赤子爱胜蚓在不同猪粪和农作物秸杆混合物中生长繁殖状况

本文研究了赤子爱胜蚓在长春市城郊不同组合的粮食猪粪、饲料猪粪和玉米、水稻秸杆混合物中生长繁殖状况,结果发现赤子爱胜蚓的质量在0 ～30 d大幅度增加,30 ～ 60 d增加减缓,60～ 90 d呈负增长趋势.赤子爱胜蚓产茧量在试验期间的总趋势是递增的.猪粪种类、秸杆种类和两者的混合比例差异对赤子爱胜蚓的质量变化产生重要影响.粮食猪粪和玉米秸杆混合物的质量比例大致在1∶1时特别适宜赤子爱胜蚓生长和繁殖.赤子爱胜蚓在饲料猪粪和玉米秸杆混合物中的日增重倍数和日产茧量多于饲料猪粪和水稻秸杆混合物.本试验结论可为城郊猪粪和作物秸杆的大规模资源化利用提供参考.     

赤子爱胜蚓对不同猪粪和秸秆的分解作用

通过赤子爱胜蚓对发酵后猪粪(粮食猪粪、饲料猪粪)和秸秆(水稻秸秆、玉米秸秆)及其不同质量比例(1∶1或3∶1)混合物料进行摄食分解后,对物料前后及不接种赤子爱胜蚓的对照样品理化性质进行分析,发现赤子爱胜蚓处理90 d后的不同物料质量均减少,减少的质量显著多于不接种蚯蚓的对照处理(P＜0.01).赤子爱胜蚓对猪粪与秸秆混合物料的分解量多于纯猪粪和纯秸秆(P＜0.01).猪粪与玉米秸秆的混合物,经蚯蚓处理后,混合物料减少的质量显著多于猪粪与水稻秸秆混合物中物料减少的质量(P＜0.05).经蚯蚓处理后,饲料猪粪混合比例为3∶1的物料质量损失显著多于混合比例为1∶1的物料质量损失(P＜0.01),而粮食猪粪混合比例为1∶1的物料质量损失与混合比例为3∶1的物料质量损失差异不显著(P＞0.05).接种赤子爱胜蚓后,物料的全氮和速效氮、全磷和速效磷以及全钾和速效钾的含量增加,有机质和pH值降低.粮食猪粪和玉米秸秆以质量1∶1的比例进行混合物料的有机质、速效氮、速效磷和速效钾含量变化幅度最大,表明赤子爱胜蚓对此混合物料的分解作用最大.本研究可为猪粪和作物秸秆的资源化利用提供理论依据.     

蚯蚓Sox2基因的克隆及其在再生进程中的表达

为了解Sox2基因在赤子爱胜蚓(Eisenia foetida)再生进程中的作用,本研究采用c DNA末端快速扩增(RACE)方法成功克隆并得到了完整的赤子爱胜蚓Sox2基因序列(Gen Bank登录号:KP637161),其c DNA序列全长2 354 bp,其中包括367 bp的5′端非翻译区,844 bp的3′端非翻译区和编码380个氨基酸残基的1 143 bp开放阅读框。通过实时荧光定量PCR检测了Sox2基因在发育成熟的赤子爱胜蚓不同体段(头部、环带和尾部)以及在尾部体段再生进程中的表达特征。结果显示:Sox2在不同体段中(头部、环带、尾部)的表达差异不显著。在尾部断肢后再生进程中,随着时间推移,Sox2的表达量明显上调,其中,截断后12 h,Sox2基因表达量达到峰值,是截断初期(0 h)的22倍。研究结果表明,Sox2基因可能与蚯蚓的再生进程有关。     

蚯蚓纤溶酶基因的克隆与序列分析

目的:获取蚯蚓纤溶酶(EFE)的基因,并对其进行序列分析。方法:从赤子爱胜蚓中提取总RNA,然后利用RT-PCR技术扩增EFE的基因,并将其插入pUCm-T载体,经PCR和酶切鉴定后进行序列测定及分析。结果:DNA序列分析表明,所克隆的EFE基因全长为738bp,其中编码区段为735bp,共编码245个氨基酸残基,成熟肽为238个氨基酸残基。与GenBank中已报道的粉正蚓的EFE序列F-Ⅲ-2的同源性最高,两者在核苷酸序列上有2处不同,即第137位(T→C)和第632位(G→T),密码子也因而分别由GTC、GGT变为GCC、GTT,导致第46位、211位的氨基酸残基分别由缬氨酸、甘氨酸变为丙氨酸和缬氨酸。结论:从赤子爱胜蚓中成功克隆了1条EFE基因F245。序列测定及同源性分析表明,蚯蚓纤溶酶F245与已报道的多条EFE基因序列具有高度同源性,且具备完整的编码区。该序列的克隆为用基因工程的方法生产单一成分的高酶活性EFE奠定了基础。     

转BPH9和Bar基因抗褐飞虱耐除草剂水稻‘H23’对非靶标生物的影响

为探明转BPH9和Bar基因抗褐飞虱耐除草剂水稻‘H23’对非靶标生物安全性的影响, 本文以‘H23’的非转基因亲本改良的‘广占63-4S’为对照, 比较二者对二化螟(Chilo suppressalis)、大型蚤(Daphnia magna)和赤子爱胜蚓(Eisenia foetida)的影响。其中, 对二化螟采用水稻稻苗饲养法, 观察接虫后6 d二化螟的存活情况, 并称取活虫体重; 对大型蚤采用水稻粉液饲喂法, 考察28 d大型蚤的存活率、繁殖数等指标; 对赤子爱胜蚓则用添加水稻秸秆的人工土壤培养法, 考察28 d内赤子爱胜蚓的存活、体重和行为情况。结果表明, 与受体对照‘广占63-4S’相比, 转BPH9和Bar基因抗褐飞虱耐除草剂水稻‘H23’对二化螟、大型蚤和赤子爱胜蚓均无显著影响, 可以认为, 短期内转BPH9和Bar基因水稻‘H23’对这3种非靶标生物无明显的生态风险。     

种植Bt水稻后的土壤对赤子爱胜蚓生长发育及酶活性的影响

【背景】转基因作物种植的安全问题一直备受关注。关于Bt蛋白对地下非靶标生物影响的研究是转基因作物安全评价的重要内容。【方法】在转Bt基因水稻收割后的稻田里分别种植豌豆、紫云英和油菜作为后茬作物。分别于2013年1、3和6月3次采集不同后茬作物田中的土壤作为材料,于室内饲养赤子爱胜蚓,4周和7周后,测定蚯蚓的生长发育指标、存活率以及体内酶活性的变化情况。此外,还测定了不同深度土壤中Bt蛋白的含量以及用Bt蛋白直接饲喂的赤子爱胜蚓的存活率。【结果】与种植过非转基因水稻MH63的土壤相比,分别种植过含cry2A和cry1C基因水稻后的土壤对赤子爱胜蚓的生长发育、存活率及体内酶活性无显著影响。1月份和3月份转cry2A基因水稻田以及1月份转cry1C基因水稻田采集的表层土样中的Bt蛋白含量显著高于地下10 cm和地下20 cm土壤中的含量,地下2层土样中的Cry2A蛋白含量之间无差异。3月份转cry1C基因水稻田以及6月份转cry2A和转cry1C基因水稻田的土壤中Bt蛋白的含量均不受土壤深度的影响。种植的后茬作物对土壤中的Bt蛋白无显著消解作用。室内模拟土壤最高Bt蛋白浓度的条件下,Cry2A蛋白处理的蚯蚓存活率为96.7%,Cry1C蛋白处理的蚯蚓存活率为95.0%,两者与对照相比无显著差异。【结论与意义】转cry2A和cry1C基因Bt水稻的种植对蚯蚓的生长发育和体内酶活性无显著影响。本研究为转基因水稻的安全评价提供了一定的依据。     

蚯蚓对废纸屑再利用及养分贫瘠土壤综合质量的影响

办公废纸屑作为常见有机废弃物,由于体积小且转化为再生纸成本高,因而再利用很难。但其含有大量有机碳(特别是纤维素)可能有助于退化土壤修复。蚯蚓对土壤有机质分解和其他土壤功能有重要影响,办公废纸屑和蚯蚓共同作用如何影响养分贫瘠土壤质量至今未知。研究以赤子爱胜蚓为接种蚯蚓,将办公废纸屑添加到养分贫瘠土壤中,分别设置纯土壤培养为对照组(S)、单独添加废纸屑(SP)、单独接种赤子爱胜蚓(SE)和添加废纸屑并接种赤子爱胜蚓的处理(SPE),比较培养90 d后各处理理化指标(pH、有机碳、全氮、全磷、全钾、碱解氮、速效磷、速效钾、交换性阳离子钾、钠、钙、镁等)、微生物磷脂脂肪酸(PLFAs)总含量和微生物结构的差异,在此基础上综合评价土壤质量,阐明废纸屑和赤子爱胜蚓在养分贫瘠土壤改良修复中的作用。结果显示：SPE处理较SP处理显著提高废纸屑的分解率89.48%。与对照相比,SP处理能够显著提高土壤pH值2.94个单位,SPE处理能够使其维持在中性水平;前者显著提高土壤有机碳(SOC)125.76%,交换性钠钙镁(Na_Ex、Ca_Ex、Mg_Ex     

HSP70单克隆抗体的制备

利用DEAE 离子交换和ATP 亲和层析法从热休克的大鼠肝脏中分离纯化了热休克蛋白 70 (HSP70 )。用它做抗原免疫小鼠 ,通过分离脾淋巴细胞和细胞融合技术 ,得到了杂交瘤细胞 ;用酶联免疫吸附测定法筛选出了阳性克隆 ;经过多次亚克隆、抗体的大量制备及分离纯化 ,得到了抗HSP70的单克隆抗体。用此一抗和Western免疫印迹技术分析了C6 大鼠神经胶质瘤细胞中HSP70的表达。     

异色瓢虫热休克蛋白70基因的克隆与分析

本研究通过RT-PCR和RACE方法,首次克隆了异色瓢虫Harmonia axyridis(Pallas)热休克蛋白70(HSP70)cDNA全序列(GenBank登录号:EF668009).获得的cDNA全长2200 bp,其中开放阅读框1956 bp,编码一个651氨基酸的蛋白,计算分子量为70 kDa,等电点(PI)为5.32.同源序列比对结果表明,异色瓢虫HSP70与其它真核生物的HSP/HSC70有着较高的序列同源性(85%～93%).其中与赤拟谷盗Tribolium castaneum热休克蛋白70(HSP70)同源性最高,达到93%.与其它真核生物HSP70一样,该序列包含真核生物HSP70高度保守的全部三个家族标签以及ATP/GTP结合位点、一个Bipartive nuclear localization signal和细胞核定位信号.     

6种重金属对赤子爱胜蚓的急性毒性效应与风险评价

【背景】近年来,土壤重金属污染问题日益凸显,对生态环境、食品安全和人体健康构成了严重威胁,其急性毒性尚未明确。【方法】采用滤纸接触法和人工土壤法测定了铜(Cu2+)、锌(Zn2+)、镍(Ni+)、镉(Cd2+)、铅(Pb2+)和锰(Mn2+)等6种重金属对赤子爱胜蚓的急性毒性效应,并参照欧盟指令91/414/EEC标准评价了其环境风险。【结果】滤纸接触法48h测定结果表明,6种重金属对蚯蚓的LC50为3.17(2.53～3.81)～90.42(69.45～140.47)μg.cm-2,其毒性次序为Cu2+>Zn2+>Ni+>Cd2+>Pb2+>Mn2+。人工土壤法14d测定结果表明,6种重金属对蚯蚓的LC50为1347(1236～1453)～6936(6144～8930)mg.kg-1,其毒性次序为Cu2+>Cd2+>Ni+>Zn2+>Mn2+>Pb2+。风险评价结果显示,Cu2+、Zn2+、Ni+、Cd2+、Pb2+和Mn2+等6种重金属的暴露比(toxicity/exposureratio,TER)分别为3.37、4.46、8.68、1428、13.87和5.85。【结论与意义】6种重金属对土壤动物蚯蚓均具有潜在的毒性效应。Cu2+、Zn2+、Ni+、Mn2+等4种重金属对赤子爱胜蚓存在急性毒性风险,而Cd2+和Pb2+对赤子爱胜蚓的急性毒性风险水平是可接受的。该评价结果可为我国制定基于风险的土壤环境质量标准提供依据。     

The chloroplast DnaJ homolog CDJ1 of Chlamydomonas reinhardtii is part of a multichaperone complex containing HSP70B, CGE1, and HSP90C

We report on the molecular and biochemical characterization of CDJ1, one of three zinc-finger-containing J-domain proteins encoded by the Chlamydomonas reinhardtii genome. Fractionation experiments indicate that CDJ1 is a plastidic protein. In the chloroplast, CDJ1 was localized to the soluble stroma fraction, but also to thylakoids and to low density membranes. Although the CDJ1 gene was strongly heat shock inducible, CDJ1 protein levels increased only slightly during heat shock. Cellular CDJ1 concentrations were close to those of heat shock protein 70B (HSP70B), the major HSP70 in the Chlamydomonas chloroplast. CDJ1 complemented the temperature-sensitive phenotype of an Escherichia coli mutant lacking its dnaJ gene and interacted with E. coli DnaK, hence classifying it as a bona fide DnaJ protein. In soluble cell extracts, CDJ1 was found to organize into stable dimers and into complexes of high molecular mass. Immunoprecipitation experiments revealed that CDJ1 forms common complexes with plastidic HSP90C, HSP70B, and CGE1. In blue native-polyacrylamide gel electrophoresis, all four (co)chaperones migrated at 40% to 90% higher apparent than calculated molecular masses, indicating that greatest care must be taken when molecular masses of protein complexes are estimated from their migration relative to standard native marker proteins. Immunoprecipitation experiments from size-fractioned soluble cell extracts suggested that HSP90C and HSP70B exist as preformed complex that is joined by CDJ1. In summary, CDJ1 and CGE1 are novel cohort proteins of the chloroplast HSP90-HSP70 multichaperone complex. As HSP70B, CDJ1, and CGE1 are derived from the endosymbiont, whereas HSP90C is of eukaryotic origin, we observe in the chloroplast the interaction of two chaperone systems of distinct evolutionary origin.     

Food-deprivation induces HSP70 and HSP90 protein expression in larval gilthead sea bream and rainbow trout

Heat shock proteins (HSPs) expression is commonly used as indicators of cellular stress in animals. However, very little is known about either the expression patterns of HSPs or their role in the stress-tolerance phenomenon in early life stages of fish. To this end, we examined the impact of food-deprivation (12 h), reduced oxygen levels (3.5 mg/L for 1 h) and heat shock (HS: + 5 °C for 1 h) on HSP70 and HSP90 protein expression in early life stages of the gilthead sea bream (Sparus aurata), a warm-water aquaculture species. Also, we investigated HSP70 and HSP90 response to food-deprivation (7 days) in early life stages of rainbow trout (Oncorhynchus mykiss), a cool-water aquaculture species, and the tolerance of this larvae to heat shock (either + 5 or + 10 °C for 1 h). Our results clearly demonstrate that food-deprivation enhances HSP70 and HSP90 protein expression in larvae of both species. In gilthead sea bream larvae, the stressors-induced HSP70 and HSP90 (only in the reduced oxygen group) protein expression returned to unstressed levels after 24 h recovery. In fed trout larvae, a + 5 °C heat shock did not elevate HSP70 and HSP90 expression, whereas 100% mortality was evident with a + 10 °C HS. However, food-deprived trout larvae, which had higher HSP70 and HSP90 protein content, survived HS and showed HS-dependent increases in HSP70, but not HSP90 expression. Overall, HSP70 and HSP90 protein expression in early life stages of fish have the potential to be used as markers of nutritional stress, while elevation of the tissue HSPs content may be used as a means to increase stress tolerance during larval rearing.     

Restoring HSP70 deficiencies improves glucose tolerance in diabetic monkeys

We evaluated heat shock protein 70 (HSP70) changes in diabetes mellitus (DM) in a nonhuman primate model. To this end, two studies were conducted in DM vervet monkeys. 1) Normal control and streptozotocin-induced DM monkeys (Stz-DM) that were differentiated into moderately or poorly controlled DM by judicious insulin administration were evaluated. Liver was collected at 4, 8, 12, 16, and 20 wk after streptozotocin, exposed to ex vivo heat shock at 42°C, and immunoblotted for heat shock factor 1 (HSF1), HSP70, and phosphorylated HSF1. 2) Spontaneous DM monkeys that were not pharmacologically induced were included in a crossover study of the HSP70-inducing drug geranylgeranylacetone (GGA). GGA at 20 mg/kg was given for 14 days with a 6-wk washout period. Glucose tolerance testing and plasma and muscle HSP70 were the primary outcome measurements. In Stz-DM, hyperglycemia reduced hepatic HSP70 in a dose-dependent fashion. HSF1 was increased in livers of monkeys with Stz-DM, but responses to ex vivo heat shock were impaired vs. normal monkeys. Activation of HSF1 appears to be important, because the phosphorylation change with heat stress was nearly perfectly correlated with HSP70 increases. Impaired HSF1 activation was also seen in Stz-DM after chronic hyperglycemia (>12 wk). In naturally occurring DM, increased circulating HSP70 resulted in significantly improved glucose tolerance and significant, positive trends in other measurements of insulin resistance. No change in muscle HSP70 content was observed. We conclude that increasing HSP70, potentially through targeting hyperglycemia-related deficits in HSF1 induction and activation in the liver, is a potent and viable strategy to improve glucose tolerance.     

Differential regulation of HSC70, HSP70, HSP90 alpha, and HSP90 beta mRNA expression by mitogen activation and heat shock in human lymphocytes

A subset of heat shock proteins, HSP90 alpha, HSP90 beta, and a member of the HSP70 family, HSC70, shows enhanced synthesis following mitogenic activation as well as heat shock in human peripheral blood mononuclear cells. In this study, we have examined expression of mRNA for these proteins, including the major 70-kDa heat shock protein, HSP70, in mononuclear cells following either heat shock or mitogenic activation with phytohemagglutinin (PHA), ionomycin, and the phorbol ester, tetradecanoyl phorbol acetate. The results demonstrate that the kinetics of mRNA expression of these four genes generally parallel the kinetics of enhanced protein synthesis seen following either heat shock or mitogen activation and provide clear evidence that mitogen-induced synthesis of HSC70 and HSP90 is due to increased mRNA levels and not simply to enhanced translation of preexisting mRNA. Although most previous studies have focused on cell cycle regulation of HSP70 mRNA, we found that HSP70 mRNA was only slightly and transiently induced by PHA activation, while HSC70 is the predominant 70-kDa heat shock protein homologue induced by mitogens. Similarly, HSP90 alpha appears more inducible by heat shock than mitogens while the opposite is true for HSP90 beta. These results suggest that, although HSP70 and HSC70 have been shown to contain similar promoter regions, additional regulatory mechanisms which result in differential expression to a given stimulus must exist. They clearly demonstrate that human lymphocytes are an important model system for determining mechanisms for regulation of heat shock protein synthesis in unstressed cells. Finally, based on kinetics of mRNA expression, the results are consistent with the hypothesis that HSC70 and HSP90 gene expression are driven by an IL-2/IL-2 receptor-dependent pathway in human T cells.     

HSP70 heat shock proteins and environmental stress in Antarctic marine organisms: A mini-review

The ability to understand and predict the effects of environmental stress on biodiversity is becoming increasingly important in our changing environment. Antarctic marine species are some of the most stenothermal on the planet and many inhabit the waters off the Antarctic Peninsula which is one of the areas where there is rapid regional climate change. Therefore these animals are highly vulnerable to changing environmental temperatures and clearly we need to understand the complexities of their response, not just at the individual species level, but also the implications for the ecosystem as a whole. Heat shock proteins have a long history of use in studies of organism stress responses and have frequently been proposed as potential universal molecular biomarkers, especially for non-model species. In this mini-review, the heat shock response and heat shock proteins (specifically the HSP70 family) are examined in Antarctic marine species alongside their physiological capabilities and limits to answer a series of questions: do these animals have a heat shock response which includes the expression of HSP70 genes? What is the relationship between their heat shock response and physiological capabilities? Can HSP70 genes be used as molecular biomarkers for these species?     

Characterization of the 90 kDa heat shock protein (HSP90)-associated ATP/GTPase

The 90 kDa heat shock protein (HSP90) is an ATP-binding molecular chaperone with an associated ATPase activity having nucleoplasmin and HSP70-binding homology domains and containing Ca-binding EF-hands and a nuclear localization signal. Here we characterize the HSP90-associated ATPase and show that it is (i) a P-type ATPase inhibited by molybdate and vanadate, (ii) able to hydrolyze methylfluorescein phosphate with a 5–6-fold higher affinity, (iii) a 3-times better GTPase than ATPase in the presence of calcium and (iv) HSP27 and F-actin, but not HSP10 can “convert” the HSP90-associated ATPase activity to HSP90 autokinase activity. The HSP90-associated ATP/GTPase may participate in the regulation of complex formation of HSP90 with other proteins, such as F-actin, tubulin and heat shock proteins.