Water and carbohydrate levels at different developmental stages and dynamics in hibernating pupae of Pieris melete (Lepidoptera: Pieridae)

For insight into the physiological indicators of diapause in Pieris melete, water and carbohydrate (glycogen and trehalose) levels were measured under both natural and laboratory conditions. The highest water content (3.71–3.79 mg/mg dry weight) was found in larvae and developing pupae, which was substantially higher than in diapausing pupae (2.59 mg/mg dry weight). Water content was almost stable during diapause, except for individuals approaching diapause termination (3.43–3.58 mg/mg dry weight). The total carbohydrate level was significantly higher in pre‐pupae (47.41 μg/mg) compared to larvae (22.80 μg/mg) and developing pupae (21.48 μg/mg). The highest level of trehalose was detected in winter diapausing pupae, and no trehalose was found in larvae or developing pupae. Levels of glycogen were highest in pre‐pupae and lowest in diapausing pupae. Levels of total carbohydrate decreased as diapause proceeded, and no significant changes were found in trehalose levels for diapausing pupae under natural conditions or treated for 60–90 days at 5°C. Pupae treated at 20°C for 60–90 days had significantly lower levels of trehalose than those treated for 30 days. Glycogen content was relatively stable at 5°C, but increased after treatment under natural conditions and 20°C for more than 60 days. These results suggest that the dynamics of water and carbohydrate levels are potential physiological diapause indicators, which show metabolic differences between trehalose and glycogen during diapause development.     

The role of Hsp90 in cell response to hyperthermia

(1) Preincubation of SKOV3 human ovarian carcinoma cells with a non-toxic dose of Geldanamycin resulted in exacerbation of hyperthermia-induced cytotoxicity and re-distribution of dying cells toward necrosis. (2) Exposure of primary human ovarian carcinoma cells to mild hyperthermia (42 °C for 2 h) led, after a recovery period of 16 h, to several-fold increase in the levels of Hsp90 and ErbB2, to a moderate decrease in the levels of phospho-Erk1/2, whereas the level of β-catenin appeared to be unchanged. (3) The inhibitors of Hsp90 (Geldanamycin and Novobiocin) significantly affected the cell signaling of heat-pretreated cultures. (4) The results suggest that Hsp90 plays a pivotal role in cell response to hyperthermia. (5) A combination of Hsp90 inhibitors with hyperthermia may considerably increase the efficacy of thermotherapy.     

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。     

Cloning and expression profiles of tumor suppressor QM homologue in response to granulovirus in Pieris rapae

The tumor suppressor, QM, has been cloned and characterized from various model organisms such as human, plant and invertebrates. Yet, it has not been seriously investigated for its role in conjunction with antiviral mechanisms involving innate insect immunity. From the expressed sequence tag (ESTs) project, conducted with larval cDNA library of cabbage butterfly, Pieris rapae, a partial fragment (718 bp) of QM homologue, termed PrQM containing 660 bp long open reading frame (ORF) encoding protein of 219 amino acids was identified. In silico analysis of PrQM ORF revealed the presence of ribosomal protein L10a/L10e type domain. Phylogenetic analysis of the P. rapae QM‐like protein showed high amino acid sequence similarity with other PrQM polypeptides identified from Heliothis virescenes (95%), Plutella rapae (92%), Bombyx mori (92%), Drosophila melanogaster (89%), and Polyrhachis vicina (85%). The butterfly QM has the closest phylogenetic relationship to a moth (Hv) QM homologue. Further investigations revealed the expression of PrQM at all developmental stages, with pronounced presence at the egg stage. In addition, spatial pattern analysis indicated its high expression in the head, salivary gland, integument and fat body with visible presence in Malpighian tubule and gut. Time course expression studies conducted after immune‐challenge with lipoteichoic acid (LTA) showed the induction of PrQM mRNA at 12 h and 24 h after challenge and also in response to granulovirus (GV). Results of this investigation therefore suggest possible role of QM‐like proteins from Pieris rapae to be involved in innate antiviral immune responses. Further elucidation on the precise function of PrQM during antiviral immune responses by using RNA interference remains a viable research front.     

Molecular cloning and expression patterns of FK506‐binding protein 12, an immunophilin from the cabbage butterfly,Pieris rapae

FK506‐binding protein (FK506BP) class belonging to immunophilin protein family has been known to play key roles in modulating T‐cell activation, regulation of cell cycle and protein folding. However, little is known about the involvement of FK506BP during viral pathogenesis in insect host. In this study, an attempt has been made to focus on the involvement of FK506BP in antiviral innate immunity, by cloning the full‐length cDNA of FK506BP12 (PrFK506BP12) from the cabbage butterfly, Pieris rapae. It comprised of 532 bp (excluding poly‐A tail) with a longest open reading frame (ORF) of 327 bp encoding 108 amino acids. In silico analysis of PrFK506BP12 ORF revealed a highly conserved FK506‐binding domain (FKBD). As expected, it showed high homology to other FK506BPs identified from Bombyx mori (92%), Manduca sexta (91%), Suberites domuncula (82%), Tribolium castaneum (81%) and Aedes aegypti (74%) . Expression of PrFK506BP12 was observed during developmental stages of P. rapae, but was pronounced in late pupal and adult stage. In addition, spatial expression pattern analysis indicated its high expression in the head and fat body. Furthermore, PrFK506BP12 mRNA was induced 12 h after LTA, Poly I:C treatment and 3h after Pieris rapae granulovirus (PrGV) treatment in carcass. It suggests that PrFK506BP12 appears to be involved in immune responses and also play an important role in the fat body, although it remains to be clarified about their precise role in response to granulovirus.     

Molecular characterization of DnaJ 5 homologs in silkworm Bombyx mori and its expression during egg diapause

A comparison of the cDNA sequences （1 056 bp） of Bombyx mori DnaJ 5 homolog with B. mori genome revealed that unlike in other Hsps, it has an intron of 234 bp. The DnaJ 5 homolog contains 351 amino acids, of which 70 contain the conserved DnaJ domain at the N-terminal end. This homolog orB. mori has all desirable functional domains similar to other insects, and the 13 different DnaJ homologs identified in B. mori genome were distributed on different chromosomes. The expressed sequence tag database analysis of Hsp40 gene expression revealed higher expression in wing disc followed by diapause-induced eggs. Microarray analysis revealed higher expression of DnaJ 5 homolog at 18th h after oviposition in diapause-induced eggs. Further validation of DnaJ 5 expression through qPCR in diapause-induced and nondiapause eggs at different time intervals revealed higher expression in diapause eggs at 18 and 24 h after oviposition, which coincided with the expression of Hsp70 as the Hsp 40 is its co-chaperone. This study thus provides an outline of the genome organization of lisp40 gene, and its role in egg diapause induction in B. mori.     

朱砂叶螨HSP90基因克隆及原核表达

采用RT-PCR及RACE技术克隆朱砂叶螨Tetranychus cinnabarinus的热激蛋白90(HSP90)基因, 并进行序列分析, 得到一条长2 595 bp的cDNA序列, 该序列开放阅读框（open reading frame, ORF）为2 169 bp, 编码722个氨基酸, 分子量约为83.45 kDa, 理论等电点为4.81, 3′非编码区（untranslated region, UTR）为249 bp, 5′UTR为177 bp。通过Antheprot分析发现5个HSP90家族的签名序列及胞质HSP90特征序列MEEVD。同源性分析表明, 朱砂叶螨HSP90编码区核苷酸序列和其他已知的HSP90, 尤其是节肢动物昆虫的HSP90, 具有很高的相似性。将鉴定正确的原核重组表达质粒pET43a-TcHSP90, 转化大肠杆菌Escherichia coli BL21(origami) 进行原核表达, 应用SDS-PAGE和Western blotting技术分离并检测融合蛋白, 结果表明构建的原核表达质粒可以在宿主菌中稳定、正确表达。朱砂叶螨TcHSP90基因的克隆、原核表达, 为进一步研究HSP90的性质和功能的研究提供有用的实验材料。     

菜粉蝶丝氨酸蛋白酶基因Pr-SP1的克隆及其表达谱分析

昆虫主要依靠先天免疫反应来抵御外源异物的入侵, 而与血淋巴黑化及抗菌肽合成等过程密切相关的丝氨酸蛋白酶激活级联反应在其中起着重要作用。为阐明丝氨酸蛋白酶在菜粉蝶Pieris rapae免疫中的作用, 本文通过简并引物RT-PCR克隆获得了菜粉蝶丝氨酸蛋白酶家族基因Pr-SP1的cDNA片段, 并利用RACE法扩增获得其全序列。该cDNA序列长1 489 bp, 其中开放阅读框长1 059 bp, 共编码353个氨基酸残基。Pr-SP1含一长度为20个氨基酸残基的信号肽序列, 其蛋白理论分子量为36.85 kDa, 理论等电点为6.41。多序列比对结果表明, Pr-SP1与其他昆虫的同源蛋白基因序列上存在较高一致性, 在N端有一个发夹结构域, 而C端是一个具有催化活性的结构域。实时荧光定量RT-PCR及免疫印迹结果表明, 蛹期Pr-SP1主要在颗粒血细胞内进行转录, 其蛋白产物主要定位在血浆; Pr-SP1在不同虫态及虫龄都有转录, 其蛋白产物在不同虫态及虫龄都有表达, 其中5龄幼虫最高, 卵期最低; Pr-SP1的转录水平及其蛋白产物的表达水平均会被大肠杆菌Escherichia coli、 藤黄微球菌Micrococcus luteus和巴斯德毕赤酵母Pichia pastoris诱导。根据这些结果分析认为, Pr-SP1属于Spätzle蛋白酶前体激活酶, 并参与菜粉蝶的先天免疫反应。     

Evolution of Hsp90 expression in Tetrahymena thermophila (Protozoa, Ciliata) populations exposed to thermally variable environments

Evolutionary consequences of thermally varying environments were studied in the ciliated protozoan Tetrahymena thermophila. Replicated lines were propagated for 60 days, a maximum of 500 generations, in stable, slowly fluctuating (red spectrum), and rapidly fluctuating (blue spectrum) temperatures. The red and blue fluctuations had a dominant period length of 15 days and two hours, respectively. The mean temperature of all time series was 25 degrees C and the fluctuating temperatures had the same minimum (10 degrees C), maximum (40 degrees C), and variance. During the experiment, population sizes and biomasses were monitored at three-day intervals. After the experiment, carrying capacity and maximum growth rate were measured at low (15 degrees C), intermediate (25 degrees C), and high (35 degrees C) temperatures for each experimental line. Physiological changes in the lines were assessed by measuring the expression of stress-induced heat shock protein Hsp90 at 25 degrees C, 35 degrees C, and 39 degrees C. Population sizes and biomasses showed no differences between stable, blue, or red temperature treatments during the experiment. Also, after the experiment, mean carrying capacities and maximum growth rates were comparable in the stable, blue, and red temperature treatments. The expression of Hsp90 was higher in lines from the blue environment than in lines from the stable environment. Lines from the red environment had an intermediate level of Hsp90 expression. This supports the hypothesis that inducible thermotolerance and expression of canalizing genes can evolve in response to rapidly varying environments. Furthermore, we found correlative evidence of benefits and disadvantages of high Hsp90 expression. Lines with high expression of Hsp90 had an increased growth rate at the highest temperature when food resources were not limiting growth. At low and intermediate temperatures the same lines had the lowest carrying capacities.     

苹果蠹蛾热激蛋白Hsp90基因的克隆及热胁迫下的表达分析

世界检疫性害虫苹果蠹蛾Cydia pomonella是一种温度耐受可塑性很高的物种。本研究针对温度波动可能导致其耐热性增强的科学问题, 采用生测法鉴定了苹果蠹蛾实验种群的高温耐受阈值, 采用同源克隆、 RACE和实时荧光定量PCR (RT-qPCR)等方法研究了苹果蠹蛾热激蛋白Hsp90基因的应激表达对耐热性的重要作用。高温耐受阈值研究结果表明, 苹果蠹蛾实验种群的死亡率随温度的升高和时间的延长显著性升高, 1-5龄幼虫分别经50℃和52℃高温处理2, 5和10 min后, 3龄幼虫耐热性最差, 5龄幼虫最强。50℃和52℃分别处理10 min和5 min均可导致1-4龄幼虫全部死亡, 而5龄幼虫在这两种处理下仍有25.0%和11.1%的存活率。以35℃处理的5龄雌幼虫为材料克隆苹果蠹蛾Hsp90基因全长cDNA, 结果显示该基因全长为2 470 bp, 完整开放阅读框为2 148 bp, 共编码716个氨基酸, 预测分子量为82.07 kDa, 命名为Cphsp90 (GenBank登录号JN624775)。该基因编码的氨基酸序列与亚洲玉米螟Ostrinia furnacalis和甘蓝夜蛾Mamestra brassicae等昆虫的Hsp90的氨基酸序列一致性高达96%, 表明了Hsp90家族的保守特性。Cphsp90 mRNA的相对表达量在32~44℃高温胁迫下随温度的升高而显著增高, 证实Cphsp90是诱导型热激基因, 且mRNA相对表达量与胁迫程度正相关。Cphsp90基因的表达还具有组织特异性, 35℃处理幼虫的表皮中Cphsp90相对表达量显著高于血淋巴、 脂肪体和中肠, 应激响应最为活跃。与未经温热预处理的昆虫相比, 35℃温热预处理3 h后的5龄幼虫在40, 45和50℃更高的温度胁迫下, Cphsp90 mRNA达到最高表达量所需要的胁迫温度有所提升, 由未经预热处理的40℃处理10 min提高到45℃处理10 min, 这与温热预处理会增强5龄幼虫耐热性的现象相符, 表明Cphsp90基因的响应表达在苹果蠹蛾耐热性及其可塑性过程中发挥重要的作用。     

Molecular analysis of Hsp70 mechanisms in plants and their function in response to stress

Studying the strategies of improving abiotic stress tolerance is quite imperative and research under this field will increase our understanding of response mechanisms to abiotic stress such as heat. The Hsp70 is an essential regulator of protein having the tendency to maintain internal cell stability like proper folding protein and breakdown of unfolded proteins. Hsp70 holds together protein substrates to help in movement, regulation, and prevent aggregation under physical and or chemical pressure. However, this review reports the molecular mechanism of heat shock protein 70 kDa (Hsp70) action and its structural and functional analysis, research progress on the interaction of Hsp70 with other proteins and their interaction mechanisms as well as the involvement of Hsp70 in abiotic stress responses as an adaptive defense mechanism.     

葱蝇热激蛋白DaHSP23基因的克隆及在冬滞育和夏滞育蛹中的表达分析

[目的]低分子量(12 ～43 kDa)热激蛋白(sHSPs)具有抗逆应答的功能,滞育是昆虫抵抗不良环境的特殊发育形式,但sHSPs在昆虫滞育发育过程中的作用仍不清楚.本研究克隆和特征化葱蝇Delia antiqua sHSP基因,并研究它在夏滞育和冬滞育发育过程中的表达模式,为阐明sHSPs在滞育发育上的功能奠定基础.[方法]通过RACE-PCR方法克隆了葱蝇HSP23基因,通过相似性比较分析了其特征、结构域及与双翅目代表性同源基因的系统发育关系;采用实时荧光定量PCR研究了该基因在葱蝇冬滞育蛹和夏滞育蛹发育过程中的表达情况,通过表达的差异比较揭示了该基因与滞育发育的关系.[结果]克隆出了葱蝇HSP23基因,命名为DaHSP23(GenBank登录号:HQ392521.1),其cDNA全长序列为904 bp,编码186个氨基酸,推测蛋白分子量为20.9 kDa,等电点为6.42.该基因的编码蛋白与其他双翅目昆虫的sHSPs有超过66％的氨基酸序列一致性,与已报道的其他双翅目昆虫的滞育相关HSP23基因同源.基因组测序显示该基因无内含子.DaHSP23基因在葱蝇非滞育蛹的发育过程中一直保持在较低的水平,各发育阶段间的表达量不存在显著差异.但在冬滞育和夏滞育蛹中,该基因从滞育起始期开始逐渐显著升高表达,到滞育维持期的中后期达到峰值,在滞育终止期逐渐降到较低的水平.[结论]DaHSP23基因在葱蝇冬滞育和夏滞育发育过程中明显上调表达,但存在差异,它在滞育期的调控可能是种专化的.DaHSP23可能在葱蝇两种类型的滞育上起重要作用.     

Molecular cloning and characterization of novel heat shock protein 90 gene from a wild <Emphasis Type="Italic">Vitis pseduoreticulata</Emphasis> native to China

Heat shock protein 90 (Hsp90), known as molecular chaperone, is involved in protein folding and assembly in the cell. In the present study, a full-length cDNA named Vitis pseduoreticulta heat shock protein 90 (VpHsp90) (GenBank accession Number:EU239815), encoding a heat shock protein 90, was obtained by degenerated primers and 3′-and 5′-RACE from Vitis pseudoreticulata according to our previously obtained EST sequence (GenBnak accession number:DV182112), putatively known as Hsp90. Comparison of VpHsp90 sequence has revealed that an open reading frame (ORF) consists of 2,100 bp nucleotides and the translated proteins of 699 amino acid residues. The molecular mass of VpHsp90 calculated from the deduced amino acid sequence was 80.2 kDa, Isolectric Point was 4.893, which is in close proximity of Hsp90. The maximum similarity of VpHsp90 at nucleotides level (85%) and protein level (96%) was found to be with Nicotiana tabacum. Phylogenetic tree analysis at both the nucleotides and amino acids levels indicates that Vitis pseduoreticulata, Nicotiana tabacum, and Arabidopsis thaliana Hsp90 sequences comprise one clade, which is closely related to Oryza sativa, Hordeum vulgare and Triticum aestivum Hsp90s. It may be reasonably concluded that VpHsp90 possesses the ancestral gene of Hsp90 similar to that of higher plant species.     

Tissue-Specific Induction of Hsp90 mRNA and Plasma Cortisol Response in Chinook Salmon following Heat Shock, Seawater Challenge, and Handling Challenge

In studying the whole-body response of chinook salmon (Oncorhynchus tshawytscha) to various stressors, we found that 5-hour exposure to elevated temperature (mean 21.6°C; + 10.6°C over ambient) induced a marked increase in Hsp90 messenger RNA accumulation in heart, brain, gill, muscle, liver, kidney, and tail fin tissues. The most vital tissues (heart, brain, gill, and muscle) showed the greatest Hsp90-mRNA response, with heart tissue increasing approximately 35-fold. Heat shock induced no increase in plasma cortisol. In contrast, a standard handling challenge induced high plasma cortisol levels, but no elevation in Hsp90 mRNA in any tissue, clearly separating the physiological and cellular stress responses. We saw no increase either in tissue Hsp90 mRNA levels or in plasma cortisol concentrations after exposing the fish to seawater overnight. Received October 1, 1999; accepted January 21, 2000