The mRNA Expression Profiles of Five Heat Shock Protein Genes from Frankliniella occidentalis at Different Stages and Their Responses to Temperatures and Insecticides

The westem flower thrips, Frankliniella occidental＆ （Pergande） is a highly invasive pest that is able to exploit many crops across a wide range of environmental conditions. Five full-length cDNAs of heat shock protein （HSP） genes （Fo-HSP90, Fo-HSP70, Fo-HSP60, Fo-HSP40 and Fo-HSP28.9） were cloned from F. occidentalis, and their expression profiles were investigated under conditions of thermal stress and insecticide exposure, and at different stages during development, using real-time quantitative PCR. All five gene sequences showed high similarity to homologs in other species, indicating the conserved fimction of this gene family. HSP60 represents an informative phylogenetic marker at the ordinal taxonomic level within Insecta, but HSP90, which has two homologous copies in Hymenoptera, was not informative. The expression of Fo-HSPs under thermal stress suggests that Fo-HSP90, Fo-HSP70, and Fo-HSP28.9 are inducible by both cold and heat stress, Fo-HSP40 is only heat-inducible, and Fo-HSP60 is thermally insensitive. There were two patterns of cold induction of Fo-HSPs： one is from 0 to 4℃ and the other is around -8℃. All five Fo-HSPs genes were induced by exposure to sublethal concentrations of the insecticide avermectin. The expression of the five Fo-HSPs during different developmental stages suggests that they all play a role in development of F. occidentalis.     

Effect of RNA Interference Hsp72 Gene Expression on Development of Mouse Preimplantation Embryos

The method of RNAi was used to inhibit the expression of induced heat shock protein 70 （Hsp72） in the 4-cell stage mouse embryos and the embryo development competence was analyzed to identify the functions of Hsp72 on embryonic heat resistance. The results indicated that the inhibition rates of siRNA1 for Hsp72 mRNA and Hsp72 protein were 87.1 and 78.5%, respectively. The blastocysts development rates were 41, 86, and 84% for the siRNA1 group, the LipofectamineTM 2 000 exposed group, and the 37℃ group, respectively, and the hatched blastocysts development rates for the above three groups were 35, 72, and 68%, respectively. The data suggest that the siRNAI has a significant inhibiting effect on Hsp72 gene, and Hsp72 gene silence reduces the blastocysts development rate and hatched blastocysts rate after heat shock during the development of mouse preimplantation embryos.     

热应激条件下小鼠睾丸组织HSP70基因表达谱的研究

本试验建立了不同温度、不同时间热应激实验小鼠动物模型,采用Real-time PCR和Western-blot方法检测了小鼠睾丸组织热应激蛋白70(HSP70)在mRNA和蛋白质水平的表达规律。结果表明:25℃时,HSP70在mR-NA和蛋白质表达水平没有变化;在31℃、34℃和37℃时,随应激时间的延长,HSP70mRNA和蛋白质的表达均呈现先升高后下降的趋势;40℃时,HSP70mRNA和蛋白质始终持续高表达。结论:小鼠受热应激后,睾丸组织HSP70mRNA和蛋白质均随着温度和时间的增加呈规律性变化,本研究为探索HSP70对精液品质的影响奠定了基础。     

Inhibition of HSP70 Gene Expression by Modified Antisense and Its Effects on Embryonic Sensitivity to Heat Shock

Experiments were performed to evaluate the efficiency of inhibition of HSP70 gene expression by antisense oligonucleotides complementary to the mRNA of HSP70 and to test the effects of inhibition of HSP70 gene expression on subsequent embryonic sensitivity to heat shock. The results showed that transfection of pre-implantation embryos at 4-cell stage with 5 μM antisense oligo had no effect on in vitro blastocyst development. However, transfection with 10 to 40 μM antisense oligo had reduced in vitro blastocyst development to 15, 10% and 0; For the embryos which exposed to 40 μM As arrested at the 16-cell stage, there was no blastocyst formation within the heat shock groups. In contrast, transfection had no effect on embryonic sensitivity to heat shock, above 25% of embryos developed to blastocyst stage in control groups.     

运输应激猪骨骼肌中热应激蛋白HSP70和HSP90家族的表达

利用Western blot技术，检测长途运输试验猪骨骼肌（背最长肌和臀长肌）中分属于HSP70家族和HSP90家族的4种应激蛋白（HSP70，HSP72，HSP86和HSP90）的表达，所有运输应激猪和对照猪肌肉组织 中均检测到了上述4种HSP，对照猪组织中HSP的表达说明HSP除了在受应激 的细胞内行使生理功能外，还具有独立于应激刺激应答以外的其它作用，6h长途运输后，HSP的表达量明显不同，HSP70和HSP72在肌肉组织中的表达虽然有一定的增加，但统计学分析差异不显（P＞0．05），而HSP86和HSP90在骨髓肌中的表达明显下降，尤以HSP90下降最显（P＜0．01），提示HSP90家族成员与肉品品质相关，可能作为判应激损伤的的指征。     

热休克对小鼠胚胎成纤维细胞CaM mRNA和HSF活性的影响

试验分别以38、40和42℃对原代培养的小鼠胚胎成纤维细胞进行热休克处理,采用半定量PCR方法和凝胶迁移阻滞(EMSA)对CaM mRNA和HSF活性进行检测,并与对照组(37℃)比较,以确定热休克对小鼠胚胎成纤维细胞CaM mRNA和HSF活性的影响。结果表明,38℃热休克处理组小鼠胚胎成纤维细胞的CaMmRNA表达...     

春小麦经热锻炼后热休克蛋白表达及对产量的影响

热休克蛋白(heat shock proteins,HSP)是细胞或生物体受到热胁迫后新合成的一类遗传上高度保守的蛋白,在干旱条件下对小麦的热休克蛋白70(HSP70)表达进行了初步的探讨.发现作物经过热锻炼(Heat Acclimation)后,热休克70的基因和蛋白表达均显著升高,其作物抗逆性有望明显提高,即通过热锻炼可以提高组织的抗逆性,进而提高农作物在干旱条件下种植时的产量.     

Characterization of T-complex polypeptide 1 (TCP-1) from the Chilo suppressalis HSP60 family and its expression in response to temperature stress

Many proteins require assistance from molecular chaperones at various stages to attain correctly folded states and functional conformations during protein synthesis. In this study, the gene encoding T-complex polypeptide 1(TCP-1), which belongs to the heat shock protein 60(HSP60) family, was isolated and characterized from the rice stem borer, Chilo suppressalis, by RACE and q PCR, respectively. The full-length c DNA of Tcp-1 was 2 144 bp and encoded a 1 635-bp ORF; the deduced translational product contained 545 amino acids with 5′-and 3′-UTRs and an isoelectric point of 5.29. Cluster analysis confirmed that the deduced amino acid sequence shared high identity(60–99%) with TCP-1 from other insects. To investigate Tcp-1 expression in response to abiotic stress, q PCR was used to analyze expression levels of Tcp-1 m RNA in C. suppressalis larvae exposed to temperatures ranging from –11 to 43°C. With respect to heat shock, Tcp-1 expression was higher than the control after a 2-h exposure to 30 and 36°C and declined at 39 and 43°C. Difference in Tcp-1 expression was observed at temperatures ranging from –11 to 27°C. q PCR analyses revealed that Tcp-1 expression was the highest in hindgut tissue as compared to heads, epidermis, fat body, foregut, midgut, and malpighian tubules. Our results indicated that Tcp-1 expression was differentially expressed in C. suppressalis tissues, and was impacted by temperature stress.     

A Heat Shock-Switchable Gene Expression System

The expression of β-D-glueuronidase (Gus) gene under the control of soybean heat shock protein(HSP) promoter Gmhsp17. 5-C was investigated in transgenic Nicotiana plumbaginifolia plants after heatshock treatment. Results of histochemical stain assay showed that the Gus activity increased markedly duringthe recovery phase (21℃) after heat shock treatment at 42℃ for 2 h. Results of quantitative fluorometric as-says indicated that maximal Gus activity after heat shock treatment could reach 5 - 6 times higher than that be-fore heat shock treatment in transgenic lines HG6 and HG9. Based on several advantages of the heat shock pro-moter such as easy to induce, sensitivity to heat shock and high inductive activity, it can be potentially used tocontrol expression of gene of interest in transgenic plants.     

Expression and Purification of Goose HSP70 and Compound Formation with Virus Polypeptide

The experiment was conducted to study the specific expression of HSP70 caused by heat shock, HSP70 purification and the characteristics of coalescence with antigenic peptide in the formation of the complex. Sixty healthy 6-week-old male Wulong geese were selected and randomly divided into three groups. The control group was slaughtered without heat treatment. Treatment group 1 was shocked with an acute heat treatment at （42 ± 1）℃ for 5 h before they were immediately slaughtered. Treatment group 2 was kept for 12 h after the heat treatment under normal conditions in order to recover and was then slaughtered. Cardiac tissue was taken in order to make paraffin sections for the immunohistochemistry experiment and the liver tissue was used to purify HSP70. The geese heart HSP70 expression differences in the three groups were determined and at the same time the experiments of HSP70 purification and appraisal in the liver tissue were carried on. HSP70 purification and synthesis of HBV PreS1 multi-peptides unified the complex, which was determined by bi-specific antibody enzyme-linked immune sandwich assay. The results indicated that widespread HSP70 positive pellets in the cardiac muscle were found under hot shock conditions. HSP70 expression in the treatment group 1 was centered in the karyotheca and its periphery, but in treatment group 2, it was centered in the surrounding cell membrane. The HSP70 purification could be obtained through two sets of purification plans; both the synthesis peptide and the HSP70 purification form the complex under certain conditions. The double antibody sandwich ELISA technique was applied to detect if the complex had been formed. Positive results showed that the complex was formed. The specific expression of HSP70 under heat shock shifted with time, suggesting that HSP70 possibly had some function in cell protection. High-purity HSP70 protein can be obtained under low-pressure chromatography conditions, and in comparison with each other, it was better in the flow of the molecular     

Characterization of two novel heat shock protein 70s and their transcriptional expression patterns in response to thermal stress in adult of Frankliniella occidentalis (Thysanoptera: Thripidae)

Heat shock protein 70(HSP70) is one of the most important members in the heat shock protein family, and plays important roles in the thermotolerance of insect. To explore the molecular mechanism of thermotolerance of Frankliniella occidentalis adults, the difference in the expression of HSP70s in F. occidentalis male or female adults under the thermal stress was studied under the laboratory conditions. Two full length c DNAs of HSP70s gene(Fohsc704 and Fohsc705) were cloned from F. occidentalis by using RT-PCR and RACE. The genomic sequence was demonstrated by genomic validation, and the position and size of the intron were analyzed by sequence analysis of c DNA. Real-time PCR was used to analyze the HSP70 expression patterns. The c DNA of Fohsc704 and Fohsc705 possessed 2 073 and 1 476 bp which encoded 690 and 491 amino acids(aa) with a calculated molecular weight of 75 and 54 k Da, respectively. Four introns in Fohsc704 and six introns in Fohsc705 protein were found. However, the HSP70 protein sequences in our study were ended with EKKN and GIFL, which were different from the reported Fo HSP70s. Various expression patterns of Fohsc704 and Fohsc705 were found in both genders of F. occidentalis under thermal stress. The expression of Fohsc704 and Fohsc705 reached to the highest level at –12 and –8°C in male adults, respectively, and Fohsc705 expressed the highest level at 33°C in female adults. In conclusion, HSP70s of F. occidentalis in our study are novel heat shock proteins. There were difference in expression patterns of the two hsc70s in genders of F. occidentalis, and the two HSP70s play important roles in the thermotolerance of F. occidentalis.     

Artificial induction of androgenetic diploid in Yellow catfish （Pelteobagrus fulvidraco）

Androgenesis was the especial zoogamy that the germ plasma of offspring was from the agnate.In this study the eggs of Yellow catfish(Pelteobagrus fulvidraco)were irradiated by UV suspending in the synthetic ovarian fluid(OF),and the total dosage of UV irradiation was 220 mJ·cm~(-2).Diploid could be induced by heat shock(40℃,2 min,3 min)with different time period after fertilization(15-37 min).The result showed that heat shock with 2 min was better than 3 min;there were two apices of induction in 17-21 min and 27-31 min after fertilization.The highest hatching rate was 3.30% at 29 min after fertilization,and the difference between two apices of induction was unobvious.     

蚯蚓提取液对家蚕热激蛋白水平及免疫功能的影响

为探讨蚯蚓提取液对家蚕热激蛋白的表达及免疫功能的影响,家蚕5龄幼虫饲喂蚯蚓提取液(50、100和200倍原液稀释)处理的桑叶,饲喂6 d后解剖获取家蚕中肠及血淋巴组织,利用实时荧光定量PCR检测中肠组织中HSP70和HSP90基因的表达;利用ELISA试剂盒检测中肠和血淋巴中HSP70、HSP90、溶菌酶(lysozyme)和抗菌肽(attacin)的水平。结果表明:1/100和1/200稀释组家蚕中肠组织中HSP70基因表达分别是对照组的2.97和1.86倍(P<0.05),1/100稀释组家蚕中肠组织中HSP90基因表达是对照组的2.2倍(P<0.05);1/50稀释组中肠HSP70和血淋巴抗菌肽显著高于对照组(P<0.05);各处理组中肠和血淋巴中HSP90差异不显著(P>0.05)。由此可见,桑叶中添食蚯蚓提取液可促进热激蛋白的表达,提高抗菌肽的水平,进而提高蚕体的抗性和免疫功能。     

热激处理和施用脱落酸对大田棉花生长的效应

在高温干旱条件下,研究了热激萌动种子(42℃、2h)、脱落酸(ABA)浸种、二叶期叶面喷施和根部灌施对棉花生长的效应.结果表明:①在高温干旱期间,与对照相比,除叶面喷施处理,其他处理都合成了一组差异蛋白质.ABA浸种和根部灌施比热激处理合成的热激蛋白(HSP)更丰富.②ABA浸种处理显著提高了棉株SOD、CAT、POD的活性;热激处理显著提高了棉株SOD、CAT的活性;根部灌施处理显著提高棉株CAT的活性;叶面喷施处理显著提高棉株SOD的活性.③ABA浸种、根部灌施和热激处理的棉株MDA含量显著高于对照,而叶面喷施处理棉株MDA含量与对照差异不显著.     

金鱼HSC70和HSP40基因克隆及其原核表达

[目的]克隆金鱼热激同源蛋白70(HSC70)和热休克蛋白(HSP40)基因,构建原核表达载体并通过IPTG诱导表达获得目的蛋白,明确HSC70和HSP40蛋白的生物学功能,为揭示金鱼的高温应答机制打下基础.[方法]提取金鱼鳃组织总RNA,利用RT-PCR扩增金鱼HSC70和HSP40基因,将目的基因片段克隆至线性空表达载体pET-32a上构建原核表达载体pET-32a-HSC70和pET-32a-HSP40,转化大肠杆菌Rosetta表达菌株后用IPTG进行诱导表达,并以SDS-PAGE和Western blotting检测分析表达获得的融合蛋白.[结果]金鱼HSC70基因CDS序列全长1950 bp,编码650个氨基酸;金鱼HSP40基因CDS序列全长996 bp,编码332个氨基酸.金鱼HSC70氨基酸序列与斑马鱼、人类、家鼠、金线鲃和草鱼的相似度分别为96.28%、95.50%、95.35%、98.15%和98.61%;HSP40氨基酸序列与斑马鱼的完全一致(相似度100.00%),与普通鲤鱼、金线鲃、人类和家鼠的相似度均为94.12%.原核诱导表达获得的融合蛋白Trx-HSC70和Trx-HSP40为可溶性表达,其大小约91.5和55.0 kD,均能与Anti-Trx抗体发生特异性反应.[结论]HSC70和HSP40蛋白在鱼类及哺乳动物中高度保守,经原核诱导表达获得的金鱼融合蛋白Trx-HSC70和Trx-HSP40为可溶性表达,且携带有Trx标签,能与Anti-Trx抗体发生特异性反应,可作为互作蛋白用于RNA pull down试验.     

Cd(Ⅱ)、Cu(Ⅱ)胁迫对双齿围沙蚕热休克蛋白70(HSP70)基因表达的影响

为研究重金属浓度的变化对双齿围沙蚕Perinereis aibuhitensis热休克蛋白70(heat shock proteins 70,HSP70)基因的表达变化,采用实时荧光定量PCR方法,分析了重金属Cu(Ⅱ)、Cd(Ⅱ)单一诱导及联合作用下双齿围沙蚕HSP70基因的表达规律。结果表明:Cu(Ⅱ)单独作用时,沙蚕HSP70 mRNA表达量随时间的增加而增大,在诱导的第3天时不同浓度组沙蚕HSP70 mRNA表达量依次为22.86μg/L浓度组44.50μg/L浓度组4.45μg/L浓度组,而在诱导的第7天和第14天时HSP70 mRNA表达量与Cu(Ⅱ)浓度呈正相关;Cd(Ⅱ)单独作用时,沙蚕HSP70 mRNA表达量则随Cd(Ⅱ)浓度和诱导时间的增加而增大,但较Cu(Ⅱ)单独作用下变化趋势平缓;Cu(Ⅱ)和Cd(Ⅱ)联合作用下,沙蚕HSP70 mRNA表达量亦与暴露浓度和时间呈正相关,在诱导的第3天和第7天时45.70μg/L Cu(Ⅱ)+10μg/L Cd(Ⅱ)浓度组HSP70 mRNA表达量高于Cu(Ⅱ)和Cd(Ⅱ)单一作用,表明Cu(Ⅱ)和Cd(Ⅱ)存在一定的协同作用。     

Genes encoding heat shock proteins in the endoparasitoid wasp,Cotesia chilonis,and their expression in response to temperatures

Five genes encoding heat shock proteins (HSPs), Cchsp40, Cchsp60, Cchsp70, Cchsc70 and Cchsp90, were cloned and sequenced from Cotesia chilonis using RT-PCR and RACE. The cDNA sequences of Cchsp40, Cchsp60, Cchsp70, Cchsc70 and Cchsp90 were 1 265, 2 551, 2 094, 2 297 and 2 635 bp in length, respectively, with a molecular weight (MW) of 39.1, 60.6, 71.45, 70.19 and 82.92 kDa, respectively. The predicted amino acid sequences of these proteins showed high similarities with published HSPs of other insects in Hymenoptera. Analysis of genomic DNAs indicated that Cchsp40, Cchsp60, Cchsp70 and Cchsp90 lacked introns, but Cchsc70 contained an intron. The results also suggested that CcHSP40 in C. chilonis was the Type II HSP40, CcHSP60 was a member of the mitochondrial HSP60 family, and CcHSP90 was a part of cytoplasmic HSP90A family. Expression patterns varied in the five Cchsps in response to temperature. Expression of Cchsp40 and Cchsp60 was induced significantly by cold but not heat stress. Cchsp70 and Cchsc70 showed similar response to the thermal stress and could be induced by both cold and heat, but their expression levels were consistently lower than that of Cchsp40 and Cchsp60. Cchsp90 could be induced by heat stress and mild cold, but not cold stress. In addition, the results demonstrated Cchsc70 might be constitutive and inducible protein that was expressed during normal cell functioning and also up-regulated in response to stressful stimuli while Cchsp70 was solely inducible protein induced by temperature changes. Overall, results generated from this study could significantly advance the understanding of Cchsps in response to temperature and provide important biological information for C. chilonis insects that reared under different temperatures.     

植物热激蛋白研究进展

热激蛋白是一类在有机体受到高温等逆境刺激后大量表达的蛋白, 是植物对逆境胁迫短期适应的必需组成成分, 对减轻逆境胁迫引起的伤害有很大的作用 有机体在受到逆境胁迫后, 体内变性蛋白急剧增加, 热激蛋白可以与变性蛋白结合, 维持它们的可溶状态, 在有Mg2+和ATP的存在下使解折叠的蛋白质重新折叠成有活性的构象 文中主要综述和讨论植物热激蛋白的产生与类型、存在与定位, 论述了热激反应的特点、基因表达调控及其功能的研究进展, 并提出热激蛋白与树木耐旱性关系的研究展望     

公牛HSP基因多态性与其精液品质及后裔性能的相关分析

以142头公牛为研究对象,以HSP为耐热性状的候选基因,分别扩增125和233 bp 2个片段,采用PCR-SSCP方法检测HSP基因多态性,并对PCR产物测序和序列分析。结果表明:HSPA8存在T3358C转换突变,该突变使缬氨酸(Val)变为异亮氨酸(Ile),产生2种基因型AA和AB,频率分别为0.577 5和0.422 5;HSPA1A发现A6201C和T6202A颠换突变,其中T6202A突变使苯丙氨酸(Phe)变为异亮氨酸(Ile),产生了5种基因型AA、AB、BB、AC和BC,频率分别为0.345 1、0.493 0、0.084 5、0.035 2和0.042 2。应用SPSS17.0软件采用最小二乘拟合线性模型,分析基因座位不同标记基因型与后裔测定性能和精液品质的相关性,结果表明:1)在HSPA8,AA基因型个体产量育种值显著高于AB型(P<0.05);在HSPA1A,BB基因型个体产量育种值显著高于AC型(P<0.05),AB基因型个体蛋白育种值显著高于AC型(P<0.05)。2)在HSPA8,精子密度、精子活力和顶体完整率AA均显著高于AB(P<0.05),而精子畸形率AA显著低于AB(P...