高温对家蚕三品系血淋巴中糖水平的影响（英文）

家蚕Bombyx mori的两个二化性品系热耐受型NB4D2和热敏感型CSR2均适合于温带气候,而多化性的PM（Pure Mysore) 品系适合于热带气候,将这3种品系5龄幼虫分别置于32℃和36℃的高温下,观察高温对其5龄幼虫至蛹期血淋巴中糖含量及海藻糖酶活性的影响。结果表明： PM幼虫和蛹的死亡率均小于NB4D2和CSR2。在蜕皮期间血淋巴海藻糖水平较高,而葡萄糖水平及海藻糖酶活性较低。32℃和36℃的高温下,幼虫蜕皮期间血淋巴中糖含量及海藻糖酶活性仅在其各自的水平上表现为小幅度的增加。蜕皮后幼虫血淋巴中海藻糖含量显著下降,而葡萄糖含量和海藻糖酶活性显著上升。在较高温度下,蜕皮后幼虫血淋巴中海藻糖含量下降幅度更大,而葡萄糖含量及海藻糖酶活性上升水平也更加显著。25±1℃下取食幼虫血淋巴中葡萄糖含量显著下降,海藻糖含量显著上升;3℃和36℃下PM 和NB4D2取食幼虫血淋巴葡萄糖和海藻糖含量以及海藻糖酶活性增加,而CSR2均减少或降低。吐丝幼虫血淋巴中葡萄糖含量及海藻糖酶活性显著下降,海藻糖小幅度下降。而在较高温度下,耐热型PM 和NB4D2吐丝家蚕血淋巴糖含量含量和海藻糖酶活性明显增加,而热敏感型CSR2的则明显下降。这3种品系蛹发育期的血淋巴糖含量及海藻糖酶活性均下降。在两较高温度下,PM蛹期血淋巴糖和海藻糖酶活性增加,而NB4D2 36℃时增加幅度小于32℃时。对于CSR2,32℃时观察到其血淋巴葡萄糖含量增加,但当环境温度增加到36℃时其血淋巴葡萄糖含量降至正常水平下。然而,当CSR2的蛹置于32℃和36℃时血淋巴海藻糖含量及其酶活性下降,且36℃时下降幅度更大。因此,桑蚕对高温的适应取决于家蚕的品系及发育阶段,并可通过其血淋巴糖及海藻糖酶活性水平进行验证。     

桑叶营养对家蚕血淋巴阳离子水平的调节作用

调查了取食不同品种桑树（M-5, S-36和V-1）叶片的家蚕（多化性品种Pure Mysore、 二化性品种NB4D2和CSR2）5龄幼虫血淋巴中阳离子的变化。结果表明: 家蚕幼虫血淋巴中Na+浓度低, K+和Mg2+浓度很高, Ca2+浓度较高。在幼虫活跃取食期间, 血淋巴中阳离子水平显著提高。血淋巴中阳离子水平与排泄物的量呈负相关。血淋巴中阳离子水平受阳离子外泌的控制。与非取食阶段相比, 取食阶段的阳离子清除速率低。家蚕二化性品种的血淋巴阳离子水平比多化性品种PM高出30%。结果说明家蚕品种对家蚕血淋巴阳离子变化的影响显著大于桑树品种或家蚕个体发育的影响。     

家蝇幼虫血淋巴蛋白组及血淋巴中热稳定蛋白研究

目的 初步研究家蝇幼虫血淋巴蛋白组学特征及其中的热稳定蛋白.方法 使用破壁法提取三龄幼虫血淋巴原液,设置全血淋巴组、沸水浴处理组,采用不同pH范围双向电泳胶条,分析血淋巴中蛋白的pH、分子量分布特点,采用二级质谱对蛋白点进行分子鉴定.结果 家蝇幼虫血淋巴中蛋白的分子量主要分布在10 kDa～66kDa之间,采用pH3～10的IPG胶,检测到286个蛋白点,采用pH4～7的IPG胶,测到601个蛋白点;血淋巴经沸水浴处理后,检测到41个蛋白点,分子量低于30 kDa,比未处理组减少了93.1％,对其中11个蛋白点进行质谱分析,鉴定出4个功能蛋白,包括存储蛋白、气味结合蛋白、铁蛋白重链样蛋白和铁蛋白2轻链蛋白同系物.结论 双向电泳能很好地分析家蝇幼虫血淋巴中的蛋白,血淋巴中的蛋白等电点主要分布在pH4～7之间;血淋巴经沸水浴处理后可去掉大量变性蛋白,其上清中可能包含耐热性较好的功能蛋白.     

家蚕丝氨酸蛋白酶抑制剂Bmserpin2对酚氧化酶原激活和抗菌肽基因表达的抑制作用

【目的】丝氨酸蛋白酶抑制剂家族蛋白是昆虫中调控自身免疫反应的重要蛋白酶抑制剂,本研究旨在研究家蚕Bombyx mori丝氨酸蛋白酶抑制剂2(Bmserpin2)在家蚕2个重要的自身免疫通路即酚氧化酶原(prophenol oxidase, PPO)激活通路和革兰氏阳性菌诱导抗菌肽的TOLL通路中的调控作用。【方法】PCR扩增家蚕Bmserpin2基因片段后原核表达并通过镍柱纯化。利用纯化后的重组Bmserpin2蛋白分别与胰蛋白酶、胰凝乳蛋白酶、弹性蛋白酶和蛋白酶K反应,检测Bmserpin2对上述蛋白酶活性的影响。通过RT-qPCR检测Bmserpin2在家蚕5龄第3天幼虫头、中肠、脂肪体、血淋巴、丝腺和表皮组织中表达的模式。往家蚕5龄第3天幼虫注射Bmserpin2重组蛋白,检测Bmserpin2对其血淋巴中PPO活性的影响。通过滕黄微球菌Micrococcus luteus诱导家蚕5龄第3天幼虫产生抗菌肽并注射Bmserpin2重组蛋白后,RT-qPCR检测其血淋巴中抗菌肽基因gloverin2和moricin表达量。【结果】成功构建重组质粒并表达纯化目的蛋白Bmserpin2。通过与不同蛋白酶反应得出Bmserpin2可极显著抑制消化酶胰蛋白酶和弹性蛋白酶活性,对胰凝乳蛋白酶和蛋白酶K活性影响不显著,提示Bmserpin2对不同蛋白酶具有生物学活性和催化特异性。基因表达模式显示Bmserpin2在家蚕5龄幼虫血淋巴和脂肪体中表达量最高。家蚕5龄幼虫注射重组Bmserpin2蛋白后发现目的蛋白能有效抑制血淋巴中PPO活性。利用滕黄微球菌诱导家蚕5龄幼虫产生抗菌肽后,滕黄微球菌和Bmserpin2混合注射组中血淋巴中抗菌肽基因gloverin2和moricin的转录表达与只注射滕黄微球菌的比较被显著下调。【结论】Bmserpin2可能参与家蚕酚氧化酶原激活和TOLL途径的胞外级联反应的免疫通路。     

家蚕丝氨酸蛋白酶抑制剂Bmserpin2对酚氧化酶原激活和抗菌肽基因表达的抑制作用

【目的】丝氨酸蛋白酶抑制剂家族蛋白是昆虫中调控自身免疫反应的重要蛋白酶抑制剂,本研究旨在研究家蚕Bombyx mori丝氨酸蛋白酶抑制剂2(Bmserpin2)在家蚕2个重要的自身免疫通路即酚氧化酶原(prophenol oxidase, PPO)激活通路和革兰氏阳性菌诱导抗菌肽的TOLL通路中的调控作用。【方法】PCR扩增家蚕Bmserpin2基因片段后原核表达并通过镍柱纯化。利用纯化后的重组Bmserpin2蛋白分别与胰蛋白酶、胰凝乳蛋白酶、弹性蛋白酶和蛋白酶K反应,检测Bmserpin2对上述蛋白酶活性的影响。通过RT-qPCR检测Bmserpin2在家蚕5龄第3天幼虫头、中肠、脂肪体、血淋巴、丝腺和表皮组织中表达的模式。往家蚕5龄第3天幼虫注射Bmserpin2重组蛋白,检测Bmserpin2对其血淋巴中PPO活性的影响。通过滕黄微球菌Micrococcus luteus诱导家蚕5龄第3天幼虫产生抗菌肽并注射Bmserpin2重组蛋白后,RT-qPCR检测其血淋巴中抗菌肽基因gloverin2和moricin表达量。【结果】成功构建重组质粒并表达纯化目的蛋白Bmserpin2。通过与不同蛋白酶反应得出Bmserpin2可极显著抑制消化酶胰蛋白酶和弹性蛋白酶活性,对胰凝乳蛋白酶和蛋白酶K活性影响不显著,提示Bmserpin2对不同蛋白酶具有生物学活性和催化特异性。基因表达模式显示Bmserpin2在家蚕5龄幼虫血淋巴和脂肪体中表达量最高。家蚕5龄幼虫注射重组Bmserpin2蛋白后发现目的蛋白能有效抑制血淋巴中PPO活性。利用滕黄微球菌诱导家蚕5龄幼虫产生抗菌肽后,滕黄微球菌和Bmserpin2混合注射组中血淋巴中抗菌肽基因gloverin2和moricin的转录表达与只注射滕黄微球菌的比较被显著下调。【结论】Bmserpin2可能参与家蚕酚氧化酶原激活和TOLL途径的胞外级联反应的免疫通路。     

苹果蠹蛾热激蛋白Hsp90基因的克隆及热胁迫下的表达分析

世界检疫性害虫苹果蠹蛾Cydia pomonella是一种温度耐受可塑性很高的物种。本研究针对温度波动可能导致其耐热性增强的科学问题, 采用生测法鉴定了苹果蠹蛾实验种群的高温耐受阈值, 采用同源克隆、 RACE和实时荧光定量PCR (RT-qPCR)等方法研究了苹果蠹蛾热激蛋白Hsp90基因的应激表达对耐热性的重要作用。高温耐受阈值研究结果表明, 苹果蠹蛾实验种群的死亡率随温度的升高和时间的延长显著性升高, 1-5龄幼虫分别经50℃和52℃高温处理2, 5和10 min后, 3龄幼虫耐热性最差, 5龄幼虫最强。50℃和52℃分别处理10 min和5 min均可导致1-4龄幼虫全部死亡, 而5龄幼虫在这两种处理下仍有25.0%和11.1%的存活率。以35℃处理的5龄雌幼虫为材料克隆苹果蠹蛾Hsp90基因全长cDNA, 结果显示该基因全长为2 470 bp, 完整开放阅读框为2 148 bp, 共编码716个氨基酸, 预测分子量为82.07 kDa, 命名为Cphsp90 (GenBank登录号JN624775)。该基因编码的氨基酸序列与亚洲玉米螟Ostrinia furnacalis和甘蓝夜蛾Mamestra brassicae等昆虫的Hsp90的氨基酸序列一致性高达96%, 表明了Hsp90家族的保守特性。Cphsp90 mRNA的相对表达量在32~44℃高温胁迫下随温度的升高而显著增高, 证实Cphsp90是诱导型热激基因, 且mRNA相对表达量与胁迫程度正相关。Cphsp90基因的表达还具有组织特异性, 35℃处理幼虫的表皮中Cphsp90相对表达量显著高于血淋巴、 脂肪体和中肠, 应激响应最为活跃。与未经温热预处理的昆虫相比, 35℃温热预处理3 h后的5龄幼虫在40, 45和50℃更高的温度胁迫下, Cphsp90 mRNA达到最高表达量所需要的胁迫温度有所提升, 由未经预热处理的40℃处理10 min提高到45℃处理10 min, 这与温热预处理会增强5龄幼虫耐热性的现象相符, 表明Cphsp90基因的响应表达在苹果蠹蛾耐热性及其可塑性过程中发挥重要的作用。     

人GDNF在甲醇酵母及家蚕幼虫中的表达

将人胶质细胞源性神经营养因子(GDNF)基因克隆入酵母分泌型表达载体pPIC9K中,酶切线性化后电穿孔导入酵母细胞进行整合,经G418筛选得到多拷贝转化子,甲醇诱导表达。将人GDNF基因克隆入昆虫病毒转移载体pBacPAK8中,与线性化Bm-BacPAK6修饰病毒基因组DNA共转染家蚕细胞,经体内重组,筛选到重组病毒。用重组病毒感染家蚕幼虫,5d后收集血淋巴。SDS-PAGE和蛋白质印迹杂交结果证实了酵母培养上清液及家蚕幼虫血淋巴中含有GDNF蛋白。活性研究表明,甲醇酵母及家蚕幼虫表达的GDNF蛋白能促进多巴胺能神经元的存活和突起生长。     

亚洲玉米螟滞育关联蛋白的分离和纯化

用6%聚丙烯酰胺凝胶电泳, 在亚洲玉米螟Ostrinia furnacalis河南种群滞育幼虫血淋巴中发现了2种滞育关联蛋白, 它们的等电点分别为5.3和4.7; 而在亚洲玉米螟上海种群滞育幼虫血淋巴中发现了1种滞育关联蛋白,等电点为4.7。亚洲玉米螟河南种群滞育关联蛋白仅在滞育幼虫血淋巴中存在。亚洲玉米螟上海种群滞育关联蛋白在滞育和非滞育幼虫血淋巴中均存在,只是它们在滞育幼虫血淋巴中的含量明显高于非滞育幼虫血淋巴中的含量。用30%～50%丙酮沉淀、SephaDex G-100凝胶过滤和MONOQ RH 5/5阴离子交换快速液相法,纯化到了高纯度(≥98%)亚洲玉米螟上海种群滞育幼虫血淋巴中的滞育关联蛋白。     

抗阿维菌素朱砂叶螨的热激反应及热激蛋白

选用朱砂叶螨Tetranychus cinnabarinus阿维菌素抗性品系和敏感品系,测定了热预刺激后其在极限高温下的存活率,并应用SDS-PAGE技术研究了热激蛋白(HSPs)的种类及其含量。结果表明：非致死的热预刺激能显著提高朱砂叶螨耐极限温度的能力。两个品系在不同温度热激处理后,其蛋白质种类和含量发生了变化。正常情况下,朱砂叶螨敏感品系与阿维菌素抗性品系相比缺失8条条带;敏感品系热激后,增加了分子量分别为97.2,74.3,62.4,53.0和30.3 kDa的5条条带; 抗性品系热激后没有特异蛋白带的产生,但进一步高温胁迫后有些蛋白表达增强。此结果有助于解释朱砂叶螨抗性品系存在高温适合度优势现象。     

利用重组杆状病毒在家蚕中表达人血管内皮细胞生长因子

家蚕作为“生物工厂”生产重组蛋白质具有很多优势 .构建携带编码人血管内皮细胞生长因子 (VEGF) 16 5个氨基酸的cDNA的重组杆状病毒 .将此重组病毒接种 5龄家蚕幼虫进行重组蛋白的表达生产 .时相表达分析表明 ,感染后大约 80h时表达水平达到最高 ,而且重组蛋白主要存在于血淋巴中 .从感染的幼虫收集血淋巴并用Nickle亲和层析纯化重组蛋白产物 .定量分析表明 ,每条家蚕幼虫的表达水平高达 4 2 6 μg左右 .通过细胞培养体外分析 ,发现与对照相比 ,加入纯化的重组VEGF(10ng ml和 10 0ng/ml)能够使人HUVEC细胞体外培养细胞数增加 1 8～ 3 3倍 ,说明家蚕幼虫表达的重组VEGF具有完全的生物活性 ,能够诱导内皮细胞在体外分裂增殖 .     

Heat shock response in mulberry silkworm races with different thermotolerances

The thermal sensitivity and heat shock response of the different races of the mulberry silkwormBombyx mori have been analysed. The multivoltine race, strainsC. Nichi andPure Mysore showed better survival rates than the bivoltine race, strainNB4D2 exposed to 41°C and above. In general, the fifth instar larvae and the pupae exhibited maximum tolerance compared to the early larval instars, adult moths or the eggs. Exposure up to 39°C for 1 or 2 h was tolerated equally whereas temperatures above 43°C proved to be lethal for all. Treatment of larvae at 41°C for 1 h resulted in a variety of physiological alterations including increased heart beat rates, differential haemocyte counts, enlargement of granulocytes and the presence of additional protein species in the tissues and haemolymph. The appearance of a 93 kDa protein in the haemolymph, fat bodies and cuticle, following the heat shocking of larvaein vivo was a characteristic feature in all the three strains examined although the kinetics of their appearance itself was different. In haemolymph, the protein appeared immediately in response to heat shock inC. Nichi reaching the maximal levels in 2–4 h whereas its presence was noticeable only after 2–4 h recovery time inPure Mysore and bivoltine races. The fat body from bothC. Nichi andNB4D2 showed the presence of 93 kDa, 89 kDa and 70 kDa proteins on heat shock. The haemocytes, on the other hand, expressed only a 70 kDa protein consequent to heat shock. The 93 kDa protein in the haemolymph, therefore could have arisen from some other tissue, possibly the fat body. The 93 kDa protein was detected after heat shock in pupae and adult moths as well, although the presence of an additional (56 kDa) protein was also apparent in the adults. The presence of 46 kDa and 28 kDa bands in addition to the 93 kDa band in the cuticular proteins immediately following heat shock was clearly discernible. The 70 kDa band did not show much changes in the cuticular proteins on heat shock. In contrast to the changes in protein profiles seen in tissues and haemolymph following heat shockin vivo, the heat treatment of isolated fat body or haemolymphin vitro resulted in protein degradation.     

Molecular characterization of heat shock proteins 90 (HSP83?) and 70 in tropical strains of Bombyx mori

Following the concept of whole organism, we have extracted total protein from the Bombyx mori for the identification and analysis of HSPs. Expression of 90 kDa HSP in first, second and third instars, 84 kDa in fourth instar and 90‐, 84‐, 62‐, 60‐, 52‐ and 33‐kDa HSPs in fifth instar larvae of tropical polyvoltine and bivoltine silkworm strains were obvious. Further, we have combined single and 2‐DE with MALDI‐TOF for analysis of BmHSPs. Ninety kilodalton band excised from 1‐DE gel was identified as HSP83 by MALDI‐TOF‐MS. The immunoblot analysis confirmed the expression of HSP90 in all the instars larvae of B. mori. Heat shock‐induced protein spots were excised from 2‐DE gels for MALDI‐TOF‐MS analysis. The Mascot search results are for HSP68, HSC70‐1 and HSP70Ba in Pure Mysore, and major HSP70Bbb, HSP68, HSC‐3 and HSP83 in NB₄D₂. Multiple sequence alignment explicit the variations in amino acid sequence between Pure Mysore and NB₄D₂. Notably, the PMF of spot 2 matched the coding sequence of B. mori and its gene annotation was determined on chromosome 9. With this novel approach, expression of BmHSP90 was confirmed in all the instars and uncovered isoforms of BmHSP70, which provided unequivocal insight to analyze and understand the biological significance in B. mori.     

Expression, purification and characterization of human GM-CSF using silkworm pupae (Bombyx mori) as a bioreactor

To date, many recombinant proteins have been expressed in Bombyx mori cells or silkworm larvae, apart from in pupae. Silkworm pupae may be more suitable for the expression of heterologous proteins as a bioreactor. If maintained at an appropriate temperature, silkworm pupae could be inoculated with recombinant baculovirus for the expression of a protein of interest. In this study, human granulocyte-macrophage colony-stimulating factor was successfully expressed in silkworm pupae using B. mori nucleopolyhedrovirus, purified and characterized with respect to its physico-chemical properties. The target protein expressed had an apparent molecular mass of 29 kDa and an isoelectric point of 5.1. The protein was purified using three chromatographic steps with a final recovery of 10.3%. Finally, approximately 3.5mg of the protein was obtained with a biological activity of up to 8.4 x 10(6) cfu mg(-1). The results of this study suggest that silkworm pupae represent a convenient and low-cost bioreactor for the expression of heterologous proteins.     

The profiles of red fluorescent proteins with antinucleopolyhedrovirus activity in races of the silkworm Bombyx mori

Partially purified red fluorescent proteins (RFPs) secured from the gut juice of 5th-instar multivoltine and bivoltine silkworm races were observed as several bands in electrophoretograms and chromatographic eluates. Interestingly, different races of silkworms had varying numbers of fluorescent protein bands: 11 in Pure Mysore (resistant), 11 in Nistari (resistant), 4 in CSR₂ (moderately susceptible) and 1 in NB₄D₂ (highly susceptible). Bioassay experiments indicated that the fluorescent bands had antinucleopolyhedrovirus (antiNPV) activity. The molar extinction coefficients and fluorescence quantum yields of all RFPs were estimated. The purified tetrapyrroles were characterized by UV–visible absorption and fluorescence spectral analyses. All tetrapyrrole moieties associated with RFPs were found to be different and characteristic of the fluorescent bands. The resulting qualitative and quantitative differences among the individual RFPs from various races of silkworm were related to the susceptibilities of the silkworms to the viral disease. Moreover, light was found to be essential for the synthesis of RFPs, and, therefore, the role of light in the synthesis of RFPs was evaluated. Thus, this work may elucidate the process of RFP synthesis in silkworm, which may be used as a biomarker to measure the degree of susceptibility of silkworm races to NPV. Therefore, the characteristic band pattern may be used as an indicator to define the relative resistance of a race towards the specific virus.     

Impact of heat shock on heat shock proteins expression, biological and commercial traits of Bombyx mori

We report the thermotolerance of new bivoltine silkworm, Bombyx mori strains NB₄D₂, KSO₁, NP₂, CSR₂ and CSR₄and differential expression of heat shock proteins at different instars. Different instars of silkworm larva were subjected to heat shock at 35°C, 40°C and 45°C for 2 hours followed by 2 hours recovery. Heat shock proteins were analyzed by SDS‐PAGE. The impact of heat shock on commercial traits of cocoons was analyzed by following different strategies in terms of acquired thermotolerance over control. Comparatively NP₂ exhibited better survivability than other strains. Resistance to heat shock was increased as larval development proceeds in the order of first instar > second instar > third instar > fourth instar > fifth instar in all silkworm strains. Expression of heat shock proteins varies in different instars. 90 kDa in the first, second and third instars, 84 kDa in the fourth instar and 84, 62, 60, 47 and 33 kDa heat shock proteins in fifth instar was observed in response to heat shock. Relative influence of heat shock on commercial traits that correspond to different stages was significant in all strains. In NB₄D₂, cocoon and shell weight significantly increased to 17.52% and 19.44% over control respectively. Heat shock proteins as molecular markers for evaluation and evolution of thermotolerant silkworm strains for tropics was discussed.     

Characterization of HSP27 and three immunologically related polypeptides during Drosophila development

The low-molecular-weight heat-shock protein HSP27 is made in the absence of heat shock during Drosophila melanogaster development. An analysis of the accumulation of HSP27 during specific stages of development is presented using an antiserum recognizing this protein. Whereas HSP27 is abundant during embryogenesis, the level of this protein begins to decrease in the 20-h old embryo and is no longer detectable in second instar larvae. A high level of HSP27 is again observed in third instar larvae and reaches a maximal level in late pupae. While still abundant in young adult flies of both sexes, a greater amount of HSP27 is found in females with the protein being highly concentrated within the ovaries. Following lysis of whole pupae, about 60% of HSP27 is found in the soluble lysate fraction in a form which sediments between 5 and 20 S. Anti-HSP27 serum also recognizes three other developmentally regulated polypeptides with apparent MW of 33, 85 and 120 kDa. The 33 kDa protein accumulates in pupae while those of 85 and 120 kDa are more abundant in third instar larvae. Unlike HSP27, these proteins are not detected in embryos or ovaries. Immunoblot analysis of V8 proteolytic fragments suggests that HSP 27 and 33 kDa are related polypeptides. Exposure of the developing insect to heat-shock treatment results in increased level of HSP27. In larvae, a small amount of the 33 kDa protein accumulates following heat shock, while in pupae and adult flies a decrease in the concentration of this protein is observed after heat shock. Finally, different cellular localizations and distributions within the pupal body have been found for these developmentally regulated polypeptides.     

人白细胞介素-11基因在家蚕培养细胞和虫体内的表达

将缺少编码信号肽序列的人白细胞介素－11（hIL-11)546核苷酸cDNA,重组于质粒pBacPAK8构建重组转移载体pBacIL-11,与经线性化修饰的家蚕核型多角体病毒（BmBacPAK)DNA共转染家蚕培养细胞株BmN,获得了插入hIL-11基因的重组病毒。Southern杂交表明重组病毒基因组中含有hIL-11基因片段,RNA斑点杂交表明hIL-11基因得到了转录。重组病毒感BmN细胞株、家蚕幼虫和蛹,在细胞培养上清、细胞抽提物、幼虫和蛹的体液样品中,SDS－PAGE电泳分析都能检测得到表达产物的特异性条带;采用IL－11依赖细胞株B9－11和MTT法测定表达产物的生物活性,表明rIL-11基因分别在培养细胞和蚕体内得到了高效表达。     

Cellular localization of HSP23 during Drosophila development and following subsequent heat shock

The low-molecular-weight heat-shock protein HSP23 is synthesized in the absence of heat shock during Drosophila development. Here, I present a quantitative analysis of this phenomenon and describe the cellular localization of this protein during normal development and after a subsequent heat shock. HSP23 is first detected in the late third instar larvae and continues to accumulate reaching a maximum level in late pupae. In a 1-week-old adult, HSP23 can no longer be detected. Following lysis of whole pupae, HSP23 is found in the soluble lysate fraction in a form which sediments between 10 and 20 S. Exposure of larvae, pupae, and the adult fly to heat stress (37 degrees C) results in an increased amount of HSP23 which, however, is recovered in an insoluble particulate form following insect lysis. During recovery from heat shock, HSP23 is again found in the soluble 10- to 20-S lysate fraction. In pupae which are exposed to a severe heat stress (41 degrees C) HSP23 remains in the pellet fraction after the heat stress and no pupae are able to emerge as adult flies. However, when pupae are first exposed to a mild heat-shock treatment prior to the 41 degrees C stress, the thermotolerance process is induced and HSP23 is again rapidly found in the soluble lysate fraction during the recovery from heat shock. These observations suggest a possible correlation between the survival of pupae after heat shock and the recovery of HSP23 in the soluble lysate fraction as 10- to 20-S structures after the heat shock.