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 3,060 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 it was also detected 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.

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 3,060 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 it was also detected 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.

We have previously investigated the proteome changes of rice leaves under heat stress (Lee et al. in Proteomics 2007a, 7:3369- 3383), wherein a group of antioxidant proteins and heat shock proteins (HSPs) were found to be regulated differently. The present study focuses on the biochemical changes and gene expression profiles of heat shock protein and antioxidant genes in rice leaves in response to heat stress (42°C) during a wide range of exposure times. The results show that hydrogen peroxide and proline contents increased significantly, suggesting an oxidative burst and osmotic imbalance under heat stress. The mRNA levels of chaperone 60, HSP70, HSP100, chloroplastic HSP26, and mitochondrial small HSP responded rapidly and showed maximum expression after 0.5 or 2 h under heat stress. Transcript levels of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and Cu-Zn superoxide dismutase (Cu-Zn SOD) showed a rapid and marked accumulation upon heat stress. While prolonged exposure to heat stress resulted in increased transcript levels of monodehydroascorbate reductase, peroxidase, glyoxalase 1, glutathione reductase, thioredoxin peroxidase, 2-Cysteine peroxiredoxin, and nucleoside diphosphate kinase 1, while the transcription of catalase was suppressed. Consistent with their changes in gene expression, the enzyme activities of APX and DHAR also increased significantly following exposure to heat stress. These results suggest that oxidative stress is usually caused by heat stress, and plants apply complex HSP- and antioxidant-mediated defense mechanisms to cope with heat stress.

Diapause duration of Paratlanticus ussuriensis is prolonged as an egg that enter both initial and final diapause stgaes. Environmental conditions, such as temperature, can modify the duration of initial diapause. Eggs enter initial diapause at 20℃, but continued early embryonic development at 30℃. Final diapause at a fully developed embryonic stage is obligatory regardless of temperature conditions. To determine temperature effects on initial diapause mechanism of P. ussuriensis eggs, we compared weights, DNA and RNA amounts of eggs incubated at either 20℃ or 30℃ for 50 days after oviposition. We identified small heat shock protein (shsp), heat shock protein 90 (hsp90) and three heat shock protein 70 (hsp70a, hap70b, hsp70c) genes of P. ussuriensis and determined those expression levels at different temperature conditions. The levels of shsp, hsp70a, hsp70b and hsp90 was not detectable until 20 days after oviposition at both temperature conditions, but highly increased at 50 and 60 days when incubated at 30℃. In contrast, hsp70c level was rapidly peaked at 20 days after oviposition, which is the time of initial diapause entrance. We analysis of temperature sensitivity of P. ussuriensis eggs. Hsp70a is expressed after the first cold treatment of mature eggs. Hsp70b is highly expressed just before hatching. Both shsp and hsp70c was highly expressed at the heat shock condition into immature egg stage. Our results suggest that high temperature breakdown initial diapause and one hsp gene, such as hsp70c, may be involved into the mechanism of initial diapause of P. ussuriensis eggs.

프로테오믹스 기법을 이용하여 벼 고온 스트레스 관련 단백질을 분리 동정하기 위하여 에서 고온처리한 벼의 줄기로부터 단백질을 분리하였다. 분리한 단백질로부터 Rubisco 단백질을 제거하기 위해 15% PEG fractionation을 실시한 후 상등액 분획의 단백질을 이차원전기 영동한 후, CBB 염색을 통해 차별적 발현을 보이는 단백질을 분석하였다. 총 46개의 단백질 spot이 발현양에 변화를 보였으며, 그 중 24개의 단백질이 고온 스트레스에 의해

Many thousands of recombinant proteins have been successfully produced in baculovirus - infected insect cells and larvae. In this study, to improve its value and the yield of recombinant protein production, we constructed transgenic silkworm using Heat shock genes with regard to protein folding. This time, we adapted GAL4/UAS system to express at necessary time point and to carry genes for foreign protein. First, we generated two transgenic cells and silkworm lines that carried the silkworm heat shock proteins, UAS-HOP and UAS-HSC70 and UAS-HSP70 and UAS-HSP40 construct plus 3xP3-DsRED. Subsequently, to drive the GAL4 gene as activatorvector, we engineered Baculoviruses that contain the GAL4 under the P10 promoter linked to the expression cassette of interest foreign genes under the polyhedron promoter. Also, activator vector linked to the GAL4 was designed expressing 6xHis and 6xHis–GST tag. Infection of silkworm larvae with recombinant virus, His-tagged human C3d gene was more efficiently produced transgenic silkworm than that of wild-type, but not His-GST tagged. We show the possibilityin use of HSPs transgenic silkworm system by GAL4/ UAS BmNPV that can generate the efficient production of foreign protein.

The genomic structure and phylogenetic relationships of HSP88 genes from P. tenuipes Jocheon-1, P. tenuipes, C. militaris and C. pruinosa are described. The HSP88 genomic DNA from P. tenuipes Jocheon-1, P. tenuipes and C. militaris all contain 5 introns and 6 exons with the length of 13, 62, 32, 1438, 306, 288 bp, encoding 713 amino acid residues. C. pruinosa HSP88 genomic DNA contains 4 introns and 5 exons encoding 713 amino acids. The length of each exon of C. pruinosa HSP88 is 13, 62, 32, 1744, 288 bp and the length of exon 4 is identical to the total length of exon 4 and exon 5 of HSP88 of P. tenuipes Jochoen-1, P. tenuipes, and C. militaris. The deduced amino acid sequence of P. tenuipes Jocheon-1 HSP88 showed 99% identity with the P. tenuipes, 97% identity with the Cordyceps militaris, and 98% identity with the C. pruinosa. Phylogenetic analysis confirmed that the P. tenuipes Jocheon-1, P. tenuipes, C. militaris and C. pruinosa HSP88 are placed together within the ascomycetes group of fungal clade.

In this study, a full-length heat shock protein88 complementary DNA (cDNA) of Paecilomyces tenuipes Jocheon-1 was obtained by screening of P. tenuipesJocheon-1 Uni-Zap cDNA library and 5' RACE polymerase chain reaction. The Paecilomyces tenuipes Jocheon-1 heat shock protein88 cDNA contains an open reading frame of 2,139 bp encoding 713 amino acid residues. The deduced amino acid sequence of the P. tenuipes Jocheon-1 HSP88 cDNA showed 77% identity to N. haematococca HSP88 and 45-76% identity to other fungi HSP88. Phylogenetic analysis and BLAST program analysis confirmed that the deduced amino acid sequences of the P. tenuipes Jocheon-1 HSP88 gene belonged to the ascomycetes group within the fungal clade and P. tenuipes Jocheon-1 HSP88 also contains the conserved ATPase domain at the N-terminal. The cDNA encoding P. tenuipes Jocheon-1 HSP88 was expressed as a 88 kDa polypeptide in baculovirus-infected insect Sf9 cells. Under different stress conditions, mRNA expression of P. tenuipes Jocheon-1 HSP88 were quantified by real-time PCR and the result showed that heat shock stress affected the mRNA expression levels of P. tenuipes Jocheon-1 HSP88.

For stable germline transformation, the promoter of B. mori cytoplasmic actin gene (BmA3) was used to ubiquitous expression of transgenes. Except for BmA3 promoter, promoters used to regulate gene expressionin all tissues and developmental stages of B. mori were not nearly developed. To identify more powerful promoter than previously reported BmA3 promoter (Mange et al., 1997), we introduced a new dot blot hybridization method, and isolated nine clones that show stronger dot signal compared to the control, BmA3by this method. Among these 9 clones, we focused on one clone which has high amino acid homology (94%) with heat shock protein 70 gene of Trichoplusia ni. This resulting positive clone, named bHsp70 (B. mori heat shock protein 70) was ubiquitiously expressed in tissues and developmental stage of fifth instar B. mori larvae,and stimulated bythermal and ER stress. As result of promoter assay using dual luciferase assay system, we found the highest transcription activity region (-1003/+147) in the 5'-flanking region of bHsp70 gene that has 264-fold more intensive promoter activity than BmA3 promoter. Moreover, transcription activity of bHsp70 promoter under heat shock condition (42 ℃, 4 hr) was increased over 2-fold than normal condition. Therefore, we suggest that bHsp70 promoter may be used more effective candidate for transgene expression in B. mori.

We investigated the effects of cadmium exposure and various stress on the transcription of heat shock protein 70 and 82 (HSP70 and HSP82) from Pardosa astrigera wolf spider. To do this, P. astrigera HSP70 and HSP82 genes were cloned and its full-length sequence determined. Female spiders were long-term exposed to cadmium or to polychlorinated biphenyl (PCB) for 2, 4 and 6 weeks and short-term exposed to endosulfan by dietary uptake. Female spiders were also exposed to various temperatures. HSP82 did not show a clear tendency of transcription induction following exposure to cadmium. On the contrary, HSP70 transcription gradually increased during the exposure to 2, 20 and 40 mM of cadmium for 2, 4 and 6 weeks. Transcript level of HSP70 was not significantly changed by endosulfan and PCB exposure. In the short-term (3 hr) temperature exposure, an increased expression of HSP70 was observed under the heat shock to 30°C and then slightly decreased at 35°C. However, induction of HSP70 transcription was not observed during the long-term (7 days) temperature exposure. Taken together, HSP70 gene appears to be up-regulated by cadmium in a time-dependent manner but little affected by other potential contaminants. Analysis of HSP70 transcript levels in P. astrigera collected from various fields revealed that levels of cadmium concentration were well correlated with HSP70 transcript levels (r2 = 0.76). Taken together, it was suggested that transcript level of HSP70 could be useful as a biomarker for the long-term cadmium exposure of P. astrigera.

Recently Transgenesis was achieved in Bombix mori. For stable and effective transgenesis in B.mori, B.mori cytoplasmic actin gene (BmA3) promoter was used to expression of marker gene, the green fluorescent protein(GFP). Green fluorescent protein expression for selection of transformants was visible in all larval, pupal, and adult tissues but, unexpectdly, was not detectable in embryos. So, it spend times and money on rearing of silkworm. Furthermore, the BmA3 promoter is predominantly active in the midgut, which makes it difficult to reliably identify transformants since autofluorescence of many insect foods can mask low-level fluorescence and only allows the detection of strongly expressing individuals with potentially multiple insertions. Therefore, we need more intensely promoter than BmA3 promoter for selected by expression of GFP in embryos and selected by reliable expression of GFP in larvae. We performed dot blot hybridization to develop strong promoter. Nine differentially expressed clones were isolated and we focused one clone of them which has high similarity with heat shock protein 70 gene from D.melanogaster. We named it as bHSP70 (Bombyx mori heat shock protein 70). Expression from the hsp70 promoter was strong and heat shock-dependent. And Drosophila hsp70 promoter appears useful for regulating expression of Exogenous DNA. So, we analyzed transcriptional activity of promoter with bHSP70 gene by using dual luciferase assay system. bHSP70 promoter has about 264 folds more intensely than BmA3 promoter. Also, when bHSP70 promoter treated heat shock(42℃), transcriptional activity incresed 2 times more than normal condition. Therefore, we suggest that bHSP70 promoter is more effective candidate for stable transformation and selection of transformants.

We examined the effects of cadmium exposure and various temperature stress on the expression of Pardosa astrigera heat shock protein 70 (HSP70). To do this, P. astrigera HSP70 gene was cloned and its sequence determined. Female spiders collected from non-contaminated region were exposed to 40mM CdCl2 for 2, 4 and 6 weeks by dietary uptake. At the end of every 2, 4 and 6 weeks of exposure, a batch of 5 spiders was collected and total RNA was extracted from each batch of whole bodies. Female spiders were also exposed to different temperatures (20, 25, 30 and 35℃) for 3h and RNA extracted likewise. Transcription profiles of HSP70 in response to cadmium and temperature were determined by quantitative real-time PCR using 18S rRNA as reference gene for data normalization. HSP70 transcription gradually increased during 2,4 and 6 weeks of exposure to cadmium. In particular, the expression level at 6-week exposure was 3.4-fold higher than that of untreated control. In the temperature response, an increased expression of HSP70 was also observed as temperature increased up to 30℃ and then slightly decreased at 35℃. The expression level at 30℃ was 2.3-fold higher than that of 25℃. Taken together, HSP70 gene appears to be up-regulated by general stress factors including cadmium exposure and temperature increase.

외부에서 투여된 열자극, 알콜 및 생리적 염과 같은 환경 스트레스는 체내 각 부위에서 스트레스단백질(열자극단백질, HSP)을 생성하게 된다. 본 연구에서는 비소가 흰쥐 대동맥의 수축에 미치는 영향을 조사하기 위해 스트레스단백질의 발현과 대동맥의 수축력의 변화와 이들과의 관계를 알아보고자 실험을 실시하였다 적출한 혈관은 organ bath에 담가 0, 0.5, 1, 2,및 4 mM As를 처리한 후 1, 3, 및 8시간 뒤에 KCI(55 mM)에 대한 수