Effect of Ultraviolet Radiation on Hsp70 Protein Expression in HaCaT Cells

Ultraviolet radiation by its wavelength is divided into: UVA, UVB and UVC. Only UVA and UVB manage to penetrate the ozone layer, but due to anthropological activities, all of them are capable of interacting with humans to a greater or lesser extent, and can generate adverse effects such as cellular stress when interacting with intra-and extracellular biomolecules. The skin is the first organ in contact with UV radiation, and the stress it generates can be analyzed by the expression of a bioindicator of cellular damage such as Hsp70. Therefore, the objective of the project was: to determine the effect of UVA, UVB and UVC radiation on HaCaT epithelial cells, by analyzing the expression of Hsp70. Materials and methods: HaCaT cells were cultured in vitro, which were irradiated with UVA, UVB and UVC light at different doses, to subsequently determine the degree of Hsp70 expression by Immunodetection by PAGE-SDS and Western Blot. Results: Basal expression of Hsp70 was observed in no irradiated HaCaT cells. When HaCaT cells were irradiated with UVA, UVB, UVC, an increase in this Hsp70 protein was observed. With UVA, a higher degree of expression was observed at a time of 30 minutes of irradiation. With UVB the highest expression shifted to a time of 20 minutes. With UVC, overexpression was observed after 10 minutes. Conclusion: UV radiation generates cellular stress on HaCaT cells, evaluated by the stress bioindicator Hsp70. According to the wavelength of UV radiation, those that have a shorter wavelength have a greater potential for cellular damage, such as UVC.

Low-Dose Gamma Radiation Fields Decrease Cell Viability, Damage DNA, and Increase the Expression of Hsp70 and p53 Proteins in Human Leukocytes

Ionizing radiations are tools in diagnosis and treatment of diseases. Leukopenia from exposure to ionizing radiation has been reported. Due to their radiosensitivity, leukocytes are a biological model to analyze cell damage. Therefore, cell viability, DNA damage, and Hsp70 and p53 expression in human leukocytes exposed to low-dose gamma radiation fields from a 137Cs source were evaluated. A decrease in cell viability, DNA damage and an increase in the expression of Hsp70 and p53 proportional to the radiation dose received was found, which was 0.2, 0.4, 0.6, 0.8 and 1.0 mGy.

Ischemic preconditioning induces chaperone hsp70 expression and inhibits protein aggregation in the CA1 neurons of rats

Objective To investigate the effect of ischemic preconditioning on chaperone hsp70 expression and protein aggregation in the CA1 neurons of rats, and to further explore its potential neuroprotective mechanism. Methods Two-vesseloccluded transient global ischemia rat model was used. The rats were divided into sublethal 3-min ischemia group, lethal 10- min ischemia group and ischemic preconditioning group. Neuronal death in the CA1 region was observed by hematoxylineosin staining, and number of live neurons was assessed by cell counting under a light microscope. Immunochemistry and laser scanning confocal microscopy were used to observe the distribution of chaperone hsp70 in the CA1 neurons. Differential centrifuge was used to isolate cytosol, nucleus and protein aggregates fractions. Western blot was used to analyze the quantitative alterations of protein aggregates and inducible chaperone hsp70 in cellular fractions and in protein aggregates under different ischemic conditions. Results Histological examination showed that ischemic preconditioning significantly reduced delayed neuronal death in the hippocampus CA1 region （P 〈 0.01 vs 10-min ischemia group）. Sublethal ischemic preconditioning induced chaperone hsp70 expression in the CA1 neurons after 24 h reperfusion following 10-min ischemia. Induced-hsp70 combined with the abnormal proteins produced during the secondary lethal 10-min ischemia and inhibited the formation of cytotoxic protein aggregates（P〈0.01 vs 10-min ischemia group）.Conelusion Ischemic preconditioning induced chaperone hsp70 expression and inhibited protein aggregates formation in the CA1 neurons when suffered secondary lethal ischemia, which may protect neurons from death.

Construction and Identification of HSP70 Antisense RNA Expression Vector for Genetic Engineering Male Sterility in Plant

[ Objective] In order to study the relation between the HSPTO gene and male sterility of plant further. [ Methods ] Anther specific expression promoter Osg6B of rice was coloned by PCR then connected with HSP70 antisense fragment to construct HSPTO antisense expression vector. The expression vector was identified by PCR experiment and enzyme digestion. [ Result] The sequence of coloned Osg6B promoter had 97% homology to the published sequence, and the cis-regulatory element in promoter area was integrated. HSP70 antisense expression vector driven by the promoter Osg6B was confired by colony PCR and enzyme digestion. [ Conclusion] The construction of expression vector would lay solid foundation for utilization of genetic engineering male sterility of plant.

Study on HSP70 Gene Expression in Different Tissue of Cyprinus carpio

[ Objective] The aim of this study was to investigate whether HSP70 can be used as a stress monitoring indicator in Cypnnus carpio breeding. [Method] Based on HSP70 sequence of Cyprinus carpio （AY120894）, one pair of primers was designed and synthesized, while the total RNA of liver tissues in Cyprinus carpio was extracted. Some cDNA fragments of Cyprinus carpio HSP70 were cloned by RT-PCR, and its differential expression in various tissues such as heart, intestine, mucus, gonad, swim bladder, gill and fin in Cyprinus carpio was also studied. [Result] The cDNA sequence of 480 bp was obtained from Cypdnus carpio HSPTO gene by RT-PCR amplification. Homology comparison between the deduced amino acid sequence after sequencing and that of other types of fish showed that the homology among Cyprinus carpio, Danio rerio, Ohcorhynehus mylciss, Paralichthys olivaceus, Xiphophoorus maculates and Carassius auratus was 96%, 98%, 98%, 96%, 98% and 96% respectively. The expression of HSP70 was detected in eight tissues of Cypnnus carpio. The expression was the highest in heart, followed by swim bladder and fin, but there was no significant difference between them （ P 〉 0.05 ）. There was no significant difference among the ex- pression in three tissues of intestine, mucus and fat （ P〉0.05）, but their expression was significantly higher than those in gonad and gill （ P〈 0.05）. [ Conclusion] HSPTO gene expression is a suitable criterion for monitoring the stress degree, stress capacity and healthy conditions in Cyprinus carpio breeding.

AAV mediated carboxyl terminus of Hsp70 interacting protein overexpression mitigates the cognitive and pathological phenotypes of APP/PS1 mice

The E3 ubiquitin ligase,carboxyl terminus of heat shock protein 70(Hsp70)interacting protein(CHIP),also functions as a co-chaperone and plays a crucial role in the protein quality control system.In this study,we aimed to investigate the neuroprotective effect of overexpressed CHIP on Alzheimer’s disease.We used an adeno-associated virus vector that can cross the blood-brain barrier to mediate CHIP overexpression in APP/PS1 mouse brain.CHIP overexpression significantly ameliorated the performance of APP/PS1 mice in the Morris water maze and nest building tests,reduced amyloid-βplaques,and decreased the expression of both amyloid-βand phosphorylated tau.CHIP also alleviated the concentration of microglia and astrocytes around plaques.In APP/PS1 mice of a younger age,CHIP overexpression promoted an increase in ADAM10 expression and inhibitedβ-site APP cleaving enzyme 1,insulin degrading enzyme,and neprilysin expression.Levels of HSP70 and HSP40,which have functional relevance to CHIP,were also increased.Single nuclei transcriptome sequencing in the hippocampus of CHIP overexpressed mice showed that the lysosomal pathway and oligodendrocyte-related biological processes were up-regulated,which may also reflect a potential mechanism for the neuroprotective effect of CHIP.Our research shows that CHIP effectively reduces the behavior and pathological manifestations of APP/PS1 mice.Indeed,overexpression of CHIP could be a beneficial approach for the treatment of Alzheimer’s disease.

In vitro Study on Role of Hsp70 Expression in DNA Damage of Human Embryonic Lung Cells Exposed to Benzo[a]pyrene

Objective Benzo[a]pyrene (B[a]P), a ubiquitous environmental pollutant, is a potent procarcinogen and mutagen that can elicit tumors, leading to malignancy. Heat shock proteins (Hsp) have been shown to protect cells against damages caused by various stresses including exposure to numerous chemicals. Whether Hsps, or more specifically Hsp70, are involved in repair of B[a]P-induced DNA damage is currently unknown. Methods We assessed the potential role of the inducible form of Hsp70 in B[a]P-induced DNA damage of human embryonic lung (HEL) cells using immunoblot and the comet assay (i.e., the single cell gel electrophoresis assay). Results Exposure to B[a]P induced a dose-dependent decrease in the level of Hsp70, but a dose-dependent +-increase in DNA damage both in untreated (control) HEL cells and in cells preconditioned by a heat treatment. Heat preconditioning prior to B[a]P exposure potentiated the effect of B[a]P at a low dose (10 μmol/L), but appeared to be protective at higher doses. There was a negative correlation between Hsp70 level and DNA damage in the non-preheated as well as in the preconditioned cells. Conclusion These data suggest that exposure of HEL cells to B[a]P may induce a dose-dependent reduction in the levels of the inducible Hsp70. The detailed mechanisms for the reduction of Hsp70 levels by B[a]P and the role of Hsp70 in DNA damage under different concentrations of B[a]P remains to be determined.

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 expressionby antisense oligonucleotides complementary to the mRNA of HSP70 and to test the effects ofinhibition of HSP70 gene expression on subsequent embryonic sensitivity to heat shock. Theresults showed that transfection of pre-implantation embryos at 4-cell stage with 5 Mantisense oligo had no effect on in vitro blastocyst development. However, transfection with10 to 40 M antisense oligo had reduced in vitro blastocyst development to 15, 10% and 0; Forthe embryos which exposed to 40 M As arrested at the 16-cell stage, there was no blastocystformation within the heat shock groups. In contrast, transfection had no effect on embryonicsensitivity to heat shock, above 25% of embryos developed to blastocyst stage in controlgroups.

Effects of aspartame on hsp70,bcl-2 and bax expression in immune organs of Wistar albino rats

Aspartame, a ＂first generation sweetener＂, is widely used in a variety of foods, beverages, and medicine. The FDA has determined the acceptable daily intake （ADI） value of aspartame to be 50 mg/kg, day, while the JECFA （Joint FAO/WHO Expert Committee on Food Additives） has set this value at 40 mg/kg of body weight/day. Safety issues have been raised about aspartame due to its metabolites, specifically toxicity from methanol and/or its systemic metabolites formaldehyde and formic acid. The immune system is now recognized as a target organ for many xenobiotics, such as drugs and chemicals, which are able to trigger unwanted apoptosis or to alter the regulation of apoptosis. Our previous studies has shown that oral administration of aspartame [40 mg/（kg, day）] or its metabolites for 90 days increased oxidative stress in immune organs of Wistar albino rats. In this present study, we aimed to clarify whether aspartame consumption over a longer period （90-days） has any effect on the expression ofhsp70, bcl- 2 and bax at both mRNA transcript and protein expression levels in immune organs. We observed that oral administration of aspartame for 90 days did not cause any apparent DNA fragmentation in immune organs of aspartame treated animals; however, there was a significant increase in hsp70 expression, apart from significant alteration in bcl-2 and bax at both mRNA transcript and protein expression level in the immune organs of aspartame treated animals compared to controls. Hence, the results indicated that hsp70 levels increased in response to oxidative injury induced by aspartame metabolites; however, these metabolites did not induce apoptosis in the immune organs. Furthermore, detailed analyses are needed to elucidate the precise molecular mechanisms involved in these changes.

Effects of Salinity Fluctuation Frequency on the Osmolarity,Na^+-K^+-ATPase Activity and HSP70 Expression in Juvenile Chinese Shrimp,Fenneropenaeus chinensis

Experiments were conducted to examine the effects of salinity fluctuation frequency on the osmolarity, Na^＋-K^＋-ATPase activity and HSP70 of Chinese shrimp Fenneropenaeus chinensis ruth initial wet body weight of 1.460g±0.091 g. The salinity in the control group （DO） was 28 throughout the experiment, whereas treatments D2, D4, D6 and D8 were subjected to different salinity fluctuation frequencies of 2, 4, 6 and 8d, respectively. The salinity in treatments D2, D4, D6 and D8 was kept at 28 for 2, 4, 6 and 8d, respectively, decreased abruptly to salinity 24, lasted for another 2 d, and then was raised to its initial value 28. This was a complete salinity fluctuation cycle that afterwards repeated itself. After 32 days, the osmolarity in treatments D2, D4, D6 and D8 was significantly lower than that in treatment DO （P〈0.05）. There were significant differences in both muscle and eyestalks HSP70 expression among groups. The HSP70 expressions in muscle and eyestalks in group D4 were 61.4% and 57.0% higher, respectively, than that in the control group DO （P〈0.05）. There were, however, no significant differences in gill or hepatopancreas Na^＋-K^＋-ATPase activity between the treatments and the control.

Heat Shock Protein 40 (Hsp40) and Hsp70 Protein Expression in Oral Squamous Cell Carcinoma (OSCC)

Introduction: As a chaperone, heat shock protein acts as central integrators of protein homeostasis in cell. The form of these functions is to help setting up a complex protein molecular fold (folded protein) in many important settings, such as growth, differentiation, and the ability to live. It has become clear that the control system plays an important role if the folding process fails or an error occurs, causing folding abnormalities and targeted functionality to accumulate. The accumulation of faulty protein folding would harm cells and can result in death. Apparently, there is a correlation between protein folding error with various diseases, such as diabetes mellitus and cancer. Method: We examined protein levels in all samples using Dotblott with monoclonal antibody anti-Hsp40 and anti-Hsp70. Levels of the protein content was read using a densitometer. Modification of Dot Blot was as follows: treatment was conducted with 3 × SSC, added with 20 mL blocking solution, add with total protein samples of 10 mg/ml on nitrocellulose paper, prehybridized, incubated at 70° for 30 seconds, incubated at 70° for 30 seconds with primary antibody anti-Hsp40 or Hsp70 protein and then added with second antibody HRP anti-Hsp40 or Hsp70 protein, treated with 3 × SSC and visualized with TSA HRP, and then administered with streptavidin, biothynil tyramide, and, finally, added with chromogen (DAB) in a confined space. Result: From the analysis of the data using Manova test with Wilk’s Lambda, there were significant differences in the levels of Hsp40 between Benign Oral Lesion (mean 688.31 area) and OSCC (mean 1354.59 area) patients (p 0.070), there was also a highly significant difference in Hsp70 levels between patients who experienced Benign Oral Lesion (mean 529.82 area) and OSCC (mean 1346.32 area) patients (p 0.006). Conclusion: OSCC patients have increased Hsp70 levels, so it is possible that something is going wrong in protein folding. Errors in protein folding result in a new homeostasis or inhibition of apoptosis and increasing cell proliferation that triggers carcinogenesis. Hsp40 acts as co-chaperones.

Effects of Batroxobin on Spatial Learning and Memory Disorder of Rats with Temporal Ischemia and the Expression of HSP32 and HSP70

The effect of Batroxobin on spatial memory disorder of left temporal ischemic rats and the expression of HSP32 and HSP70 were investigated with Morri`s water maze and immunohistochemistry methods. The results showed that the mean reaction time and distance of temporal ischemic rats in searching a goal were significantly longer than those of the sham-operated rats and at the same time HSP32 and HSP70 expression of left temporal ischemic region in rats was significantly increased as compared with the sham-operated rats. However, the mean reaction time and distance of the Batroxobin-treated rats were shorter and they used normal strategies more often and earlier than those of ischemic rats. The number of HSP32 and HSP70 immune reactive cells of Batroxobin-treated rats was also less than that of the ischemic group. In conclusion, Batroxobin can improve spatial memory disorder of temporal ischemic rats; and the down-regulation of the expression of HSP32 and HSP70 is probably related to the attenuation of ischemic injury.

Effect of flunarizine on HSP70 gene expression for following transient ischemia

Objective To investigate the effects of flunarizine on HSP70 gene expression for following transient ischemia.Methods Northern blot and immunohistochmistry (IHC) were used to determine the expression of the HSP70 during different periods after post ischemic reperfusion with or without Flunarizine treatment in the gerbil. Results The HSP70mRNA expression was increased in the forebrain,but the HSP70 protein only expressed at the 1st day of reperfusion(P<0.05);Flunarizine treatment could increase the expression of HSP70 protein, but couldn’t significantly increase the expression of HSP70 mRNA (P >0.05). Conclusion Flunarizine could protect neurons from ischemic damage through increasing the HSP70 expression.

Effects of Low Temperature Stress on the Morphology and hsp70 and hsp90 Gene Expression of Phascolosoma esculenta

Phascolosoma esculenta is an intertidal organism that has recently attracted attention because of its ability to survive at relatively low temperatures.However,the gene regulation in P.esculenta in relation to its response to low temperatures is unclear.To explore the low temperature adaptability of P.esculenta,this study analyzed the changes in the morphology and hsp70 and hsp90 gene expression of P.esculenta exposed to a low temperature gradient.At 5℃,P.esculenta stretched and softened,and some individuals moved apart from the group.Histological analysis revealed cuticle breaches,myofiber scattering,disruption of the body wall,and epithelial layer dispersion and muscle fiber rupturing in the nephridium.Furthermore,the mRNA expression levels of hsp70 and hsp90 increased under acute low temperature stress,suggesting that these genes function in low temperature tolerance.Overall,low temperature stress causes morphological changes and histological damage in P.esculenta,and hsp70 and hsp90 potentially function in the low temperature adaptability of P.esculenta.Our results provide new insights into the adaptive strategies of P.esculenta under low temperature environments.

Molecular characterization and expression of HSP70, HSF and HSBP genes in Octopus vulgaris during thermal stress

Temperature is an important environmental factor that affects the growth and survival of Octopus vulgaris, the common octopus. To understand the protective mechanism that O. vulgaris exhibits under heat stress, we used rapid amplification of cDNA ends （RACE） to obtain full-length sequences of three heat stress response related genes： （1） the heat shock protein 70 （OvHSPT0）, （2） the heat shock transcription factor （OvHSF）, and （3） the heat shock factor-binding protein （OvHSBP） of O. vulgaris. The OvHSP70, OvHSF, and OvHSBP proteins contained 2 222 bp, 2 264 bp, 841 bp that encoded for 635, 458 and 90 amino acids, respectively. The results of multiple sequence alignment showed that the amino acid sequences of OvHSP70 were highly conserved with respect to other species. Similarly, the DNA binding domain, the trimerization domain of OvHSF, and the coiled coil region of OvHSBP also had highly conserved regions. The real-time polymerase chain reaction （PCR） results indicated that OvHSP70 was temperature-dependent and time-dependent, showing a positive response to heat stress. On exposure to 26℃ and to 30℃, the mRNA expression levels of OvHSF and OvHSBP were higher than those in the control group at 24℃. The mRNA expression of OvHSBP significantly increased with heat treatment at 26℃, while the mRNA expression of OvHSF decreased. The experimental results indicated that the expression of OvHSP70, OvHSF and OvHSBP were all sensitive to heat stress, which suggests that these three genes may play an important role for O. uulgaris in responding to environmental stress. Thus, this study sets a theoretical foundation for further in-depth studies on the molecular protective mechanisms of the heat response in O. vulgaris.

Protective Effects of Focal Ischemic Preconditioning and HSP70 Expression on Middle Cerebral Artery Occlusion in Rats

Summary： To systematically evaluate the importance of protein synthesis in ischemic preconditioning （PC）-induced ischemic tolerance （IT）, temporary middle cerebral artery occlusion （MCAO） by Longa （20 min） was used for PC （ischemic precondioning）. Twenty-four hours of reperfusion was allowed after PC and before permanent MCAO to establish ischemic tolerance （IT） to compare with non-PC （sham-operated） rats （n=5 for each group）. Infarct size and neurological deficits were measured 24 h after PMCAO. Samples of brain were taken for the determination of HSP70 expression by Western blot analysis. The effects of the protein synthesis inhibitor cycloheximide administered just before PC or administered long after PC but just before PMCAO on IT were also determined （n=5 for each group）. Our results showed that hemispheric infarct was significantly reduced （P〈0.01） only if PC was performed after 24 h, and PC significantly （P〈0.05） reduced neurological deficits （similar to reductions in infarct size）. Cycloheximide eliminated ischemic PC-induced IT effects on both brain injury and neurological deficits if administered before PC but not if administered long after PC but before PMCAO. PC produced no brain injury but did increase HSP70 protein 24 h after PC. Cycloheximide eliminated that effect. The results suggest that PC is a powerful inducer of ischemic brain tolerance as reflected by the preservation of brain tissue and motor function. PC induces IT that is dependent on de novo protein synthesis.

Effect of Different Diet Level on the Physiological Adaptability, Biochemical and Endocrine Responses and Relative Hepatic HSP70 and HSP90 Genes Expression in Osmanabadi Kids

The study was conducted to investigate the impact of different levels of feed on the adaptive capability based on physiological, blood biochemical, endocrine and molecular mechanisms in growing Osmanabadi kids. The primary objective of the study was to identify if HSP70 and HSP90 can be a nutritional stress marker for goat. The study was conducted for a period of two months. The animals were randomly divided into three groups as GI （n = 6; ad libitum feeding）, GII （n = 6; 20% less than ad libitum） and GIII （n = 6; 40% less than ad libitum）. The animals were fed with feed consisting of 50% roughage and 50% concentrate. Blood collection was carried out at fortnightly intervals. Body weights were recorded at weekly interval. Physiological responses, biochemical responses, plasma tri-iodo-thyronine （T3）, thyroxin （＇1＂4） and cortisol were recorded at fortnightly interval. At the end of study period, only GI and Gill animals were slaughtered and different organs were collected for histopathological studies as well as for hepatic HSP70 and HSP90 mRNA transcript expression. Body weight recorded showed significant （P 〈 0.01） differences between the groups. Physiological responses showed significant （P 〈 0.01） variation among the groups. Among the biochemical parameters, plasma glucose and total plasma protein and globulin showed significant （P 〈 0.01） differences between the groups. Plasma T3 （P 〈 0.01）, T4 （P 〈 0.01） and cortisol （P 〈 0.05） also differed significantly between the groups. The relative hepatic HSP70 mRNA transcript expression was significantly （P 〈 0.05） higher in Gill （2.8 fold） as compared to GI （1 fold） kids. Similar result was obtained for hepatic HSP90 mRNA transcript expression. From the results, it can be concluded that Osmanabadi kids possessed the ability to alter their adaptive mechanisms to maintain homeostasis. Further, the study revealed the significance of providing the optimum nutrition for these animals to adapt to existing environmental conditions. The study also established that respiration rate （RR）, rectal temperature （RT）, T3, T4 and cortisol are considered as nutritional stress markers for goat. Further, the results revealed that probably this is the first study to establish the nutritional stress impact on heat shock protein （HSP） expression in goats. The study identified both HSP70 and HSP90 to be the ideal molecular markers for feed deficit in goats.

Batroxobin Against Anoxic Damage of Rat Hippocampal Neurons in Culture:Morphological Changes and Hsp70 Expression

Batroxobin,the thrombin-like enzyme,is used for therapeutic defibrination. We have found that batroxobin has good therapeutic effect in ischemic reperfusion rats and clinical practices in vivo. But we have not studied the neuroprotective effect of batroxobin on anoxic hippocampal neurons in vitro. The purpose of this study was to obtain further information on the mechanism of the batroxobin-induced neuroprotection and examine the neuroprotective effect on neurons exposed to anoxia. The effect of batroxobin on anoxic damages in cultured hippocampal neurons of neonatal rats was investigated by using morphological changes and heat shock protein 70Kd (Hsp70) immunoreactive expression as indicators. The results indicate that batroxobin, besides its defibrination, may have a direct neuroprotective effect on anoxic damage of hippocampal neurons.

基于Hsp70基因的绵羊肺炎支原体TaqMan检测方法的建立及其遗传演化分析

热休克蛋白70 (heat shock protein 70,Hsp70)是绵羊肺炎支原体(Mycoplasma ovipneumoniae, Movi)的重要膜蛋白,是机体内高度保守的生物分子,但在种间差异大,可作为分子生物学检测的候选靶区。为建立基于Hsp70基因的Movi通用型TaqMan实时荧光定量PCR(qPCR)检测方法,并进一步了解其遗传变异情况,本研究基于GenBank中Movi的Hsp70基因特征,设计特异性的引物及探针,建立了基于Hsp70基因的绵羊肺炎支原体qPCR检测方法。应用建立的检测方法对88份山羊鼻拭子样品及43份疑似羊支原体性肺炎(Mycoplasmal pneumonia of sheep and goats, MPSG)病料进行检测。将检测结果为Movi阳性的肺组织样品进行分离鉴定,并对分离株Hsp70基因进行序列分析。结果显示,建立的qPCR检测方法其相关系数为1.00,扩增效率为96.0%,斜率为-3.411,Y轴截距为37.29。特异性强,与丝状支原体山羊亚种(Mycoplasma mycoides subsp.capri, Mmc)、山羊支原体山羊肺炎亚种(Mycoplasma capricolum subsp.capripneumoniae, Mccp)、莱氏无胆甾原体(Acholeplasmalaidlawii, AL)、无乳支原体(Mycoplasma agalactiae, Maga)、伪结核棒状杆菌(Corynebacterium pseudotuberculosis, CP)、羊口疮病毒(orf virus, ORFV)、牛支原体(Mycoplasma bovis, Mb)等牛羊常见病原均无交叉反应;敏感性高,最低检测限为5.72 copies·μL^(-1);重复性优,组内变异系数和组间变异系数均小于1.00%。6株Movi分离株Hsp70基因全长均为1 818 bp,与其它Movi参考株核苷酸和氨基酸同源性分别为96.0%~99.4%和98.0%~100.0%;进一步分析发现,山羊源Movi均比绵羊源多1个N-糖基化位点。遗传演化分析表明,其均处于ClusterⅠA亚分支(均为山羊源)。综上,本研究建立了特异性强、敏感性高、重复性优的基于Hsp70基因的Movi的qPCR检测方法。序列分析发现,不同来源Movi的Hsp70基因核苷酸同源性高;遗传演化分析证实,Movi福建株与山羊源分离株遗传关系较近。本研究不仅为Movi临床诊断提供了技术支持,更为进一步了解Movi遗传演化规律提供参考。

油桐HSP70基因家族的全基因组鉴定与表达分析

为探究油桐HSP70基因的功能和进化关系,本研究对HSP70基因家族成员进行了生物信息学鉴定和分析,包括理化性质、进化关系、基因结构、保守基序及染色体定位。同时分析了HSP70基因家族成员在不同组织中的表达差异。结果表明,油桐HSP70基因家族包含24个家族成员,24个HSP70蛋白的理论等电点为4.57~8.70,且大部分属于稳定的、亲水性蛋白质,系统进化树分析结果表明HSP70家族成员分为5个聚类,油桐与毛果杨亲缘关系较近。24个VfHSP70蛋白氨基酸序列都包含HSP70保守结构域。22条HSP70基因分布在10条染色体上。分析不同时空的VfHSP70基因的表达量发现,VfHSP70-5在开花前30 d、开花前25 d、开花前10 d的花蕾中表达量均较高。VfHSP70-4在油桐根、茎、叶和种子中均有较高表达量。以上结果表明,VfHSP70基因可能在调控油桐生长发育中具有重要作用。