热应激和吡虫啉对大豆蚜hsp70和hsc70基因mRNA表达的影响

【目的】昆虫在高温或农药的胁迫下,通过高效表达热休克蛋白(HSP)等建立应激自我保护机制。本研究为从转录组水平上认识大豆蚜Aphis glycines在热应激和吡虫啉胁迫下hsp70和hsc70 mRNA表达分子机制,进而寻找自我保护应激反应中的薄弱环节,为大豆蚜的生物防治提供理论基础。【方法】采用同源克隆、RACE技术和实时荧光定量PCR等方法研究不同热激时间和热激后不同恢复时间及不同吡虫啉浓度对大豆蚜4龄若虫hsp70和hsc70的表达影响。【结果】37℃热激后,大豆蚜4龄若虫中hsp70表达量先上调,1 h时升至对照组的10.36倍（P<0.05）,然后逐渐下降。同样热激后恢复时间的长短对大豆蚜若蚜中hsp70的表达具有显著影响。热激处理后,大豆蚜若蚜中hsp70立即大量表达,表达量为对照组的8.78倍（P<0.05）,随后表达量下降至对照组水平,而hsc70的表达量并没有显著变化（P>0.05）。大豆蚜若蚜受吡虫啉的胁迫时,其hsp70和hsc70的表达量受吡虫啉的浓度及胁迫的时间的影响,呈现先升高后下降的趋势,具有明显的短期效应。【结论】吡虫啉诱导大豆蚜hsp70和hsc70表达量的上调;而热胁迫对hsp70和hsc70 mRNA具有不同的表达模式,高温可以诱导hsp70的表达,但对hsc70没有明显的诱导作用。     

Selective postmortem degradation of inducible heat shock protein 70 (hsp70) mRNAs in rat Brain

Summary 1. Altered mRNA levels in postmortem brain tissue from persons with Alzheimer's disease (AD) or other neurological diseases are usually presumed to be characteristic of the disease state, even though both agonal state (the physiological state immediately premortem) and postmortem interval (PMI) (the time between death and harvesting the tissue) have the potential to affect levels of mRNAs measured in postmortem tissue. Although the possible effect of postmortem interval on mRNA levels has been more carefully evaluated than that of agonal state, many studies assume that all mRNAs have similar rates of degradation postmortem.2. To determine the postmortem stability of inducible heat shock protein 70 (hsp70) mRNAs, themselves unstablein vivo at normal body temperature, rats were heat shocked in order to induce synthesis of the hsp70 mRNAs. hsp70 mRNA levels in cerebellum and cortex were then compared to those of their heat shock cognate 70 (hsc70) mRNAs, as well as to levels of 18S rRNAs, at 0 and at 24 hr postmortem.3. Quantiation of northern blots after hybridization with an hsp70 mRNA-specific oligo probe indicated a massive loss of hsp70 mRNA signal in RNAs isolated from 24-hr postmortem brains; quantitation by slot-blot hybridization was 5- to 15-fold more efficient. Even using the latter technique, hsp70 mRNA levels were reduced by 59% in 24-hr-postmortem cerebellum and by 78% in cortex compared to mRNA levels in the same region of 0-hr-postmortem brain. There was little reduction postmortem in levels of the hsp70 mRNAs or of 18S rRNAs in either brain region.4.In situ hybridization analysis indicated that hsp70 mRNAs were less abundant in all major classes of cerebellar cells after 24 hr postmortem and mRNAs had degraded severalfold more rapidly in neurons than in glia. There was no corresponding loss of intracellular 18S rRNA in any cell type.5. We conclude from these results that the effect of postmortem interval on mRNA degradation must be carefully evaluated when analyzing levels of inducible hsp70 mRNAs, and perhaps other short-lived mRNAs, in human brain.     

Cloning of heat shock protein genes from the brown planthopper,Nilaparvata lugens,and the small brown planthopper,Laodelphax striatellus,and their expression in relation to thermal stress

Three heat shock protein (HSP) genes (hsp70, hsc70, hsp90) were partially cloned from the brown planthopper Nilaparvata lugens and the small brown planthopper Laodelphax striatellus (Homoptera: Delphacidae), which are serious pests of the rice plant. Sequence comparisons at the deduced amino acid level showed that the three HSPs of planthoppers were most homologous to corresponding HSPs of dipteran and lepi‐dopteran species. Identities of both heat shock cognate 70 and HSP90 were higher than HSP70 in both species. Identity of the HSP70 between the two planthopper species was only 81%, a value much lower than seen among fly and moth groups. Effects of heat and cold shocks were demonstrated on expression of the three hsp genes in the two planthopper species. Heat shock (40 °C) upregulated the hsp90 level but did not change the hsc70 level in either the nymph and adult stages of either species. On the other hand, the hsp70 level was only upregulated in L. striatellus. This heat shock response was prompt and lasted only for 1 h after treatment. In contrast, cold shock at 4°C did not change the expression levels of any hsp in either species.     

Expression and localization of Drosophila melanogaster hsp70 cognate proteins.

Monoclonal antibodies have been used to identify three proteins in Drosophila melanogaster that share antigenic determinants with the major heat shock proteins hsp70 and hsp68. While two of the proteins are major proteins at all developmental stages, one heat shock cognate protein, hsc70, is especially enriched in embryos. hsc70 is shown to be the product of a previously identified gene, Hsc4. We have examined the levels of hsp70-related proteins in adult flies and larvae during heat shock and recovery. At maximal induction in vivo, hsp70 and hsp68 never reach the basal levels of the major heat shock cognate proteins. Monoclonal antibodies to hsc70 have been used to localize it to a meshwork of cytoplasmic fibers that are heavily concentrated around the nucleus.     

Stress-induced, tissue-specific enrichment of hsp70 mRNA accumulation in Xenopus laevis embryos

In this study, we have employed whole-mount, in situ hybridization to study the spatial pattern of hsc70 and hsp70 mRNA accumulation in normal and heat shocked embryos during Xenopus laevis development. Our findings revealed that hsc70 mRNA was constitutively present in a global fashion throughout the embryo and was not heat inducible. Accumulation of hsp70 mRNA, however, was detected only in heat shocked embryos. Furthermore, hsp70 mRNA accumulation was enriched in a tissue-specific manner in X. laevis tailbud embryos within 15 minutes of a 33 degrees C heat shock. Abundant levels of heat shock-induced hsp70 mRNA were detected in the head region, including the lens placode, the cement gland, and in the somitic region and proctodeum. Preferential heat-induced accumulation of hsp70 mRNA was first detected at a heat shock temperature of 30 degrees C. Placement of embryos at 22 degrees C after a 1-hour, 33 degrees C heat shock resulted in decreased hsp70 mRNA with time, but the message persisted in selected tissues, including the lens placode and somites. Treatment of tailbud embryos with either sodium arsenite or zinc chloride induced a tissue-specific enrichment of hsp70 mRNA in the lens placode and somitic region. These studies reveal the complex nature of the heat shock response in different embryonic tissues and suggest the presence of regulatory mechanisms that lead to a stressor-induced, tissue-specific enrichment of hsp70 mRNA.     

Cloning and nucleotide sequencing of three heat shock protein genes (hsp90, hsc70, and hsp19.5) from the diamondback moth, Plutella xylostella (L.) and their expression in relation to developmental stage and temperature

Heat shock protein genes, hsp90, hsc70, and hsp19.5, were cloned and sequenced from the diamondback moth, Plutella xylostella (L.) by RT-PCR and RACE method. The cDNA sequence analysis of hsp90 and hsp19.5 revealed open reading frames (ORFs) of 2,151 and 522 bp in length, which encode proteins with calculated molecular weights of 82.4 and 19.5 kDa, respectively. Analysis of cDNA from hsc70 revealed an ORF of 1,878 bp coding a protein with a calculated molecular weight of 69.3 kDa. Furthermore, the analysis of genomic DNA from hsc70 confirmed the presence of introns while no introns were apparent in hsp90 and hsp19.5. Southern blot analysis suggested the presence of multiple copies of each gene family in the DBM genome. Detectable expression of hsp19.5 was observed at the pupal stage while expression of hsp90 and hsc70 was detected at both pupal and adult stages. At adult stage, females showed a higher expression of hsp90 and hsc70 than males. An increased expression was observed in all three genes after exposure to a high temperature in both sexes. These results suggest that in addition to a heat shock response, these HSP genes might be involved in other functions during the course of development in DBM.     

hsp47 and hsp70 gene expression is differentially regulated in a stress- and tissue-specific manner in zebrafish embryos

We have examined differences in the spatial and temporal regulation of stress-induced hsp47 and hsp70 gene expression following exposure of zebrafish embryos to heat shock or ethanol. Using Northern blot analysis, we found that levels of hsp47 and hsp70 mRNA were dramatically elevated during heat shock in 2-day-old embryos. In contrast, ethanol exposure resulted in strong upregulation of the hsp47 gene whereas hsp70 mRNA levels increased only slightly following the same treatment. Whole-mount in situ hybridization analysis revealed that hsp47 mRNA was expressed predominantly in precartilagenous cells, as well as several other connective tissue cell populations within the embryo following exposure to either stress. hsp70 mRNA displayed a very different cell-specific distribution. For example, neither stress induced hsp70 mRNA accumulation in precartilagenous cells. However, high levels of hsp70 mRNA were detectable in epithelial cells of the developing epidermis following exposure to heat shock, but not to ethanol. These cells did not express the hsp47 gene following exposure to either of these stresses. The results suggest the presence of different inducible regulatory mechanisms for these genes which operate in a cell- and stress-specific manner in zebrafish embryos. Dev. Genet. 21:123–133, 1997. © 1997 Wiley-Liss, Inc.     

盐芥热激同源蛋白70基因(Thhsc70)的分子克隆及鉴定

热激蛋白70(hsp70s)具有分子伴侣的功能,其中在非胁迫条件下表达的hsp70s称为热激同源蛋白70(hsc70).为更好地了解hsc70基因的特性,鉴定了盐芥(Thellungiella halophila(C.A.Mey.)O.E. Schulz)中编码胞质hsc70蛋白的基因Thhsc70.实验结果表明:在非胁迫条件下,Thhsc70基因具有组织特异性表达;Thhsc70基因受热胁迫和冷胁迫的诱导表达,但几乎不受盐诱导和干旱诱导.Thhsc70基因在拟南芥中过量表达后提高了转基因拟南芥的热耐受性和冷耐受性.     

盐芥热激同源蛋白70基因(Thhsc70)的分子克隆及鉴定(英文)

热激蛋白70(hsp70s)具有分子伴侣的功能,其中在非胁迫条件下表达的hsp70s称为热激同源蛋白70(hsc70)。为更好地了解hsc70基因的特性,鉴定了盐芥(Thellungiella halophila(C. A. Mey. )O. E. Schulz)中编码胞质hsc70蛋白的基因Thhsc70。实验结果表明:在非胁迫条件下,Thhsc70基因具有组织特异性表达;Thhsc70基因受热胁迫和冷胁迫的诱导表达,但几乎不受盐诱导和干旱诱导。Thhsc70基因在拟南芥中过量表达后提高了转基因拟南芥的热耐受性和冷耐受性。     

An intron-containing,heat-inducible stress-70 gene in the millipede Tachypodoiulus niger (Julidae,Diplopoda)

The highly conserved part of the nucleotide-binding domain of the hsp70 gene family was amplified from the soil diplopod Tachypodoiulus niger (Julidae, Diplopoda). Genomic DNA yielded 701, 549 and 540 bp sequences, whereas cDNA from heat shocked animals produced only one distinct fragment of 543 bp. The sequences could be classified as a 70 kDa heat shock protein (hsp70), the corresponding 70 kDa heat shock cognate (hsc70) and a glucose-related hsp70 homologue (grp78). Comparisons of genomic and cDNA sequences of hsc70 identified two introns within the consensus sequence. Generally, stress-70 expression levels were low, which hampered successful RT-PCR and subsequent subcloning. Following experimental heat shock, however, the spliced hsc70 was amplified predominantly, instead of its inducible homologue hsp70. This finding suggests that microevolution in this soil-dwelling arthropod is directed towards low constitutive stress-70 levels and that the capacity for stress-70 induction presumably is limited. hsc70, albeit having introns, apparently is inducible and contributes to the stress-70 response.     

Cryptic Expression of the 70-kDa Heat Shock Protein,hsp72, in Gerbil Hippocampus after Transient Ischemia

The 70 kDa heat shock protein, hsp72, is known to be induced following transient global ischemia in brain, as detected by immunocytochemistry and in situ hybridization techniques. However, while hsp72 mRNA is expressed rapidly following postischemic recirculation, immunocytochemistry fails to detect hsp72 protein for many hours after such insults, even in cell populations that readily express Fos and other proteins encoded by ischemia-induced mRNAs. In the present study, hsp72 expression in gerbil hippocampus was compared by immunocytochemistry and immunoblot methods at several intervals following 10 min ischemia. As established in previous studies, hsp72 immunoreactivity remained undetectable in postischemic neurons at 6 h following such insults. In contrast, immunoblots of dissected gerbil hippocampus demonstrated nearly maximal accumulation of hsp72 at this time point. These results indicate that the protein is present, but cryptic to detection in perfusion-fixed sections, during early recirculation. The constitutively expressed heat shock cognate protein, hsc70, did not show significant changes in level or distribution by either method, except for a decrease in CA1 staining at 48 h. These results confirm that hsp72 rapidly accumulates to high levels in postischemic hippocampus, and suggest that further studies of its subcellular localization during this interval may offer insight into its functional role as a component of the stress response in neurons after such insults.