsp70 chaperone system. The mechanism of this "bi-chaperone" network, however, remains elusive. Here we review the current understanding of the structure-function relationship of the ClpB/Hsp104 molecular chaperone and its role in protein disaggregation.     

The elusive middle domain of Hsp104 and ClpB: location and function

Hsp104 in yeast and ClpB in bacteria are homologous, hexameric AAA+ proteins and Hsp100 chaperones, which function in the stress response as ring-translocases that drive protein disaggregation and reactivation. Both Hsp104 and ClpB contain a distinctive coiled-coil middle domain (MD) inserted in the first AAA+ domain, which distinguishes them from other AAA+ proteins and Hsp100 family members. Here, we focus on recent developments concerning the location and function of the MD in these hexameric molecular machines, which remains an outstanding question. While the atomic structure of the hexameric assembly of Hsp104 and ClpB remains uncertain, recent advances have illuminated that the MD is critical for the intrinsic disaggregase activity of the hexamer and mediates key functional interactions with the Hsp70 chaperone system (Hsp70 and Hsp40) that empower protein disaggregation.     

Role of Hsp104 in the propagation and inheritance of the [Het-s] prion

The chaperones of the ClpB/HSP100 family play a central role in thermotolerance in bacteria, plants, and fungi by ensuring solubilization of heat-induced protein aggregates. In addition in yeast, Hsp104 was found to be required for prion propagation. Herein, we analyze the role of Podospora anserina Hsp104 (PaHsp104) in the formation and propagation of the [Het-s] prion. We show that DeltaPaHsp104 strains propagate [Het-s], making [Het-s] the first native fungal prion to be propagated in the absence of Hsp104. Nevertheless, we found that [Het-s]-propagon numbers, propagation rate, and spontaneous emergence are reduced in a DeltaPaHsp104 background. In addition, inactivation of PaHsp104 leads to severe meiotic instability of [Het-s] and abolishes its meiotic drive activity. Finally, we show that DeltaPaHSP104 strains are less susceptible than wild type to infection by exogenous recombinant HET-s(218-289) prion amyloids. Like [URE3] and [PIN(+)] in yeast but unlike [PSI(+)], [Het-s] is not cured by constitutive PaHsp104 overexpression. The observed effects of PaHsp104 inactivation are consistent with the described role of Hsp104 in prion aggregate shearing in yeast. However, Hsp104-dependency appears less stringent in P. anserina than in yeast; presumably because in Podospora prion propagation occurs in a syncitium.     

Structure and function of Hsp78, the mitochondrial ClpB homolog

The cellular role of Hsp100/Clp chaperones in maintaining protein stability is based on two functional aspects. Under normal growth conditions they represent components of cellular protein quality control machineries that selectively remove damaged or misfolded polypeptides in cooperation with specific proteases. After thermal stress, proteins of the ClpB subfamily have the unique ability to directly resolubilize aggregated polypeptides in concert with Hsp70-type chaperones, leading to the recovery of enzymatic activity. Hsp78, the homolog of the bacterial chaperone ClpB in mitochondria of eukaryotic organisms, participates in both protective activities. Hsp78 is involved in conferring thermotolerance to the mitochondrial compartment but also participates in protein degradation by the matrix protease Pim1. Despite the high sequence conservation between Hsp78 and ClpB, an analysis of the structural properties revealed significant differences. The identified mitochondrial Hsp78s do not contain N-terminal substrate-binding domains. In addition, formation of the oligomeric chaperone complex was more variable as anticipated from the studies with bacterial ClpB. Hsp78 predominantly formed a trimeric complex under in vivo conditions. Hence, mitochondrial Hsp78s form a distinct subgroup of the ClpB chaperone family, exhibiting specific structural and functional properties.     

Mapping the road to recovery: the ClpB/Hsp104 molecular chaperone

The AAA(+)-ATPases are a family of molecular motors which have been seconded into a plethora of cellular tasks. One subset, the Hsp100 molecular chaperones, are general protein remodellers that help to maintain the integrity of the cellular proteome by means of protein destruction or resurrection. In this review we focus on one family of Hsp100s, the homologous ClpB and Hsp104 molecular chaperones that convey thermotolerance by resolubilising and rescuing proteins from aggregates. We explore how the nucleotide binding and hydrolysis properties at the twelve nucleotide-binding domains of these hexameric rings are coupled to protein disaggregation, highlighting similarities and differences between ClpB and Hsp104.     

ClpB/Hsp100 proteins and heat stress tolerance in plants

High-temperature stress can disrupt cellular proteostasis, resulting in the accumulation of insoluble protein aggregates. For survival under stressful conditions, it is important for cells to maintain a pool of native soluble proteins by preventing and/or dissociating these aggregates. Chaperones such as GroEL/GroES (Hsp60/Hsp10) and DnaK/DnaJ/GrpE (Hsp70/Hsp40/nucleotide exchange factor) help cells minimize protein aggregation. Protein disaggregation is accomplished by chaperones belonging to the Caseinolytic Protease (Clp) family of proteins. ClpB/Hsp100 proteins are strikingly ubiquitous and are found in bacteria, yeast and multi-cellular plants. The expression of these proteins is regulated by heat stress (HS) and developmental cues. Bacteria and yeast contain one and two forms of ClpB proteins, respectively. Plants possess multiple forms of these proteins that are localized to different cellular compartments (i.e. cytoplasm/nucleus, chloroplast or mitochondria). Overwhelming evidence suggests that ClpB/Hsp100 proteins play decisive roles in cell adaptation to HS. Mutant bacteria and yeast cells lacking active ClpB/Hsp100 proteins are critically sensitive to high-temperature stress. Likewise, Arabidopsis, maize and rice mutants lacking cytoplasmic ClpB proteins are very sensitive to heat. In this study, we present the structural and functional attributes of plant ClpB forms.     

Connexin45 directly binds to ZO-1 and localizes to the tight junction region in epithelial MDCK cells

The molecular chaperone protein Hsp78, a member of the Clp/Hsp100 family localized in the mitochondria of Saccharomyces cerevisiae, is required for maintenance of mitochondrial functions under heat stress. To characterize the biochemical mechanisms of Hsp78 function, Hsp78 was purified to homogeneity and its role in the reactivation of chemically and heat-denatured substrate protein was analyzed in vitro. Hsp78 alone was not able to mediate reactivation of firefly luciferase. Rather, efficient refolding was dependent on the simultaneous presence of Hsp78 and the mitochondrial Hsp70 machinery, composed of Ssc1p/Mdj1p/Mge1p. Bacterial DnaK/DnaJ/GrpE, which cooperates with the Hsp78 homolog, ClpB in Escherichia coli, could not substitute for the mitochondrial Hsp70 system. However, efficient Hsp78-dependent refolding of luciferase was observed if DnaK was replaced by Ssc1p in these experiments, suggesting a specific functional interaction of both chaperone proteins. These findings establish the cooperation of Hsp78 with the Hsp70 machinery in the refolding of heat-inactivated proteins and demonstrate a conserved mode of action of ClpB homologs.