高温胁迫下绿豆象内参基因筛选及其Hsp70基因的表达特性

为明确Hsp70蛋白在绿豆象Callosobruchus chinensis高温耐受性中的作用,该研究筛选并克隆β-actin、α-tubulin、β-tubulin、EF1-α、GAPDH、Hsc70、AK和RPL40八个候选内参基因,采用实时荧光定量PCR(quantitative real-time PCR,qPCR)技术测定其在绿豆象雌雄成虫体内的表达量,通过geNorm、BestKeeperr和NormFinde软件对这8个候选内参基因的表达稳定性进行评价,选择最适宜的内参基因对不同高温胁迫下绿豆象体内Hsp70基因的表达特性进行测定。结果表明,8个候选基因引物均有良好的特异性和引物扩增效率,引物扩增效率介于91.6%～108.1%之间。高温胁迫下,β-tubulin基因较其他7个候选基因有较小的平均变异度、稳定值和标准差,为最适宜的内参基因。39、42和45℃高温处理1 h后绿豆象雌雄成虫体内Hsp70表达水平较对照显著上调,当处理时间延长至3 h后,Hsp70基因的相对表达量较对照也有不同程度上调,但差异不显著,表明绿豆象Hsp70基因可以响应高温胁迫,Hsp70蛋白在绿...     

三叶斑潜蝇热激蛋白Hsp90基因的克隆及其在高温胁迫下的表达分析

采用RT-PCR及RACE技术克隆了三叶斑潜蝇Hsp90基因全长cDNA序列,并用实时定量RT-PCR的方法检测其在不同发育阶段受到高温胁迫后的表达水平。该基因的cDNA序列全长2 408 bp,开放阅读框为2 145 bp,编码714个氨基酸;5′非编码区为151 bp,3′非编码区为112 bp。该基因推导的氨基酸序列与其他昆虫同源序列比较有很高的相似性（80%~99%）。聚类分析结果显示三叶斑潜蝇与美洲斑潜蝇和南美斑潜蝇的亲缘关系最近。实时荧光定量PCR检测结果表明三叶斑潜蝇Hsp90基因的表达受到热胁迫的诱导,诱导3龄幼虫最大表达量的温度比诱导其他发育阶段的温度低,在43 ℃时预蛹和蛹的表达量在整个生命周期中最高,在检测的高温胁迫条件下,雄虫比雌虫的表达量更高。该结果为阐明三叶斑潜蝇胁迫耐受能力及其对其他潜蝇种群的取代机制奠定了分子基础。     

目录

为了表达牛环形泰勒虫（Theileria annulata）截短HSP70基因编码蛋白并研究该蛋白的结构与功能特性,本研究扩增牛环形泰勒虫HSP70目的基因并构建重组质粒pMD18-T-HSP70,选取其他种的同源HSP70蛋白序列构建系统进化树;利用生物信息学方法分析HSP70基因编码蛋白的氨基酸组成、基本理化性质、亲疏水性、跨膜区结构、信号肽、可能的磷酸化位点、亚细胞定位及蛋白的二级结构和三级结构;对重组蛋白HSP70进行蛋白互作网络分析;构建原核表达载体pET28a-HSP70,筛选诱导表达条件,镍柱纯化重组蛋白及检测反应原性。结果显示,牛环形泰勒虫HSP70蛋白序列与小泰勒虫的序列同源性较高,蛋白分子质量为42 ku,理论等电点（pI）为5.61,属于酸性亲水性蛋白,无跨膜区及信号肽;蛋白功能预测结果显示,HSP70包含32个可能的磷酸化位点,亚细胞定位分析显示该蛋白主要分布于细胞质。蛋白质二级结构中α-螺旋、β-转角、无规则卷曲、延伸链分别占39.18%、8.51%、30.41%和21.91%。蛋白互作网络构建结果显示,与HSP70相互作用的蛋白主要为HSP90家族成员,另外还有伴侣蛋白GrpE同系物,预示着HSP70可能在细胞内与HSP90形成复合体发挥作用。本试验成功构建原核表达载体,获得了大小约为48 ku的融合蛋白,以0.6 mmol/L IPTG于37 ℃诱导5 h,蛋白表达较好;点状印迹及Western blotting结果表明,表达产物可被自然感染的牛环形泰勒虫阳性血清识别,具有良好的反应原性。本试验结果为进一步探讨牛环形泰勒虫HSP70功能机制提供了理论依据。     

Extracellular heat shock protein 70 levels in tumour-bearing dogs and cats treated with radiation therapy and hyperthermia

Hyperthermia is a form of a cancer treatment which is frequently applied in combination with radiotherapy (RT) to improve therapy responses and radiosensitivity. The mode of action of hyperthermia is multifactorial; the one hand by altering the amount of the blood circulation in the treated tissue, on the other hand by modulating molecular pathways involved in cell survival processes and immunogenic interactions. One of the most dominant proteins induced by hyperthermia is the major stress-inducible heat shock protein 70 (Hsp70). Hsp70 can be found in the blood either as a free-protein (free HSP70) derived from necrotic cells, or lipid-bound (liposomal Hsp70) when it is actively released in extracellular vesicles (EVs) by living cells. The aim of the study was to evaluate the levels of free and liposomal Hsp70 before and after treatment with RT alone or hyperthermia combined with radiotherapy (HTRT) in dogs and cats to evaluate therapy responses. Peripheral blood was collected from feline and canine patients before and at 2, 4, 6 and 24 h after treatment with RT or HTRT. Hsp70 enzyme-linked immunosorbent assays (ELISAs) were performed to determine the free and liposomal Hsp70 concentrations in the serum. The levels were analysed after the first fraction of radiation to study immediate effects and after all applied fractions to study cumulative effects. The levels of free and liposomal Hsp70 levels in the circulation were not affected by the first singular treatment and cumulative effects of RT in cats however, after finalizing all treatment cycles with HTRT free and liposomal Hsp70 levels significantly increased. In dogs, HTRT, but not treatment with RT alone, significantly affected liposomal Hsp70 levels during the first fraction. Free Hsp70 levels were significantly increased after RT, but not HTRT, during the first fraction in dogs. In dogs, on the other hand, RT alone resulted in a significant increase in liposomal Hsp70, but HTRT did not significantly affect the liposomal Hsp70 when cumulative effects were analysed. Free Hsp70 was significantly induced in dogs after both, RT and HTRT when cumulative effects were analysed. RT and HTRT treatments differentially affect the levels of free and liposomal Hsp70 in dogs and cats. Both forms of Hsp70 could potentially be further investigated as potential liquid biopsy markers to study responses to RT and HTRT treatment in companion animals.     

中国杧果科学研究70年

新中国成立70年来,我国杧果科学研究在人才培养与团队建设、硬件平台、基础理论创新与技术研发等方面均取得了重要进展,有力支撑了我国杧果产业的创建和可持续发展。本文在概述世界和我国杧果地位、分布、规模的基础上,从科学研究发展历程、种质资源与遗传育种、土壤与肥水管理、耕作模式与花果管理、病虫害防控、采后贮运保鲜、产业经济等方面全面梳理了我国杧果研究取得的成就,分析了存在的不足并提出了未来的重点发展方向。     

Chronic otitis externa with heat shock protein 70-positive intranuclear inclusion bodies in the ceruminous gland epithelium of a Chihuahua dog

A Chihuahua dog showed persistent itching in the right ear canal. Anti-inflammatory medicines and prednisolone were ineffective and total ear canal ablation was performed. Histological diagnosis was chronic otitis externa. Eosinophilic intranuclear inclusion bodies (Cowdry type A and full-type) were occasionally observed in the ceruminous gland epithelium. The inclusion bodies were negative for nucleic acid and ultrastructurally composed of fibrous structures (approximately 10 nm in width). Viral infection was initially suspected, but polymelase chain reaction tests did not detect the expected viral genes. Immunohistochemistry revealed that the inclusion bodies were positive for heat shock protein 70 (HSP70), suggesting that these bodies could be protein aggregates including HSP70. The etiology of this lesion has not been elucidated, but chronic inflammation may influence the cytoplasm-to-nuclear transportation of HSP70. To the best of our knowledge, this is the first report of canine chronic otitis externa with HSP70-positive intranuclear inclusion bodies.     

The effects of starving and feeding on Dover sole (Solea solea,Soleidae, Linnaeus, 1758) stress response and early larval development

In the view of an urgent necessity to improve the quantity and the quality of farmed fish species, there is a strong need to improve our basic knowledge on the effects of first feeding during the developmental stages of fish larvae. High mortality, mainly due to food deprivation or inappropriate food quality, has been observed in many larval fish species, but knowledge about the morphological, biochemical and molecular processes related to this topic is still poorly understood. The understanding of the early larval ontogeny as well as the larval nutritional requirements and the molecular and cellular mechanisms elicited by fish larvae during food deprivation and starvation are thus of primary importance. At this regard, this study investigates, in Dover sole larvae, the effects of starvation and starving/re‐feeding procedures at a morphological, histological, biochemical and molecular level. The results evidenced that starved larvae progressively decrease in growth, lipid content, affected their gastrointestinal tract and muscle development and increased cortisol and heat shock protein 70 levels. On the contrary, starved and re‐fed larvae showed, after the restoration of a favourable feeding condition, a compensatory growth. In conclusion, this is the first study analysing through a multidisciplinary approach the effects of food deprivation on the development of an important economic species, the Dover sole.     

Heat shock protein 70 expression in different tissues of Cirrhinus mrigala (Ham.) following heat stress

Members of heat shock protein 70 (HSP70) are highly conserved proteins of about 70 kDa and play important roles in protein folding. Levels of these proteins increase when cells are under stress. Environmental temperature influences both the basal and induced levels of HSPs. However, studies on HSPs in fishes from a tropical country such as India are lacking. In the present study, Indian major carp (IMC) Cirrhinus mrigala (Ham.) acclimatized at 25±2°C had high levels of HSP70, viz., 1.2–1.3 ng μg^?1 total protein in kidney and gill and 4.2–5.3 ng μg^?1 total protein in liver and brain tissues, indicating the presence of biochemically significant levels of stress. However, maintenance of acclimatized fish at 17°C for up to 48 h did not lead to a significant decrease in stress protein levels. A heat shock at 37°C for up to 48 h resulted in only two to threefold increase in HSP70 levels in these organs. Although the increase in HSP70 levels was apparent from the first hour of heat stress in all these tissues, the increase was significant from the second hour in the brain, the sixth hour in liver and kidney and the 20th hour in the gills.     

Induced thermotolerance and expression of heat shock protein 70 in sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>

ABSTRACT:   Thermal limits, induced thermotolerance and the expression of heat shock protein 70 (Hsp70) in an echinoderm Apostichopus japonicus were studied. The sublethal and lethal temperatures for the juveniles were 30 and 34°C, respectively; a previous sublethal heat shock exposure (30°C, 2 h) could increase the survival rates of the sea cucumbers when they were exposed to 34°C. This induced thermotolerance could last for at least 2 days. Levels of Hsp70 increased substantially after sublethal heat shock exposure and linearly decreased with time. This result indicated that a close relationship existed between the induction of thermotolerance and the levels of Hsp70 in A. japonicus.