Molecular Cloning the Gene of Small Heat Shock Protein in the Mitochondria and Endoplasmic Reticulum of Tomato

A cDNA Library was constructed with the heat shocked tomato ( Lycopersicon esculentum Mill.) flowers and then was screened with the probes of mitochondrial and endoplasmic reticulum conservative regions that were cloned by using RT-PCR. The complete cDNAs of mitochondrial and endoplasmic reticulum small heat shock protein ( shsp) were selected out from the cDNA library. Furthermore, the temperature responses of these shsp genes were determined. Northern hybridization showed that the heat response temperatures of both genes in tomato flower were lower than that in leaf and that mitochondria shsp in leaf was cold-inducible. In this paper, the molecular features of the cloned genes, the causes of the uncommon heat response temperatures of sHSP in newer and the cold inducible character of mitochondria shsp gene in leaf were discussed.

Expression analysis of two reverse duplicated small heat shock protein genes in rice(Oryza sativa L.)

supported by grants from the National Natural Science Foundation of China （30671178）;the Shanxi Province Science Foundation for Youths, China （2014021029-2）

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.

Expression profiles of two small heat shock proteins and antioxidant enzyme activity in Mytilus galloprovincialis exposed to cadmium at environmentally relevant concentrations

Small heat shock proteins encompass a widespread but diverse class of proteins, which play key roles in protecting organisms from various stressors. In the present study, the full-length cDNAs of two small heat shock proteins （MgsHSP22 and MgsHSP24.1） were cloned from Mytilus galloprovincialis, which encoded peptides of 181 and 247 amino acids, respectively. Both MgsHSP22 and MgsHSP24.1 were detected in all tissues examined by real-time PCR, with the highest expression being observed in muscle and gonad tissues. The real-time PCR results revealed that Cd significantly inhibited MgsHSP22 expression at 24 h and MgsHSP24.1 at 24 and 48 h under 5 ug/L Cd2＋ exposure. MgsHSP24.1 expression was also significantly inhibited after 50 ug/L Cd2＋ exposure for 48 h. With regard to antioxidant enzymes, increased GPx and CAT activity were detected under Cd2＋ stress （5 and 50 ug/L）, while no significant difference in SOD activity was observed throughout the experiment. Overall, both MgsHsps and antioxidant enzymes revealed their potential as Cd stress biomarkers in M. galloprovincialis.

Expression of Foreign Gene in Mycobacterium Regulated by Human Mycobacterium Tuberculosis Heat Shock Protein 70 Promoter

The DNA fragments of 150bp length promoter 0f human Mycobacterium(M.) tuberculosis heat shock protein (hsp)7O and 650bp length foreign gene, the Schistosoma japonicum glutathione S-transferase(Sj26GST)gene,were obtained by amplification with polymerase chain reaction. And the 150p DNA sequence upstream initiation codon ATG of the human M. tuberculosis hsp7O promoter that contains the sequence TTGAG and ATCATA which consensus with E. coli promoter's -35 and-10 region respectively, as well as ribosome binding site GGAGG at position-12-8 upstream the ATG were determined by SangerDideoxyribonucleotide-mediated chain-termination method-Then, the human M. tuberculosis hsp70 promoter and Sj26GST cDNA were cloned into E. coli-mycobacteria shuttle plasmid pBCG-2000 to construct E. coli-Mycobacterium expression shuttle plasmid pBCG- Sj26 that can express Sj26GST gene.The M. smegmatis were electroporated and the positivecolonies were selected by kanamycin-The M.smegmatis containing the vector pBCG-Sj26 can be induced by heating and hydrogen peroxide (H2O2) to express GST. The molecular weight of the recombinant GST(rGST) was 26000. The rGST contents that were about 10 percent of the total bacterial protein were analyzed by density scanning after running SDS-PAGE. This study would provide scientific evidences for application of hsp70 promoter in expressing foreign gene in mycobacterium and development of mycobacterium as multiple-valent vectoral vaccine.

Expression of Heat Shock Protein 70 and 27 in Non-small Cell Lung Cancer and Its Clinical Significance

The heat shock proteins （HSPs） 70 and HSP 27 expression in patients with non-small cell lung cancer （NSCLC） was studied and the relation.ship between HSP 70 and HSP 27 with the clinicopathological features of NSCLC was investigated. The expression of HSP 70 and HSP 27 was detected in tumor tissues from 60 patients with NSCLC by S-P immunohistochemistry. The findings were analyzed in combination with the histological types, histopathological differentiation, lymph node metastasis, patients＇ clinical stages, smoking history and gender. The results showed that of the 60 NSCLC tissue specimens studied, the immunoreactivity of HSP 70 and HSP 27 was detected in 47 （78.3 %） and 43 （71.7 %） specimens, respectively. A positive correlation was found between the overexpression of HSP 70 and HSP 27. The histopathological differentiation, lymph node metastasis, clinical stages and smoking history were correlated to the expression of HSP 70, but not to the expression of HSP 27. No statistical significance was observed in histological types and gender with respect to both HSP 70 and HSP 27 expression. It is suggested that the HSP 70 expression is a powerful and significant prognostic indicator and is related to histopathological differentiation, lymph node metastasis, patients＇ clinical stages, smoking history, whereas HSP 27 expression is not.

Effects of temperature on the respiratory metabolism,feeding and expression of three heat shock protein genes in Anadara broughtonii

Anadara broughtonii is one of the main commercially important species of shellfish in northern China.A.broughtonii lives in relatively stable subtidal zone where the clam could respond to environmental changes with minimum energy.Therefore,slight fluctuations in water environment may have a great impact on physiological processes such as feeding and metabolism.Large-scale mortality often occurs during the intermediate rearing processes,and high temperatures in summer are considered the leading cause of mortality.To understand the physiological and molecular response patterns of A.broughtonii at high temperatures and to estimate the appropriate metabolism temperature for A.broughtonii,the effects of temperature on the respiratory metabolism and food intake at different growth stages were studied.The response patterns of heat shock protein genes to heat stress and post-stress recovery were also explored.Results show that the temperature and growth stage of A.broughtonii were two important factors affecting metabolism and feeding.The optimum temperature for feeding and physiological activities in each shell-length group was 24℃.The temperature at which the peak metabolic rate occurred in each shell-length group increased with the increase in shell length.With the increase in heat stress,the expression of three heat shock protein genes(Abhsp60,Abhsp70,and Abhsp90)in the tissues of two size groups of A.broughtonii increased significantly when temperature was above 24℃and reached a peak at 30℃.After heat shock at 30℃for 2 h,the expression of the three heat shock protein genes rapidly increased,peaked at 2 h after the heat shock,and then gradually decreased to the level of the control group at 48 h after the heat shock.The three heat shock protein genes respond rapidly to heat stress and can be used as indicators to the cellular stress response in A.broughtonii under an environmental stress.

The polymorphism of heat shock protein 70 genes in Chinese Han population in Fujian province

To understand the polymorphism of the heat shock protein 70 （HSPTO） genes in Chinese Han population and to explore the co-relations between HSP70 polymorphism and disease, three polymorphic loci of HSP70 genes in 127 healthy Chinese Han population in Fujian province were analyzed by PCR and restriction enzyme analysis, and the genotypes and allele frequencies of HSPTO in different populations from various area were compared. It was found that the proportions of HSPTO-1 genotypes GG, GC and CC among Chinese Han population in Fujian province were 55.1%, 40.2% and 4.7% respectively, while those of HSP70-2 genotypes AA, AG and GG were 44.1%, 48.8 % and 6.9% respectively, and those of HSP70-hom genotypes TF, TC and CC were 59.8%, 37.0% and 3.2% respectively. The allele frequencies of G and C in HSP70-1 were 75.2% and 24.8% ; those of A and G in HSP70-2 were 68.5% and 31.5% and those ofT and C in HSP70-hom were 78.3% and 21.7% respectively. The distribution of the HSPTO-1 polymorphisms in Chinese Han population was almost the same as those in Japanese and Mexican populations, but it was rather different from those of American and Spanish populations with a significant differences. Meanwhile, the frequency of GG homozygote in HSPTO- 1 was signifi- cantly higher than those in American and Spanish populations. No significant difference was found in the distribution of HSPTO-2 polymorphism between Chinese and Japanese populations, in which the differences among American, Mexican and Spanish populations were quite obvious. The frequency of AA homozygote in HSPTO-2 was significantly higher than those in Mexican, American and Spanish populations, while, the distribution of HSPTO-hom genotype and allele frequency in Chinese Han population was almost just the same as those in Japanese and Mexican populations. Furthermore, it was also found that the genotype distribution and allele frequencies of the HSPTO genes in Han population of Fujian province were almost the same as those in Han population in Taiwan, but they were different in certain loci from those of Han population in Wuhan area. It is evident that the distribution of HSPTO gene polymorphisms among Chinese Han population are different from other regions in the world.

Cloning and Expression Analysis of Heat Shock Protein Gene DpHsp70 in Dahlia

[Objectives]This study was conducted to investigate the relationship between HSPs and the response of high temperature stress.[Methods]Molecular biological techniques were applied to clone and analyze the gene sequence of DpHsp70 gene.The changes in the expression of DpHsp70 gene under high temperature stress and exogenous salicylic acid(SA)were observed and further analyzed by qRT PCR.[Results]The coding region of the Dahlia DpHsp70 gene was 705 bp,encoding 234 amino acid residues(GenBank accession number:MH102288).Aligned with Compositae plants,the Dahlia DpHsp70 gene shared more than 83%homology in gene sequence while 99%-100%homology in amino acid sequence.Under the 35℃high temperature stress,the expression of DpHsp70 gene in Dahlia petals significantly increased.Meanwhile,the expression of DpHsp70 gene further increased under SA at 35℃temperature,which was significantly higher than those of the control group and the 35℃high temperature stress treatment group.It was demonstrated that the Hsp70 gene in Compositae is with highly conservative property and its expression could be up-regulated in response to high temperature stress.It can also be concluded that applying exogenous SA can improve the high temperature resistance of Dahlia.[Conclusions]This study provides a new experimental basis for elucidating the physiological function and mechanism of Dahlia in response to high temperature stress.

Construction, Expression and Identification of a Recombinant BCG Vaccine Encoding Human Mycobacterium Tuberculosis Heat Shock Protein 65

Heat shock protein 65 (HSP65) is one of the most important protective immunogens against the tuberculosis infection. The signal sequence of antigen 85B and the whole HSP65 DNA sequence of human Mycobacterium tuberculosis (M. tuberculosis) were amplified from BCG genome and plasmid pCMV-MTHSP65 respectively by polymerase chain reactions (PCR). These two sequences were cloned into the plasmid pBCG-2100 under the control of the promoter of heat shock protein 70 (HSP70) from human M. tuberculosis, yielding the prokaryotic shuttle expression plasmid pBCG-SP-HSP65. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis showed that the two cloned DNA sequences were consistent with those previously reported, and the direction of their inserting into the recombinant was correct and the reading frame had been maintained. The recombinants were electroporated into BCG to construct the recombinant BCG vaccine and induced by heating. The induced expression detected by SDS-PAGE showed that the content of 65 kD protein expressed in recombinant BCG was 35.69 % in total bacterial protein and 74.09 % in the cell lysate supernatants, suggesting that the recombinant HSP65 gene could express in BCG with high efficiency and the expressed proteins were mainly soluble. Western-blot showed that the secretive recombinant proteins could specifically combine with antibody against M. tuberculosis HSP65, indicating that the recombinant proteins possess the biological activity of HSP65.

Characterization of a TaJ Gene from Wheat

A novel J-domain protein gene was cloned from wheat （Triticum aestivum L.） using RT-PCR technology and named as TaJ. The J-domain protein is defined by the presence of a J-domain. The cDNA of T. aestivum gene, TaJ （GenBank accession number： DQ789026）, was 1263 bp and contained a complete open reading frame （ORF） encoding a J-domain protein of 420 amino acid residues. The predicted amino acid sequence of TaJ possesses three functionally essential domains： the Nterminal J-domain which includes the highly conserved HPD tripeptide, an adjacent domain that is rich in glycine and phenylalanine residues （G/F） and a Cysteine-rich zinc-finger domain with four repeats of CxxCxGxG that is important for protein interactions. The C-terminal of TaJ was -CAQQ, a farnesylation motif. The full-length deduced amino acid sequence of TaJ is highly homologous to J-domain proteins from various plant species. Southern blot analysis indicated that a single copy of TaJ existed in wheat genome. The expression pattern of TaJ performed by real-time PCR demonstrated that heat shock （HS） at 37℃ induced the expression of TaJ rapidly and strongly, but the response of the TaJ gene to cold stress was much slower than that to HS. Tissue-specific expression analysis showed that the expression level of TaJ gene was much higher in leaves than that in roots.

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 lepidopteran 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 ℃） 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℃ did not change the expression levels of any hsp in either species.

番茄转ER-sHSP基因植株构建及其抗冷性研究

将CaMV35S启动子驱动的内质网小分子热激蛋白基因导入番茄,比较转基因、未转基因和转空载体番茄的抗冷能力。冷胁迫下,同对照相比,转基因番茄的冷害症状轻,叶绿素含量的损失少,体内积累的丙二醛(MDA)含量少,电解质外渗程度低,具有较高的净光合速率和最大光化学效率,并易于恢复由低温所引起的光抑制,表明转基因番茄具有较强的冷胁迫耐性,说明内质网小分子热激蛋白在植物抗冷过程中发挥重要作用。

坛紫菜两种小分子热激蛋白(sHSP)基因的克隆及表达特征分析

小分子热激蛋白(s HSP)不仅在应激条件下高效表达,还在正常状态的细胞中广泛存在,参与一些重要细胞生理活动的调节。为研究坛紫菜应答高温和失水等逆境胁迫的分子机制,本研究以坛紫菜转录组测序获得的unigene序列为基础,采用RACE或直接PCR扩增,克隆获得了坛紫菜2种s HSP的全长基因:Ph Hsp22和Ph Dna J。序列分析结果表明,Ph Hsp22序列全长857 bp,包含一个519 bp的开放阅读框,所编码的多肽包含172个氨基酸,分子量为19.1 ku,等电点为5.24(收录号:KM102540);Ph Dna J序列全长1 616 bp,包含一个1 290 bp的开放阅读框,所编码的多肽包含429个氨基酸,分子量为46.1 ku,等电点为6.43,属于Hsp40亚家族(收录号:KM102541)。基因表达水平的定量分析结果表明,两个基因在高温胁迫不同时间水平和不同失水胁迫程度下的表达均呈现出一致的表达模式,即在胁迫初期表达水平显著上调,但随着胁迫的持续,这两个基因的表达水平开始逐渐下调。结果表明,这2个基因在逆境胁迫下的表达可能存在一个反馈调节机制。

高温(34℃)条件下家蚕热激蛋白基因BmsHSP27.4的表达变化

热激蛋白(heat shock protein,HSP)在保护细胞免受损伤或降低受损程度等方面发挥重要作用。BmsHSP27.4(GenBank登录号:AK382044)是从家蚕幼虫脂肪体中克隆的一个热激蛋白基因,蛋白质同源性分析发现BmsHSP27.4区别于以往发现的小热激蛋白,单独处于一个分支。以耐高温家蚕品种7532和对高温较敏感的普通家蚕品种皓月为材料,运用半定量RT-PCR和实时荧光定量PCR技术检测高温(34℃)条件下,BmsHSP27.4基因在不同家蚕品种以及同一家蚕品种不同性别5龄幼虫的中肠、丝腺和脂肪体组织中的表达水平。结果显示:高温处理后该基因表达水平在2个家蚕品种雌雄幼虫的3个组织中均呈快速上升的趋势,说明该基因是高温敏感基因;耐高温品种7532雄蚕脂肪体和中肠组织中该基因的表达水平高于雌蚕。该基因的表达可能与家蚕的耐热性有关。

松材线虫Bx-sHSP16A基因克隆及蛋白质结构研究

分子伴侣小热激蛋白(sHSPs)是线虫度过高温环境的关键因子。为开发直接针对松材线虫的防治技术提供理论依据,对松材线虫小热激蛋白Bx-sHSP16A基因进行了研究。采用SL法进行基因全长克隆,STRING9.0进行蛋白质互作网络分析,NAMD2.9进行分子动力学模拟。本研究克隆到松材线虫小热激蛋白基因Bx-sHSP16A。Bx-sHSP16A具α-crystallin保守结构域"F-polar-R-polar-aromatic-x-L-P"序列和"I/L-x-I"序列,初步判断具备分子伴侣功能。Bx-sHSP16A与多个蛋白发生互作,深入研究这些蛋白有助于了解Bx-sHSP16A在应激反应路径中的作用。Bx-sHSP16A二聚体中A亚基的"I/L-x-I"序列可以嵌入B亚基的去水合位点,契合后氢键稳定,说明Bx-sHSP16A可以形成多聚体。在应激反应过程中,去水合位点与多聚体聚合相关,Bx-sHSP16A以多聚体形式发挥分子伴侣功能。

sHsps在小鼠正常腭发育和腭裂发生过程中的表达

目的 研究sHsps在正常腭发育及腭裂发生过程中的表达情况。方法 在GD10经口一次分别给予实验组孕小鼠80mg kg的视黄酸(RA)、对照组孕鼠等体积的植物油,并分别于GD15 GD17取两组胎鼠的腭,利用RT- PCR方法半定量检测实验和对照组中sHsps的表达丰度。结果 正常腭除Hspb10在GD17不表达外,其余Hsps基因在GD15 - GD17均有明显表达,Hsp2 0、Hsp2 5、Hsp2 7、Hsp32、Hspb2、Hspb3、Hspb7表达丰度基本恒定,Hsp30、Hspb5随胚龄的增加而表达丰度增高,Hsp10、Hsp2 2、Hspb4、Hspb9在GD16出现表达高峰。Hsp30、Hsp32、Hspb4、Hspb10在GD15 - GD17的表达丰度腭裂组明显高于正常组。Hsp10、Hsp6 0、Hspb5、Hspb9在GD15 - GD17的表达丰度腭裂组明显低于正常组。Hspb9、Hspb10在正常腭发育和腭裂发生过程中的表达模式明显不同。结论 15种Hsps除Hspb10在GD17不表达外,其余在GD15 - GD17正常腭发育过程中均有明显表达,但不同Hsps的表达特点有所不同;Hsp30、Hsp32、Hspb4、Hspb10在腭裂发生中可能主要起应激保护反应,Hsp10、Hsp6 0、Hspb5、Hspb9、Hspb10可能与腭裂的发生有关。

转ER-sHSP基因番茄植株的耐盐性检测

旨在检测内质网小分子热激蛋白(ER-sHSP)的过量表达对植物耐盐性的影响。利用Hoagland营养液配制200mmol/LNaCl溶液并处理转ER-sHSP基因番茄2周,测定植物的生物量、脯氨酸含量、SOD酶活、MDA含量及相对电导率。结果显示:与对照相比,转基因番茄抗盐表型明显,植物干鲜重分别比对照增加57%和53%,脯氨酸含量和SOD酶活性的平均值分别是对照番茄的1.7倍和3倍,但MDA含量和相对电导率却显著降低(P<0.01)。综上所述,转基因番茄具有较高的耐盐能力,说明ER-sHSP在植物的耐盐机制中发挥重要作用。

家蝇小热休克蛋白(sHsp20.6)的生物信息学分析

研究家蝇小热休克蛋白sHsp20.6的生物学功能。方法应用生物信息学的方法和工具对家蝇sHsp20.6的理化性质、疏水性、跨膜区和信号肽、膜体分析、二级结构功能域、蛋白质的功能分类预测、多重序列比对与系统发育树构建、三级结构建模进行分析。结果表明:家蝇sHsp20.6是一个亲水蛋白,分子量为20.64kD,等电点为5.66,不具有跨膜区和信号肽,包含有一个HSP20的结构域,主要构成原件为α螺旋和无规则卷曲,三维结构预测显示该蛋白为棒状结构,C端结构域具有7个片层结构。聚类分析显示,家蝇sHsp20.6蛋白与昆虫中的直系同源小热休克蛋白(orthologoussmallheatshockprotein)聚为一类。