The Expression Characteristics and Potential Functions of Heat Shock Factors in Diatom Phaeodactylum tricornutum

Heat shock transcription factor(HSF)are essential regulators of heat shock protein(HSP)gene expression in plants and algae,contributing to their resilience against biotic and abiotic stresses.However,the localization,structure,phylogenetic relationship,and characteristics of PtHSF genes in microalgae,especially in diatom Phaeodactylum tricornutum,remain largely unexplored.This study presents a comprehensive analysis of the PtHSF gene family in P.tricornutum.A genome-wide analysis identified 68 PtHSF genes,which were classified into two distinct subfamilies:traditional and untraditional.Motif and structure analyses revealed evidence of multiple duplication events within the PtHSF gene family.Expression profiling revealed diurnal patterns,with 34 genes being downregulated during the light period and upregulated during the dark period,while 19 genes exhibited the opposite pattern.These findings suggest that PtHSF genes may have specialized functions during the diurnal cycle and play a crucial role in maintaining cellular homeostasis in response to various stresses.Notably,PtHSF16,30,and 43 genes exhibited higher expression levels,suggesting their potential importance.This study provides a valuable foundation for future investigations into the specific functions of HSFs under different stress conditions and their regulatory mechanisms in P.tricornutum and other microalgae.

Prokaryotic Expression and Purification of Heat Shock Factor HSF1 in Arabidopsis thaliana

[ Objective ] This study was to express and purify Arabidopsis thaliana heat shock factor HSF1. [ Method ] Using Escherichia coli M15 harboring HSF1 （pQE32/His6-HSF1, pREP4） as experimental materials, HSF1 was induced to express with isopropyl-β-D-galactoside （IPTG） ; then the expression product was purified using Ni-NTA-agarose affinity chromatography and analyzed by SDS-PAGE. [Result] HSF1 of Arabidopsis thaliana was successfully expressed and purified. [ Conclusion] This study provides materials for understanding the blinding site of HSF1 on Arabidopsis thaliana chromosome, further laying a good foundation for revealing the regulatory mechanism and physiological function of HSF1.

Construction of Plant Expression Vector of Heat Shock Factor Gene AtHsfA1a from Arabidopsis

[ Objective ] Heat shock factors （HSFs） are the major transcription factors of eukaryotic heat shock responses. This study aims to investigate the adversity stress tolerance functions of Arabidopsis heat shock factor AtHsfAla, which has important significance for in-depth understanding of adversity stress tolerance mechanisms of plants and further utilization of heat shock factor genes. [Method] Genomic DNA of Arabidopsis was extracted with CTAB method and purified to obtain Arabidopsis DNA samples for in vitro site-specific recombination cloning （ Gateway cloning） to construct plant expression vector of heat shock factor AtHs- fAla. Firstly, donor vector pDONR 201/AtHsfAla was constructed based on attB and attP site-specific recombination method （BP reaction）, to identify E. coli transformants harboring correct sequence of AtHsfAla by sequencing; secondly, plant expression vector pBTWG2/AttlsfAla overexpressing Arabidopsis heat shock factor AtHsfAla was constructed based on attL and attR site-specific recombination method （LR reaction）, to screen E. coli transformants harboring target plasmid. [ Result] Plant expression vector of Arabidopsis heat shock factor gene AtHsfAla was constructed successfully. [ Conclusion] This study not only provided experimental materials for acquiring transgenic plants overexpressing heat shock transcription factor AtHsfAla, but also laid the foundation for further investigation of the diversity of adversity stress tolerance functions reanlated by HSFs.

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 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.

Effect of the C.–1388 A>G polymorphism in chicken heat shock transcription factor 3 gene on heat tolerance

Heat stress is one of the main factors that inlfuence poultry production. Heat shock proteins (HSPs) are known to affect heat tolerance. The formation of HSPs is regulated by heat shock transcription factor 3 (HSF3) in chicken. A DNA pool was established for identifying single nucleotide polymorphisms (SNPs) of the chicken HSF3, and 13 SNPs were detected. The bioinformatic analysis showed that 8 SNPs had the capacity to alter the transcription activity of HSF3. The dual luciferase report gene assay showed that there was a signiifcant difference (P<0.01) in the Firelfy luciferase/Renil a luciferase ratio (F/R) of C.–1 703 A>G (S1) and C.–1 388 A>G (S4) sites at the 5′-untranslated region (UTR) of chicken HSF3. The elec-trophoretic mobility shift assay showed that the S4 site was a transcription binding factor. The analysis of the association of the S1 and S4 sites with heat tolerance index revealed that the S4 site was signiifcantly correlated with the CD3+T cel , corticosterone, and T3 levels in Lingshan chickens and with the heterophil/lymphocyte value in White Recessive Rock. These results showed that the S4 site at the 5′ UTR of chicken HSF3 might have an impact on heat tolerance in summer and could be used as a potential marker for the selection of chicken with heat tolerance in the future.

Influence of High Temperature on the Expression of Arabidopsis Heat Shock Transcription Factor AtHsfA1a

[ Objective] This study ~med to investigate the influence of high temperature on the expression of heat shock transcription factor AtHsfAla in different genotypes of Arabidopsis. [ Method ] Arabidopsis plants overexpressing heat shock transcription factor AtHsfA1 a were used as experimental materials and treated un- der high temperature at 39℃ for 1 rain and 5 min; total RNA of AtI-IsfAla was extracted, and the reverse transcription and amplification were conducted using RT- PCR technology, the amplification products were detected by electrophoresis. [ Result ] The expression levels of AtHsfA1 a in Arabidopsis plants overexpressing heat shock transcription factor AtHsfAla at high temperature and room temperature were higher than wild-type Arabidopsis; the expression levels of AtHsfAla in both wild-type Arab/dops/s and transgenic Arabidopsls plants overexpressing heat shock transcription factor AtHsfAla at high temperature of 39 ~C were higher than that at room temperature of 25 ~C, but the expression levels of AtHsfAla in wild-type Arab/dops/s and transgenic Arab/dops/s plants overexpressing heat shock transcription factor AtHsfAla varied little after high temperature treatment at 39 ~C for 1 rain or 5 rain. [ Conclusion] The expression of AtHsfAla is induced rapidly by high tem- perature, thus regulating the expression of early adversity-resistant genes. This study will lay the foundation demonstrating the mechanism of Arabidopsis heat shock transcription factor AtHsfAla.

Microarray-based Screening of Target Genes Regulated by Heat Shock Factor AtHsfA1a in Arabidopsis thaliana

[ Objective] This study aimed to screen target genes regulated by heat shock factor AtHsfAla in Arabidopsis thaliana. [ Method] Using AtHsfAla-in- serted mutant athsfala （SALK-068042） and wild-type A. thaliana seedlings as experimental materials, target genes regulated by heat shock factor AtHsfAla were screened by microarray assay. Differentially expressed genes were screened by multiple method. Specific functions of differentially expressed genes were analyzed by gene ontology （GO） analysis. Signal transduction pathways, in which differentia｜ly expressed genes were involved, were analyzed by pathway analysis. Gene-gene interaction network was constructed by Signal-Net. [ Result] A total of 3 672 differentially expressed genes were screened out. Up-regulated differentially expressed genes were involved in 198 functions and 7 signal transduction pathways; down-regulated differentially expressed genes were involved in 94 functions and 10 signal transduction pathways. In the signal transduction network, it was found that cwlNV4 and HXK3 had relatively high ability of mediation; AT1 G14240 and cwlNV4 ex- hibited the most interactions with other genes, which were located in key positions throughout the gene-gene interaction network. [ Conclusion] Heat shock factor AtHsfAla regulates a large number of target genes in A. thaliana.

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.

Establishment of a tetracycline-off and heat shock-on gene expression system in tobacco

The tetracycline （Tet）-off gene expression regulation system based on the TetR-VP16/Top10 construct has not been widely utilized in plants, for its highly expressed TetR-VP16 activator is toxic to some plants and repeatedly replenishing tetracycline to turn off the constitutively active system is a tedious process. To solve these problems, a Tet-off and heat shock （HS）-on gene expression regulation system was constructed in this study. This system is composed of a chimeric transactivator gene TetR-HSF that is derived from a Tet repressor （TetR） and a HS transcription factor （HSF） controlled by a HS promoter HSP70m, and a Tet operator containing hybrid promoter, Om35S, that drives expression of the β-glucuronidase （GUS） gene. The resultant system yields a GUS expression pattern similar to that of the HSP70m promoter under inducing temperatures and at 35 and 40℃ drives GUS expression to a similar level as the Cauliflower mosaic virus （CaMV） 35S promoter. Further examination revealed that the TetR-HSF and GUS genes were induced by HS, reaching peak expression after 1 and 6 h treatment, respectively, and the HS induction of the expression system could be inhibited by Tet. This system will provide a useful tool for transgenic studies of plants in the laboratory and in the field, including transgene function analysis, agronomic trait improvement, biopharmaceutical protein production and others.

THE ROLE OF INTRONIC HSE IN THE HEAT SHOCK INDUCED EXPRESSION OF HUMAN hsp 90β GENE

The first intron of human hsp90β gene is not only essential in maintaining high constitutive expression but also critical for heat shock inducibility of the hsp90β gene. Typical HSEs in the first intron play a vital role in the heat induced expression of human hsp90β gene. Slot blot analysis shows that hsp90β gene mRNA transcripts initiated from the 3’ of the first intron dominates over that of the first exon. The intronic HSEs of the hsp90β gene show much higher binding affinity toward the recombinant heat shock factor HSF1 than that of the recombinant heat shock factor HSF2.

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.

Constitutively Expressed αB—Crystallin in Heat Schock Transcription Factor 1 Knockout Mice Myocardium

Objective-To investigate the effects of heat shock transcription factor 1) gene on the constitutivety expressed αB-CrystaUin (aBC) in mice myocardium. Methods-The expression levels of constitutive aBC in HSF1 knockout (hsf1 - /- ) and HSFl wild type (As/1 + /+) mice myocardium were evaluated by western blot and immunohistochemistry. Results : The αBC levels in hsfl -/- and hsfl +/+ were 68. 42±4. 16, 100. 00±7. 58, respectively (P<0. 05, cytoso-lic fraction) , and 20. 53±1. 01, 37. 55±1. 91, respectively (P<0. 05, pellet fraction). The aBC signals decreased significantly in hsfl -/- myocardium when compared with those in hsfl +/+ myocardium stained with fluorescence immunohistochemistry. Conclusion-HSF1 is an important, but not the only factor, which mediates the constitutively expressed aBC.

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.

Real-time cell analysis and heat shock protein gene expression in the TcA Tribofium castaneum cell line in response to environmental stress conditions

The rust red flour beetle, Tribolium castaneum （Herbst, 1797） （Coleoptera： Tenebrionidae）, is a pest of stored grain and one of the most studied insect model species. Some of the previous studies involved heat response studies in terms of survival and heat shock protein expression, which are regulated to protect other proteins against environ- mental stress conditions. In the present study, we characterize the impedance profile with the xCELLigence Real-Time Cell Analyzer and study the effect of increased temperature in cell growth and viability in the cell line BCIRL-TcA-CLG 1 （TcA） of T castaneum. This novel system measures cells behavior in real time and is applied for the first time to insect cells. Additionally, cells are exposed to heat shock, increased salinity, acidic pH and UV-A light with the aim of measuring the expression levels of lisp27, Hsp68a, and Hsp83 genes. Results show a high thermotolerance of TeA in terms of cell growth and viability. This result is likely related to gene expression results in which a significant up-regulation of all studied Hsp genes is observed after 1 h of exposure to 40 ~C and UV light. All 3 genes show similar expression patterns, but Hsp27 seems to be the most affected. The results of this study validate the RTCA method and reveal the utility of insect cell lines, real-time analysis and gene expression studies to better understand the physiological response of insect cells, with potential applications in different fields of biology such as conservation biology and pest management.

Transcript analysis and expression profiling of three heat shock protein 70 genes in the ectoparasitoid Habrobracon hebetor （Hymenoptera： Braconidae）

Heat shock proteins （HSPs） are known as chaperones that help with folding of other proteins when cells are under environmental stresses. The upregulation of HSPs is essential for cold survival during insect diapause. The ectoparasitoidHabrobracon hebetor, a potential biological control agent, can enter reproductive diapause when reared at low temperature and short photoperiod. However, the expression of HSPs during diapause of H. hebetor has not been studied. In this study, we sequenced and characterized the full-length complementary DNAs of three Hsp70 genes （HhHsp70I, HhHsp70II and HhHsp70IIl） from 11. hebetor. Their deduced amino acid sequences showed more than 80% identities to their counterparts from other insect species. However, the multiple se- quence alignment among the three deduced amino acid sequences of HhHsp70s showed only 46% identities. A phylogenetic analysis of the three HhHsp70s and all other known Hsp70 sequences from Hymenoptera clustered all the Hsp70s into four groups, and the three HhHsp70s were distributed into three different groups. Real-time quantitative poly- merase chain reaction analysis showed that the expression of the three HhHspTO genes in H. hebetor reared at different conditions was quite different. HhHspTOI showed higher relative expression when H. hebetor were reared at 27.5℃ than at two lower temperatures （17.5℃ and 20℃） regardless of the photoperiod, whereas HhHspTOII showed higher ex- pression when H. hebetor were reared at 20℃ and 10 ： 14 L ： D than when reared at 17.5℃ and either 16 ： 8 L ： D or 10 ： 14 L ： D. In contrast, HhHSP7OIIIwas expressed at similar levels regardless of the rearing conditions. These results may suggest functional differences among the three HhHspTO genes in H. hebetor.

Effect of Temperature on Gene Expression in the Pearl Oyster Pinctada fucata

In this study, we examined the effect of elevated temperature on the expression patterns of genes, i.e., nacrein, irr, n16, n19, and hsp70 in the pearl oyster Pinctada fucata. The experiment was carried out at 4 temperatures, i.e., 20℃(ambient, control), 24, 28℃, and 32℃. The expression levels of target genes in P. fucata were assayed at 0, 6, 24, 48, and 96 h via real-time polymerase chain reaction. Results showed that the expression levels of nacrein and irr had no significant variations among different time points below 28℃, but significantly increased over time at 32℃. The expression levels of n16 and n19 did not change markedly at 20℃. The former increased significantly at 6 h and 24 h while the latter substantially decreased during 6–96 h at 24, 28 and 32℃. Among different temperatures, the level of n16 was significantly lower at 20℃ than at other temperatures during 6–96 h, and the level of n19 significantly varied among different temperatures at 48 h and 96 h. The expression level of hsp70 was significantly higher at 32℃ than at 20, 24 and 28℃ at 24 h. These results demonstrated that elevated temperature impacted the physiological processes of P. fucata and potentially influenced its adaptability to thermal stress.

Cloning and expression of Hsp22.4 gene from Chaetomium globosum

A study was conducted on the molecular mechanism of small heat shock proteins （sHSPs） in Chaetomium globosum. Heat shock protein 22.4 （Hsp22.4） from C. globosum was cloned and expressed in Escherichia coli. BlastX analysis revealed that the Hsp22.4 gene from C. globosum shared the highest identity in amino acid sequence with a Hsp gene from Neurospora crassa, and the identity between them was 65%. The C. globosum Hsp22.4 gene was inserted into the expressive vector of pGEX-4T-2 and the recombinant plasmid named pGEX-HSE E. coli BL21 transformed with pGEX-HSP plasmid was induced by IPTG, and the expressed proteins were analyzed with SDS-PAGE. A 50 kD protein was specially expressed in E. coli BL21, and the result was consistent with expectation, and showed that the Hsp22.4 gene had been expressed in E. coli. Our study has made a foundation for further studying the function ofsHSPs protein.

热激转录因子HSF调控植物非生物胁迫响应的作用机制

热激转录因子(Heat shock transcription factors,HSF)是植物抵抗热胁迫最重要的转录因子家族之一,广泛存在于真核生物。HSF拥有高度保守的DNA结合域,可参与复杂的胁迫信号转导和响应网络。HSF作为信号转导链的末端组分,介导非生物逆境胁迫下靶标基因的表达。在逆境胁迫响应中,HSF受上游蛋白激酶介导磷酸化和泛素化或通过ABA信号通路结合热激元件,调控热激蛋白等下游基因的表达,从而提高植物的抗逆性。本文综述HSF的发现、结构、分类、调控机制及其在植物响应非生物逆境胁迫(极端温度、干旱、高盐碱和重金属等)中的作用机制,并对未来研究方向加以展望,以期为农作物和牧草抗逆性遗传改良提供理论依据和基因资源。