二化螟热休克蛋白70基因的克隆及热胁迫下的表达分析

热休克蛋白70是已知热休克蛋白家族中最重要的一种, 它在细胞内的大量表达可以明显改善细胞的生存能力, 提高对环境胁迫的耐受性。为探讨热胁迫对二化螟Chilo suppressalis幼虫热休克蛋白70表达的影响, 采用RT-PCR及RACE技术从二化螟血淋巴细胞中克隆了热休克蛋白70基因全长cDNA序列。该基因全长2 102 bp, 开放阅读框 (open reading frame, ORF)为1 959 bp, 编码652个氨基酸; 5′非编码区（untranslated region, UTR)为81 bp, 3′UTR为62 bp。从该基因推导的氨基酸序列与其他昆虫的同源序列比较有很高的相似性（73%~97%)。实时定量PCR显示二化螟HSP70基因能被热胁迫诱导表达, 幼虫血淋巴细胞的HSP70基因在36℃时表达量最高。流式细胞术研究发现HSP70在蛋白质水平上的表达变化与在mRNA水平上高度一致, 说明二化螟HSP70基因在转录及翻译水平上受到热应激的调节。     

甜菜夜蛾HSP90基因克隆及高温胁迫下其表达量的变化

为阐明热激蛋白90（heat shock protein 90, HSP90）在甜菜夜蛾Spodoptera exigua (Hübner)幼虫抵抗高温过程中的作用, 克隆了其HSP90基因cDNA全长序列, 并检测了在系列高温胁迫下不同龄期幼虫体内其相对表达量。根据已报道的热激蛋白90基因序列同源性设计简并引物, 利用反转录聚合酶链式反应（RT-PCR）结合cDNA末端快速扩增（RACE）技术成功克隆了甜菜夜蛾HSP90基因全长cDNA（GenBank登录号FJ862050）。该cDNA序列开放阅读框长2 154 bp, 编码717个氨基酸, 预测的相对分子量和等电点分别为82.6 kD和5.0。该序列具有HSP90家族的典型特征和特殊的功能结构域, 并且与多种生物的HSP90基因序列有较高的同源性。为了研究HSP90抵抗高温的作用, 构建荧光定量RT-PCR体系, 检测了37, 39, 41, 43和45℃胁迫下甜菜夜蛾不同龄期幼虫体内HSP90表达量的变化。结果表明, 高温胁迫对甜菜夜蛾幼虫体内的HSP90表达具有明显的诱导作用。幼虫体内HSP90表达量随着温度升高呈增加的趋势。43℃和45℃胁迫下, 各龄幼虫体内HSP90的表达量均显著高于常温（P< 0.05）, 但不同龄期之间没有显著差异。这说明HSP90在甜菜夜蛾幼虫抗高温中起到重要作用。     

小菜蛾热休克蛋白基因的鉴定及其表达模式分析

热休克蛋白(heat shock protein, HSP)在昆虫应对外界胁迫刺激时起着重要作用。为了系统研究小菜蛾Plutella xylostella HSP基因家族, 根据家蚕的HSP蛋白序列, 采用本地Blast程序对小菜蛾全基因组数据库进行同源序列检索, 从小菜蛾基因组数据库中鉴定了25个HSP基因, 包括2个HSP90、 8个HSP70和15个sHSP（small heat shock protein, sHSP）基因。小菜蛾、 家蚕Bombyx mori、 黑腹果蝇Drosophila melanogaster和赤拟谷盗Tribolium castaneum的HSP系统进化分析显示, 昆虫的小分子量热休克蛋白sHSP具有很强的种属特异性, HSP70家族的保守性比sHSP强。小菜蛾HSP基因表达模式分析显示, 与敏感品系对比, 抗性品系（抗毒死蜱和抗氟虫氰品系）中HSP基因具有不同的表达模式。小菜蛾1, 2和3龄幼虫HSP基因表达模式较为接近, 而与4龄幼虫中的表达模式相差较大; 4龄幼虫和蛹中的表达模式相近; 雌成虫和雄成虫中的表达模式显著不同, 与果蝇精子形成有关的两个热休克蛋白HSP23和HSP27基因［分别为CCG003980.1 (Px23.5)和CCG005412.2 (Px27.5)］, 在小菜蛾雄成虫中的表达量显著高于雌成虫。研究结果表明小菜蛾HSP基因不仅在杀虫剂抗性、 发育分化, 甚至在生殖上均可能起着重要的作用。本研究为深入研究小菜蛾HSP与生长发育、 抗逆行为的相互关系奠定了基础。     

热胁迫对豌豆下胚轴生理的一些影响

通过测定热驯和热胁迫下3个豌豆品种幼苗下胚轴生长、细胞膜损伤、抗坏血酸（AsA）和丙二醛（佃A）含量的变化及热激蛋白70（HSP70）表达,探讨热胁迫对豌豆生理的影响。结果表明,在48℃高温胁迫下豌豆种子萌发率下降,幼苗下胚轴生长受抑制,细胞膜受损,AsA含量下降,MDA含量升高;经37℃热驯再48℃热激处理的下胚轴长度和ASA明显高于直接热胁迫的,细胞膜受损程度和MDA含量则低于后者。HSP70测定表明,除台湾品种外,37℃热驯1h不足以诱导HSP70表达;而37℃热驯后常温恢复再48℃热激和直接48℃热激均能诱导HSP70表达,其中蒙自品种经热驯后再热激的HSP70表达量高于直接热激的。     

日灼胁迫对枇杷果皮蛋白质组的影响

为了探讨枇杷果皮响应日灼胁迫的差异表达蛋白的变化,以日灼抗性弱的‘金钟’枇杷和日灼抗性较强的‘红猴本’枇杷为研究对象,将处于转色期的枇杷果实在40 ℃下日灼处理90 min,果皮蛋白采用双向电泳分离和MALDI TOF/TOF MS进行鉴定。结果显示：（1）日灼后枇杷果皮中有64个表达丰度在2倍以上的差异蛋白,其中56个蛋白被成功鉴定,且38个在‘金钟’中表达,23个在‘红猴本’中表达。（2）这些蛋白质参与胁迫响应和防御（30.3%）、光合作用（14.3%）、呼吸作用（25.0%）、蛋白质代谢（14.3%）等生理过程。（3）相对于‘红猴本’枇杷,‘金钟’枇杷果皮细胞中多种热激蛋白（HSP）的含量上升,参与呼吸作用的蛋白含量增加以产生更多的能量,但是大部分与光合作用相关的蛋白含量下降,清除活性氧（ROS）酶如抗坏血酸过氧化物酶（APX）和多酚氧化酶（PPO）含量降低,同时PPO酶活性下降。研究表明,要提高枇杷果皮抗日灼性可能需要提高清除ROS酶的含量和活性,需要一定量的HSP,以及保持正常的呼吸作用和光合作用。     

橡胶树胶乳高表达热激蛋白HbHSP90.4基因抗逆功能分析

为了分析热激蛋白90（HSP90）基因在橡胶树（Hevea brasiliensis）逆境胁迫和激素转导中的作用,利用PCR技术从橡胶树品种热研73397胶乳中克隆得到HbHSP90.4基因全长cDNA序列,该基因含有1个2 451 bp开放阅读框（ORF）,编码816个氨基酸。生物信息学分析结果表明,HbHSP90.4含有HSP90 superfamily和HATPase superfamily结构域,属于HSP90家族成员。系统进化分析发现该蛋白与木薯MeHSP90具有较近的亲缘关系。亚细胞定位预测显示HbHSP90.4基因定位在内质网。qRT-PCR结果表明HbHSP90.4基因主要在橡胶树胶乳中表达。干旱、冷胁迫、橡胶树白粉菌侵染、H₂O₂和MeJA处理均可促进胶乳HbHSP90.4基因上调表达,而其在ETH、SA和ABA处理中均呈现显著下调表达。构建植物表达载体HbHSP90.4-mScarlet,为进一步的转基因植物的做成准备了材料。本研究为阐明胶乳HbHSP90.4基因响应橡胶树逆境胁迫过程和植物激素信号传导途径分子调控机制奠定坚实基础。     

甘菊ClHSP70与ClHSP90基因的克隆及表达分析

该研究以甘菊(Chrysanthemum lavandulifolium)为实验材料,通过RT-PCR方法从甘菊转录组数据中分离出热激蛋白合成相关基因,命名为ClHSP70和ClHSP90。序列分析表明,ClHSP70基因ORF全长为2 559bp,编码852个氨基酸,蛋白功能区预测表明含有典型的HSP70蛋白NBD和SBD保守结构域;ClHSP90基因ORF全长为2 094bp,编码697个氨基酸,含有HATPase结构域和HSP90保守结构域。生物信息学分析表明,甘菊ClHSP70与大豆(Glycine max)和烟草(Nicotiana tomentosiformis)HSP70蛋白有较高的一致性,ClHSP90基因编码的氨基酸序列与紫茎泽兰(Ageratina adenophora)HSP90高度相似;实时荧光定量表达分析表明,在42℃处理不同时间,甘菊叶片中ClHSP70和ClHSP90基因表达均在0.5h时显著增加,1h达到最大值,2h后缓慢下降;不同组织表达分析表明,甘菊在42℃处理1h后,ClHSP70在成熟叶中的表达量显著高于嫩叶和根等其他组织;ClHSP90在成熟茎中的表达量最高。研究说明,ClHSP70和ClHSP90基因具有热激蛋白特征,参与了甘菊热胁迫应答过程,该研究结果为以后深入研究其基因功能奠定了基础。     

Sestrin2对热暴露肺上皮细胞凋亡的干预作用及其机制*

目的:探讨Sestrin2蛋白对热暴露肺上皮细胞凋亡的干预作用及其作用机制。方法:体外培养的Beas-2B细胞分为对照组(37℃)和热暴露组(39℃、40℃和41℃),在上述温度中暴露不同时间(0、3、6和12 h),胰酶消化后收集细胞,分别通过Western blot、荧光分光光度计、流式细胞仪等方法检测细胞中的Sestrin2、超氧化物歧化酶(SOD)、活性氧自由基(ROS)表达水平,细胞线粒体膜电位及细胞凋亡率。基因序列克隆入高表达质粒pcDNA 3.1⁺中,采用Lipfectamine 2000方法转染Beas-2B细胞,构建Sestrin2和SOD高表达细胞,观察细胞线粒体膜电位及细胞凋亡等指标的变化。结果:随着暴露温度的升高,与对照组相比,热暴露组细胞Sestrin2蛋白表达水平下降。在41℃热暴露Beas-2B细胞,不同时间点ROS水平显著上升,线粒体膜电位显著下降,细胞凋亡率增加。Sestrin2和SOD高表达细胞,在41℃暴露条件下,与对照组比较,ROS表达水平显著降低,线粒体膜电位下降幅度减小,热暴露导致细胞凋亡率降低。结论: Sestrin2能够通过线粒体膜电位和SOD缓解热暴露引起肺上皮细胞的凋亡,对Beas-2B细胞具有保护作用。     

水稻二化螟小分子量热激蛋白21.3基因的克隆和表达分析

小分子量热激蛋白(sHSPs,small heat shock proteins)在昆虫中分布广泛,能影响昆虫的生长繁殖、参与多种生理过程以及增强昆虫的温度耐受性。水稻二化螟Chilo suppressalis(Walker)是水稻上的主要害虫之一,目前在我国局部稻区危害严重。为了深入了解sHSPs在水稻二化螟生长发育及温度耐受性中的功能,利用RT-PCR和RACE技术克隆出一个二化螟小分子量热激蛋白Cs HSP21.3基因。Cshsp21.3的cDNA序列全长为844 bp,其中开放阅读框(ORF)为555 bp,编码184个氨基酸,理论分子量为21.3 kDa,等电点为5.97;基因组结构分析发现,Cshsp21.3缺失内含子。实时定量PCR的实验结果表明:在脂肪体中Cshsp21.3的表达高于其它不同组织器官;在二化螟的不同发育阶段,3龄幼虫Cshsp21.3的表达水平最高,而且雌雄蛹的表达差异显著;高低温不能显著诱导Cshsp21.3的表达,说明该基因对温度胁迫不敏感。总之,Cshsp21.3与二化螟的温度耐受性之间没有密切的联系,但在二化螟的生长发育方面起着重要的作用。     

低温、饥饿胁迫及恢复对奥尼罗非鱼幼鱼HSP70基因表达的影响

在室内可控条件下,研究奥尼罗非鱼幼鱼在低温、饥饿胁迫后及恢复期间HSP70基因的表达变化情况。结果显示,在低温、饥饿胁迫下发现处理组的肝脏组织中热应激蛋白HSP70 m RNA表达水平上升,且在胁迫发生14 d后出现显著升高,明显高于对照组(p0.05)。在胁迫结束恢复期间(14~28 d),处理组的肝脏组织中热应激蛋白HSP70 m RNA表达水平下降,且在第21天显著下降,且与对照组无差异。研究结果表明短期的低温胁迫与急性低温应激的应答模式都能够一定程度上提高奥尼罗非鱼幼鱼肝脏组织中热应激蛋白HSP70 m RNA的表达水平。     

Effects of heavy metals on insect immunocompetent cells

The influence of the following heavy metals, copper (Cu), zinc (Zn), cadmium (Cd) and lead (Pb), on haemocytes of the house fly Musca domestica L. was studied under laboratory conditions. House fly larvae were exposed to low or high, semi-lethal concentrations of metals. These particular metals were selected because they are present in polluted environments in Poland. In addition, we studied expression of the stress proteins HSP70 and HSP72 in haemocytes collected from larvae that had been exposed to heavy metal. The obtained results showed changes in haemocytes morphology and phagocytotic plasticity in the experimental flies in comparison to control. The number of prohaemocytes, regarded as stem cells, increased, while granulocytes, responsible for phagocytosis, decreased. However, we have not detected any clear changes in expression of HSP70 or HSP72 in flies treated with low or high concentrations of the heavy metals.     

Antioxidant responses of Chilo suppressalis (Lepidoptera: Pyralidae) larvae exposed to thermal stress

High temperatures cause a variety of physiological stress responses in insects, including increased generation of reactive oxygen species (ROS), which can cause oxidative damage. This study investigated the effects of thermal stress on ROS generation, the expression of heat shock protein 70 (Hsp70) at the mRNA and protein levels, the activity of antioxidant enzymes (SOD, CAT), and apoptosis in hemocytes of Chilo suppressalis larvae. Results indicated that thermal stress significantly elevated the level of ROS and antioxidant enzyme activity in C. suppressalis larvae. Real-time quantitative PCR showed that hsp70 gene expression was induced by heat stress. Flow cytometric results revealed that the expression profile of Hsp70 at the protein level was in agreement with that at the mRNA level. The expression of Hsp70 at both the mRNA and protein levels reached a maximum at 36 °C in larval hemocytes. Exposure to tested temperatures did not cause any significant change in the rate of apoptosis in larval hemocytes. These results suggest that thermal stress leads to oxidative stress and that antioxidant enzymes and the Hsp70 play an important role in reducing oxidative damage in C. suppressalis larvae.     

Cloning and expression of a heat shock protein (HSP) 90 gene in the haemocytes of Crassostrea hongkongensis under osmotic stress and bacterial challenge

Heat shock protein 90 (HSP90) is a highly conserved and multi-functional molecular chaperone that plays an essential role in both cellular metabolism and stress response. Here, we report the cloning of the HSP90 homologue in Crassostrea hongkongensis (ChHSP90) through SSH in combination with RACE from cDNA of haemocytes. The full-length cDNA of ChHSP90 is 2459 bp in length, consisting of a 3', 5'-untranslated region (UTR) and an open reading frame of 2169 bp encoding 722 amino acids. The identity analysis of the amino acid sequence of HSP90 revealed that ChHSP90 is highly conserved. Distribution of ChHSP90 mRNA in gonad, heart, adductor muscle, mantle, gill, digestive gland, and haemocytes suggested that ChHSP90 is ubiquitously expressed. The mRNA levels of ChHSP90 under salinity and bacterial challenges were analyzed by real-time PCR. Under hypo-osmotic treatment, ChHSP90 mRNA in gonad, heart and haemocytes were significantly up-regulated on day 2 and onwards; while in gill, digestive gland and adductor muscle it was significantly down-regulated; the expression in mantle was decreased significantly on day 2 and 3 (P < 0.01), and then up-regulated on day 4 (P < 0.05). Under hyper-osmotic treatment, the mRNA level in gonad, heart, adductor muscle was increased on day 2 and onwards; in gill, it was firstly increased, and then gradually decreased, reaching a minimum on day 3. On day 4, the expression level in gill recovered to pre-treatment level; in mantle and digestive gland, the expression levels were decreased, reaching to the minimum on day 3. During Vibrio alginolyticus challenge, the mRNA level of ChHSP90 increased 3-fold at 4 h post-infection, returned to its pre-challenge level at 6 h post-infection, then was further up-regulated from 8 to 36 h post-infection. These experiments demonstrate that ChHSP90 mRNA is constitutively expressed in various tissues and apparently inducible in haemocytes under salinity and bacterial challenges, suggesting its important role in response to both osmotic stress and bacterial invasion.     

Food-deprivation induces HSP70 and HSP90 protein expression in larval gilthead sea bream and rainbow trout

Heat shock proteins (HSPs) expression is commonly used as indicators of cellular stress in animals. However, very little is known about either the expression patterns of HSPs or their role in the stress-tolerance phenomenon in early life stages of fish. To this end, we examined the impact of food-deprivation (12 h), reduced oxygen levels (3.5 mg/L for 1 h) and heat shock (HS: + 5 °C for 1 h) on HSP70 and HSP90 protein expression in early life stages of the gilthead sea bream (Sparus aurata), a warm-water aquaculture species. Also, we investigated HSP70 and HSP90 response to food-deprivation (7 days) in early life stages of rainbow trout (Oncorhynchus mykiss), a cool-water aquaculture species, and the tolerance of this larvae to heat shock (either + 5 or + 10 °C for 1 h). Our results clearly demonstrate that food-deprivation enhances HSP70 and HSP90 protein expression in larvae of both species. In gilthead sea bream larvae, the stressors-induced HSP70 and HSP90 (only in the reduced oxygen group) protein expression returned to unstressed levels after 24 h recovery. In fed trout larvae, a + 5 °C heat shock did not elevate HSP70 and HSP90 expression, whereas 100% mortality was evident with a + 10 °C HS. However, food-deprived trout larvae, which had higher HSP70 and HSP90 protein content, survived HS and showed HS-dependent increases in HSP70, but not HSP90 expression. Overall, HSP70 and HSP90 protein expression in early life stages of fish have the potential to be used as markers of nutritional stress, while elevation of the tissue HSPs content may be used as a means to increase stress tolerance during larval rearing.     

热激蛋白90与中红侧沟茧蜂的发育关系

根据热激蛋白90(the heat shock proteins90,Hsp90)基因序列的保守性,设计引物,采用PCR结合RACE扩增的方法克隆得到中红侧沟茧蜂Microplitis mediatorHaliday Hsp90cDNA全序列,并用半定量RT-PCR的方法研究Hsp90与中红侧沟茧蜂发育及滞育间的关系。结果表明,滞育条件和非滞育条件下,热激和冷激都能诱导Hsp90的表达;不论在滞育还是非滞育条件下,Hsp90表达量均在2龄初期最高,随发育时间的增加逐渐降低,成虫期表达量又显著升高;滞育褐茧在4℃保存初期,Hsp90呈现高表达且随保存时间的增加而降低。