家蚕hsp20.4启动子克隆及其驱动表达产物EGT对家蚕蛹体发育的影响

为验证家蚕Bombyx mori热休克蛋白基因hsp20.4启动子的活性以及家蚕核多角体病毒egt的表达产物对家蚕发育的影响, 本实验通过PCR扩增分别得到hsp20.4启动子片段和egt片段。利用hsp20.4的启动子和红色荧光蛋白报告基因DsRed构建重组载体, 在家蚕BmN细胞以及家蚕组织中得到了瞬时表达, 表明所克隆的hsp20.4启动子序列具有热休克蛋白基因的启动子活性。又利用hsp20.4启动子和家蚕核多角体病毒的egt构建重组载体, 通过注射到蚕蛹中进行瞬时表达, 以检测egt表达产物对家蚕发育的影响, 经42℃ 1 h热诱导后, hsp20.4启动子控制的egt表达产物可以延迟家蚕发育。     

SHR和WKY大鼠主动脉hsp70mRNA水平的比较研究

本工作应用热激蛋白７０ＫＤ（ｈｓｐ７０）核酸分子杂交方法,检测了：１．自发性高血压（ＳＨＲ）主动脉和离体培养的主动脉平滑肌细胞受热激后ｈｓｐ７０ｍＲＮＡ水平的变化;２．不同细胞培养时间（３个月与６周）的ＳＨＲ、ＷＫＹ主动脉ｈｓｐ７０ｍＲＮＡ水平。结果提示ＳＨＲ主动脉ｈｓｐ７０ ｍＲＮＡ水平高;６周的ＳＨＲ细胞较同期和３个月的ＷＫＹ细胞ｈｓｐ７０ｍＲＮＡ高。推论ＳＨＲ血管平滑肌细胞对热敏感,原癌基因     

紫茎泽兰hsp90和hsp17.66基因启动子的克隆及其序列分析

以入侵植物紫茎泽兰(Ageratina adenophora Sprengel)为材料,采用基于PCR方法的染色体步行和改良的热不对称交错PCR(thermal asymmetric interlaced PCR,TAIL-PCR)两种方法分别克隆到hsp90和hsp17.66基因的5'上游启动子序列,长度分别为864 bp和1 485 bp(GenBank登录号分别为FJ434253和FJ434252).测序结果表明:这两个启动子序列均具有hsp启动子特有的HSE元件,及其他一些启动子顺式作用元件,如TATA-box,CAAT-box等.     

稀有鮈鲫HSP70基因启动子的克隆及功能分析

热休克蛋白70(HSP70)作为一种分子伴侣,在环境毒理学中受到广泛研究。前期研究表明稀有鮈鲫HSP70基因(GrHSP70)表达量与五氯酚(pentachlorophenol, PCP)处理的浓度和时间在肝脏中呈现剂量/时间-依赖效应。为探究启动子在热休克蛋白70表达调控中的作用,根据已知的GrHSP70 cDNA序列,采用染色体步移技术克隆了GrHSP70的5'侧翼区的核苷酸序列。生物信息学分析从预测的转录起始位点(C)起的5'侧翼区域共1 487bp,潜在的转录因子结合位点包括雌激素响应元件(ERE)、Sp1结合位点(Sp1)、糖皮质激素响应元件(GRE)、TATA结合蛋白(TBP)、CCAAT/增强子蛋白结合位点(C/EBP)、Oct-1结合位点(Oct-1)、GATA转录因子结合位点(GATA-1)等。实验构建了含有启动子缺失片段的萤火虫萤光素酶(firefly luciferase)和海肾萤光素酶(renilla luciferase)报告基因表达载体,瞬时转染HeLa细胞后,利用双荧光活性检测确定获得的GrHSP70启动子具有启动活性,其核心启动位点位于转录起始点上游-1 487~-1 093bp。同时,用不同浓度PCP暴露成功转染了重组质粒(pGL-HSP70 promoter-Luc+)的HeLa细胞,培养24h后检测双荧光活性,与对照相比,随PCP浓度的增加,荧光活性均显著增加。说明在稀有鮈鲫肝脏中PCP会通过激活GrHSP70启动子来诱导GrHSP70表达,但PCP在稀有鮈鲫体内通过何种机制来调节HSP70的合成,仍然需要进一步研究。     

家蚕中肠特异启动子P56的克隆及活性分析

中肠是家蚕的消化器官,也是抵御外界病源入侵的生理屏障。为克隆和鉴定新的家蚕中肠特异启动子,首先利用RT-PCR检测家蚕组织特异表达候选基因Bm P56的表达特性,发现该基因只在中肠组织表达。进一步克隆该基因上游调控序列P56,构建由该序列驱动红色荧光蛋白基因DsRed表达的转基因载体p Bac[P56DsRed SV40,3×P3EGFP],经显微注射和荧光筛选获得转基因家蚕。表达分析显示,报告基因DsRed只在转基因家蚕中肠组织表达,与Bm P56的表达特征一致,说明克隆的上游调控序列P56是有活性的家蚕中肠特异启动子。     

SHR和WKY大鼠主动脉hsp 70mRNA水平的比较研究

本工作应用热激蛋白70KD(hsp 70)核酸分子杂交方法,检测了:1.自发性高血压(SHR)主动脉和离体培养的主动脉平滑肌细胞受热激后hsp 70mRNA水平的变化;2.不同细胞培养时间(3个月与6周)的SHR、WKY主动脉hsp 70mRNA水平。结果提示SHR主动脉hsp mRNA水平增加,SHR细胞培养受热激(42℃,15min)后2h,hsp 70mRNA水平明显高于WKY鼠者,6周较3个月的SHR细胞hsp 70mRNA水平高;6周的SHR细胞较同期和3个月的WKY细胞hsp 70mRNA高。推论SHR血管平滑肌细胞对热敏感,原癌基因c-myc和抗癌基因p~(53)可能参与hsp 70表达调控,并共同参与SHR细胞增殖的调节。     

琥珀蚕孵化酶基因AaHE的克隆及启动子活性分析

[目的]琥珀蚕Antheroea assama具有典型的野蚕特征,蚕卵孵化不齐,严重影响琥珀蚕的室内规模化饲养.本研究旨在探究对琥珀蚕卵孵化起关键作用的孵化酶(hatching enzyme)基因及其启动子序列特征,为进一步选择合适的抑制剂或促进剂调节琥珀蚕卵的孵化奠定基础.[方法]采用RACE技术克隆琥珀蚕孵化酶基因...     

烯啶虫胺、 毒死蜱和高效氯氰菊酯对烟粉虱地中海隐种hsp70 mRNA的诱导表达

为深入理解杀虫剂胁迫对烟粉虱Bemisia tabaci适应逆境能力的影响, 本研究运用实时荧光定量PCR技术测定了LC₂₅, LC₅₀和LC₇₅ 3种浓度的烯啶虫胺、 毒死蜱和高效氯氰菊酯分别处理对烟粉虱地中海隐种B. tabaci Mediterranean成虫体内热激蛋白hsp70表达水平的影响。结果表明： 在低温（15℃）条件下, LC₅₀和LC₇₅的烯啶虫胺、 毒死蜱、 高效氯氰菊酯可显著诱导烟粉虱地中海隐种hsp70的表达量增加, 此后随时间延长hsp70表达量逐渐下降, 到72 h时恢复到对照水平, 但LC₂₅的3种杀虫剂对hsp70的表达无明显影响; 在常温（25℃）下, 较高浓度（LC₅₀和LC₇₅）的烯啶虫胺、 毒死蜱和高效氯氰菊酯处理24 h同样可促进hsp70表达量的增加, 然后hsp70表达量逐渐下降, 到72 h时恢复至正常水平, 但低浓度(LC₂₅)的3种药剂处理后hsp70表达量随时间延长而增加, 到72 h时达到最高; 在高温（32℃）条件下, LC₂₅和LC₅₀的烯啶虫胺、 毒死蜱和高效氯氰菊酯处理24 h可显著增强hsp70的表达水平, 此后随时间的延长hsp70表达恢复至正常水平。杀虫药剂诱导的hsp70表达量增加增强了烟粉虱地中海隐种对杀虫药剂和高温的耐受能力, 这可能是导致其在我国快速扩张的原因之一。     

嗜热四膜虫五个hsp70基因的表达分析

鉴定得到嗜热四膜虫13个含有完整保守结构域的hsp70基因,对其中5个高度相似且无内含子的hsp70基因进行表达分析。在37、39和41℃热激条件下,实时荧光定量PCR结果表明,hsp70-2基因对热激响应最敏感。在四膜虫生长、饥饿和接合生殖这3种生理或发育状态下,Microarray结果显示,hsp70-4基因恒定且高表达;在热激条件下,hsp70-4基因的表达水平随着温度的升高而略微增加,证实hsp70-4基因为热休克相关蛋白hsc70基因;克隆的hsp70-4基因全长2208bp,开放阅读框长1959bp,编码653个氨基酸。Microarray结果提示,hsp70-3可能参与四膜虫饥饿早期(0～12h)的耐受和接合生殖后期(6～10h)的新大小核形成,老大核凋亡等事件;hsp70-5可能参与四膜虫饥饿晚期(12～15h)的耐受和接合生殖早期(0～6h)的小核减数分裂、小核交换和原核(pronuclear)融合事件。Blast2GO分析表明,与hsp70-3和hsp70-5共表达的基因分别参与不同的生物学过程,进一步反映了hsp70-3和hsp70-5这两个基因在功能上是存在差异的。     

家蚕丝素重链启动子驱动DsRed的瞬时分泌表达

根据家蚕丝蛋白基因的启动子活性高、丝蛋白具有高效分泌的特性,克隆了家蚕丝素重链基因(Fib-H)启动子及其下游的信号肽序列(FibHS),将DsRed基因与信号肽序列融合构建了分泌型瞬时表达载体;转染细胞实验显示,该载体能在家蚕BmN细胞中瞬时表达DsRed;家蚕注射载体后,可在丝腺腔中检测到红色荧光,表明瞬时表达的DsRed分泌到丝腺腔,推测所克隆的序列具有信号肽的功能。此外,本研究为家蚕丝腺生物反应器分泌表达外源基因的研究奠定了基础。     

Utilization of the Bombyx mori heat shock protein 70 promoter for screening transgenic silkworms

Silkworm transgenesis is now a routine method leading to a satisfactory yield of transformed animals and the reliable expression of transgenes during multiple successive generations. However, the screening of G1 transgenic individuals from numerous progeny has proved to be difficult and time‐consuming work. Previously, we characterized the promoter of heat shock protein 70 from Bombyx mori (bHsp70), which is ubiquitously expressed in all tissues and developmental stages. To investigate the utilization of the bHsp70 promoter to screen transgenic individuals, the EGFP marker gene was inserted into the piggyBac vector under the control of the bHsp70 promoter. Mixtures of the donor and helper vectors were micro‐injected into 3060 eggs of bivoltine silkworms (Keomokjam). EGFP fluorescence was observed in 17 broods of transgenic silkworms under a florescence stereomicroscope. Interestingly, this fluorescent marker protein was detected, not only in parts of the embryo segments on the seventh day of the G1 embryonic developmental stage, but also in a part of the body of G1 hatched larvae, in the middle silk gland of G1 fifth instar larvae, and in the wings of seven‐day‐old G1 pupae and G1 moths. Therefore, we suggest that the bHsp70 promoter can be used for the rapid and simple screening of transgenic silkworms.     

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.     

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.     

Response to natural and laboratory selection at the Drosophila hsp70 genes

Abstract.— To determine whether and how laboratory and natural selection act on the hsp70 (70-Kd heat-shock protein) genes of Drosophila melanogaster , we examined hsp70 allele frequencies in two sets of populations. First, five populations reared at different temperatures for more than 20 years differentially fixed both a large insertion/deletion (indel) polymorphism at the 87A7 hsp70 cluster ("56H8"/"122") and a single nucleotide polymorphism at the 87C1 hsp70 cluster. In both cases, the 18°C and 25°C populations fixed one allele and the 28°C populations the other, consistent with previously described evolved differences among these populations in Hsp70 expression and thermo-tolerance. Second, we examined 56H8 and 122 frequencies in a set of 11 populations founded from flies collected along a latitudinal transect of eastern Australia. The 56H8 allele frequencies are positively associated with latitude, consistent with maintenance of the 56H8/122 polymorphism by natural selection. Thermal extremes and average values are negatively correlated with latitude. These results suggest that natural selection imposed by temperature and thermal variability may affect hsp70 allele frequencies.     

Structure and expression of a rice hsp70 gene

A genomic hsp70 gene was isolated from a rice IR36 genomic library and 4 794 bp of the gene have been sequenoed. The 5' flanking region of the gene contained a putative TATA box and a typical heat shock element sequence 5'-CTcgGAAccTTCgAG-3'. The amino acid sequence of the rice HSP70 deduced from the coding region shared 84%-92% homologies with those of HSP70s from other plant species. An intron 1939bp long was identified in the coding region at the codon specifying amino acid 72 (Asp), the similar position introns occurring in other intron-containing hsp70 genes. In addition, another intron of 57 bp was found in the 3'-untranslated region in the rice hsp70 gene. Southern blot hybridization showed that rice hsp70 gene family contained at least three members. Analysis of the RNA leveis with the gene-specific and non-specific probes revealed that the rice hsp70 gene expressed at normal temperature and the expression was enhanced by heat shock treatment.