摘要
柑橘全爪螨Panonychus citri(McGregor)属蛛形纲Arachnida、蜱螨目Acarina、叶螨科Tetranychidae、全爪螨属Panonychus。该螨是一种世界性害螨,寄主广泛,大多以刺吸方式为害叶片、嫩梢、果皮,尤以叶片受害最重,严重时导致落叶、落果,影响树势和产量。柑橘是中国南方最主要的水果之一,栽培品种多,出口数量大,是重要的经济作物。由于采取清耕法的集约化栽培,果园植物单一,加之单一依赖化学防治,用药不当,大量杀灭果园害螨天敌,破坏橘园的生态平衡,且该螨世代周期短,抗药性增加迅速,使柑橘全爪螨成为柑橘栽培中头等重要的害虫(螨)之一,是柑橘园常发性、灾害性的有害生物。柑橘全爪螨每年发生多代,一年有2个高峰期(春季至初夏和秋季)和2个低谷期(夏季和冬季)。已有研究表明,柑橘全爪螨适宜的温度范围为20-30℃,高于35℃不利于其生殖发育,40℃以上死亡率高。在中国柑橘主产区夏季气温常高达35℃以上,甚至40℃,冬季温度常低至0℃以下。极端温度的存在,必将对柑橘全爪螨造成热胁迫,柑橘全爪螨在长期的进化过程中形成了一系列响应机制应对热胁迫。热激蛋白(Heat shock proteins, Hsps)是当今生物适应逆境胁迫研究的热点,Hsps是广泛存在于原核细胞和真核细胞的蛋白质,亚致死热胁迫能够诱导生物体产生热激Hsps,它们能够提高生物的热忍耐力。此外热胁迫常会引起过氧化反应,有机体的抗氧化系统在应对氧化胁迫方面发挥着重要作用。物质和能量是守恒的,在有机体为应对胁迫而合成抗胁迫物质的同时,会减少其他有机物的合成,表现为缩短寿命、生殖力下降等,导致种群数量的变化。
本学位论文以柑橘全爪螨为研究对象,瞄准生物适应逆境胁迫的机制这一国际研究热点,在生物学特性研究的基础上,开展了柑橘全爪螨Hsps基因的克隆、分子生物学特性、转录表达模式及异源表达研究,并结合抗氧化系统,综合分析柑橘全爪螨对热胁迫的响应机制,旨在理解柑橘全爪螨Hsps的分子伴侣功能及其与抗热胁迫的关系,了解柑橘全爪螨抗氧化系统在抵御热胁迫方面发挥的重要作用,揭示柑橘全爪螨乃至其他昆虫(螨)抗逆机制,为掌握昆虫(螨)种群数量的季节动态提供理论依据。取得的主要研究结果如下：
1高温对柑橘全爪螨存活及生殖的影响
在室内设置了32、35、38及41℃4个温度梯度,以25℃为对照,分别短时胁迫柑橘全爪螨卵及雌成螨,观察高温对柑橘全爪螨存活及生殖的影响。研究结果表明,随着胁迫温度升高,卵孵化率下降,当胁迫温度达35℃及以上时,各处理的卵孵化率显著低于对照。25℃时,柑橘全爪螨每雌平均总产卵量达53.7粒,在上述高温胁迫1h,柑橘全爪螨的单雌总产卵量呈显著下降趋势,且随着胁迫时间的延长,产卵量继续下降。环境温度为25℃时,柑橘全爪螨雌成螨的平均寿命为12.1天。高温胁迫1h,柑橘全爪螨寿命总体呈缩短趋势,温度上升到35℃以上,差异达显著水平,且随胁迫时间延长,缩短程度加剧。高温胁迫对柑橘全爪螨雌成螨的特定年龄存活率(lx)和特定年龄生殖力(mx)均存在不利影响,且随胁迫温度的升高及时间的延长影响程度增大。
2柑橘全爪螨对热胁迫的抗氧化反应
热胁迫是导致柑橘全爪螨发生氧化胁迫的因素之一。研究表明,热激或冷激能够干扰机体氧化还原反应平衡,从而导致氧化胁迫。柑橘全爪螨雌成螨在0、5、10、15、32、35、38及41℃条件下分别胁迫1h、2 h及3 h,以25℃为对照,测定柑橘全爪螨的抗氧化反应。结果表明,柑橘全爪螨遭受热胁迫后,其体内CAT和POD活性以及T-AOC在清除ROS的过程中,发挥的作用有限；而SOD在冷激条件下出现先上升后下降的动态趋势,暗示低温诱导SOD的抗氧化反应。GSTs的抗氧化反应无论在热激还是冷激条件下,均出现先上升然后下降的动态趋势。由此可见,SOD和GSTs在柑橘全爪螨抗氧化胁迫反应中发挥着重要作用。作为脂质过氧化的指标之一的MDA,其含量从时间动态上看能够维持在正常水平。综合分析表明,柑橘全爪螨能有效抵御热胁迫造成的氧化胁迫。
3柑橘全爪螨RT-qPCR内参基因的筛选
运用geNorm和NormFinder两种软件,对柑橘全爪螨已有的7个看家基因Pc5.8SrRNA、PcActin、PcEf-1αPcGapdh. PcRpⅡ、PcSdha及Pca-Tubulin在不同发育阶段和热胁迫下的稳定性进行了评价。发育阶段选取了卵、幼螨、若螨、雄成螨和雌成螨共5个处理,热胁迫分为冷激(0、5和10℃)和热激(35、38和41℃),处理时间均为1 h,以25℃为对照。分析结果表明,在柑橘全爪螨不同发育阶段及热胁迫1 h条件下,Pc5.8SrRNA、PcActin及PcSdha的稳定性均较差。在不同发育阶段稳定性较好的是PcRpⅡ;低温胁迫1 h稳定性较好的是PcGapdh和Pca-Tubulin;高温胁迫1 h稳定性较好的是PcRpⅡ。若使用多内参基因评价体系,不同发育阶段最适内参基因数目是2,建议选用PcEf-1α+PcRpⅡ组合；低温胁迫下最适内参基因数目也是2,建议选用PcGapdh+Pca-Tubulin组合;高温胁迫下,在现有内参基因的情况下,较适内参基因数目是3,建议选用PcEf-1 a+PcGapdh+PcRpⅡ组合。本研究采用单个内参基因评价体系,综合上述两种软件评价结果,柑橘全爪螨不同发育阶段及雌成螨高温胁迫下均选用PcRpⅡ作为内参基因；低温胁迫下,选用Pca-Tubulin作为内参基因。
4柑橘全爪螨Hsp90和Hsp70家族基因克隆和序列分析
4.1 Hsp90
利用RT-PCR结合RACE技术,从柑橘全爪螨体内成功克隆获得了1个Hsp90家族基因PcHsp90的cDNA全长序列,GenBank登录号为GQ495086。该基因cDNA全长为2,763 bp,开放阅读框长度为2,193 bp,编码730氨基酸残基。
进一步利用Protparam和Scanprosite等生物信息学软件分析了推导蛋白质的理化性质、保守基序等序列特征。PcHsp90基因编码的氨基酸序列具有5个典型的Hsp90家族特征基序(motif),它们分别是：NKEIFLRELISNSSDALDKIR、LGTIARS、IGQFGVGFYSAYLVAD、IKLYVRRVFI和GVVDSEDLPLNISRE,且具有细胞质Hsp90 C-端标志性基序“MEEVD",明确了其在细胞中的定位。采用软件MEGA 4.1用邻接法(Neighbor-joining method, N-J法),从GenBank选取12个不同物种的Hsp90与PcHsp90编码的氨基酸共同构建了系统发育树,其中PcHsp90与朱砂叶螨Hsp90亲缘关系最近,最先聚为一支。然后再与肩突硬蜱Hsp90相聚,而与昆虫纲的亲缘关系较远。应用蛋白质三维结构同源模拟工具SWISS-MODEL,构建了PcHsp90编码的蛋白质三维结构模型,可清楚地看到3个结构域：NBD (Nuleotide binding domain)、MD (Middle domain)和CTD (C-terminus domain)。
4.2 Hsp70s
同样,从柑橘全爪螨体内成功克隆获得了3个Hsp70家族基因PcHsp70-1、PcHsp70-2和PcHsp70-3的cDNA全长序列,GenBank登录号分别为：GQ495083、GQ495084和GQ495085。PcHsp70-1基因cDNA全长为2,586 bp,开放阅读框长度为1,977 bp,编码658个氨基酸残基；PcHsp70-2基因cDNA全长为2,405 bp,开放阅读框为1,968 bp,编码655个氨基酸残基；PcHsp70-3基因cDNA全长为2,300 bp,开放阅读框长度为2,028 bp,编码675个氨基酸残基。
进一步利用Protparam和Scanprosite等生物信息学软件分析了推导的蛋白质理化性质、保守基序等序列特征。上述3个基因编码的氨基酸序列均具有Hsp70家族3个典型的特征基序(motif)。PcHsp70-1和PcHsp70-2编码的氨基酸末端序列为‘'EEVD",是细胞质Hsp70所具有的特征基序,因此二者为细胞质型Hsp70。PcHsp70-3编码的氨基酸末端序列为“KDEL”,为内质网蛋白质的典型特征,判定其为内质网型Hsp70。柑橘全爪螨的3个Hsp70家族基因推导的氨基酸序列之间的相似性如下：PcHsp70-1和PcHsp70-2之间为87%；PcHsp70-1和PcHsp70-3为64.7%；PcHsp70-2和PcHsp70-3为65.8%。从GenBank中下载了来自其他物种的27个Hsp70氨基酸序列与本研究中的3个Hsp70基因推导的氨基酸序列共同构建了系统发育树。该系统发育树分为3支,分别是来自细胞质、内质网和线粒体的Hsp70。与序列分析结果一致,PcHsp70-1和PcHsp70-2首先与其它物种的细胞质型Hsp70聚为一支,PcHsp70-3与其它物种内质网型Hsp70聚为一支。应用蛋白质三维结构同源模拟工具SWISS-MODEL,分别构建了PcHsp70-1、PcHsp70-2和PcHsp70-3编码的蛋白质三维结构模型,它们均含有2个结构域：NBD (Nucleotide binding domain)和SBD (Substrate binding domain)。
5柑橘全爪螨Hsp90和Hsp70基因的mRNA表达模式解析
采用RT-qPCR技术,以PcRpⅡ做为内参基因,分别测定了PcHsp90、PcHsp70-1、PcHsp70-2和PcHsp70-3四个基因在柑橘全爪螨卵、幼螨、若螨、雄成螨及雌成螨等几个发育阶段的mRNA表达量的变化。结果显示,PcHsp90基因在卵、若螨和成螨阶段均大量表达,PcHsp70-1和PcHsp70-3基因在各发育阶段的表达量变化不大,而PcHsp70-2基因在各阶段的表达量变化非常明显。说明PcHsp90基因与维持基本生命活动和生长发育相关,而PcHsp70-1和PcHsp70-3基因可能是维持基本的生命活动所必须的,PcHsp70-2基因可能与柑橘全爪螨的生长发育密切相关。
以Pca-Tubulin做为内参基因,分别测定了柑橘全爪螨Hsp90家族和Hsp70家族的.上述4个基因在低温(0、5和10℃)胁迫1 h后mRNA表达量的变化。结果表明,4个基因在低温胁迫下mRNA表达量均呈下降趋势。说明低温短时胁迫可能诱导其它Hsps和物质表达,从而抑制这4个基因的mRNA表达水平。
以PcRpII做为内参基因,分别测定了上述4个基因在高温(35、38和41℃)胁迫1 h后mRNA表达量的变化。结果表明,高温胁迫下,4个基因的表达量随温度升高都呈现上调趋势,其中PcHsp90和PcHsp70的表达量与对照相比变化显著,如41℃处理1 h,PcHsp90基因mRNA表达量为对照的6.75倍,PcHsp70-2基因表达量为对照的28.37倍。说明二者在抵御高温胁迫方面发挥着重要作用。
综上所述,PcHsp70-1和PcHsp70-3基因主要与维持生命活动有关,与热胁迫关系不密切；PcHsp90基因参与基本生命活动,并在抵御高温胁迫方面具有重要作用;PcHsp70-2基因与生长发育密切相关,并在抵御高温胁迫方面发挥着重要作用。低温短时胁迫可能诱导其它Hsps或物质表达,从而抑制这4个基因的表达。
6柑橘全爪螨Hsp70基因异源表达载体的构建
利用BnmHI和NotⅠ的双酶切以及DNA重组技术成功构建了柑橘全爪螨PcHsp70-1、PcHsp70-2和PcHsp70-3基因基于pET 28a(+)的原核表达载体,这为进一步深入研究Hsp70蛋白质特性奠定了基础。
综上所述,本研究明确了高温短时胁迫对柑橘全爪螨存活和生殖的影响,为生化及分子水平的研究奠定了生物学基础,从抗氧化角度研究了热胁迫诱导柑橘全爪螨的抗氧化反应。在此基础上,深入分子机制研究,克隆获得了柑橘全爪螨Hsp90和Hsp70家族新基因4个,为准确解析这4个基因的nRNA水平表达模式,运用目前常用的两种软件geNorm和NormFinder软件评价7个候选内参基因,最终确定了柑橘全爪螨不同发育阶段和热胁迫下的最适内参基因。采用RT-qPCR技术,解析了这4个基因在不同发育阶段和热胁迫下的转录水平表达模式,揭示了它们在柑橘全爪螨响应热胁迫中发挥的作用。此外,成功构建了原核表达载体,为今后进一步研究Hsp70基因编码的蛋白质特性奠定了基础。本研究综合运用昆虫生理生化和分子生物学学科知识,全面解析了柑橘全爪螨的热胁迫响应机制,为理解抗氧化系统在有机体抵御热胁迫中的作用及丰富Hsps生理功能的认识等方面均具有重要的价值,同时,为明确昆虫(螨)种群季节动态规律提供了理论参考。
The citrus red mite Panonychus citri (McGregor), belonging to Arachnida, Acarina, Tetranychidae, Panonychus, is an important pest that devastates both deciduous and evergreen fruit trees such as citrus, pear, peach, and holly. When the mites are rampant, they will lead to great loss of output. Recent population outbreaks of P. citri may be attributed to a disruption in the orchard ecosystem, caused mainly by the application of broad-spectrum pesticides targeting other insects, leading to decreases in the natural enemy pressure on P. citri. In addition, the citrus red mite is often difficult to manage because of their ability to rapidly develop resistance to various acaricides due to its short life cycle. Nowadays, the P. citri has become one of the most serious pests in citrus orchard. In China, citruses have been cultivted in subtropical zones, and the mite populations on citrus trees have two infestation peaks every year, one in early summer and one in the autumn; but maintain low densities during the hot summer and cold winter. In main citrus producing areas where summer temperatures often reach or exceed 40℃, while winter temperatures may decline below 0℃. The optimum temperature zone for the citrus red mite ranges from 20-30℃, and 35℃is the proximity of the upper thermal threshold. So the citrus red mites are often exposed to the thermal stress in hot summer and cold winter. In general, tolerance mechanisms for extreme temperature are complex and act by various biochemical molecules such as polyols, lipids, and proteins. The sub-lethal hyperthermia induces heat shock responses involving increased expression of a suite of heat shock proteins (Hsps) which results in acquired thermo-tolerance of organisms. Heat or cold shock may also disturb the redox balance in animals and lead to oxidative stress. The antioxidant systems have an important role in deal with the damage caused by oxidative stress.
Based on the economic importance of the citrus red mite and the significance of thermal-stress responses, the current study was aiming at the adaption mechanisms of the mite to environment stress, and four new heat shock proteins (Hsps) genes were isolated, and their expression patterns were analyzed. In addtion, the response of antioxidant system of the citrus red mite under the thermal stress were investigated. Our results will not only provide great insights into exploring the developmental and physiological functions of the Hsps from the mite, but also provide evidence for clarifying the adaptive mechanisms of the mite to thermal stress. In the meantime, the current results enrich and develop the scientific theoretical study system for the antioxidant response in thermal stress. The study was supported in part by Special Fund for Agroscientific Research in the Public Interest (nyhyzx07-057,201103020), the Program for Innovative Research Team in University (IRT0976), Natural Science Foundation of Chongqing (CSTC,2009BA1042), and the Earmarked Fund for Modern Agro-industry (Citrus) Technology Research System.The main results are as follows:
1 Effects of thermal stress on survival and fecundity of P. citri
Temperature is one of the main factors influencing the arthropod population dynamics. The survival and fecundity of P. citri were studied following treated by 32, 35,38, and 41℃for short time, respectively. Temperature of 25℃served as a control. The results showed that the egg hatch rate decreased with the increasing of temperatures, and when temperature rose above 35℃, the hatch rate decreased significantly with comparison to control. The total average eggs laid per female was 53.7 eggs at 25℃, but it decreased significantly after stressed for 1 h under above temperatures, and the decrease tendency kept pace with the stress time. At 25℃, the average longevity of female adult was 12.1 d, but it was shortened after stressed for 1 h by high temperatures, and the longevity became shorter and shorter with stress time prolonged. In addition, high-temperature stress had negative effect on age-specific survival rage and age-specific fecundity of female adult, and the disadvantage became even worse with the length of stress time.
2 Antioxidant response of P. citri to thermal stress
Thermal stress is one of the factors that have the possibility to generate oxidative stress products in the citrus red mites. It has been confirmed that heat or cold shock may disturb the redox balance in this species and lead to oxidative stress. Catalase (CAT) and peroxidase (POD) activity as well as total antioxidant capacity (T-AOC) changed slightly in removal of ROS when the citrus red mites exposed to thermal stress. Nevertheless, superoxide dismutase (SOD) and glutathione-S-transferases (GSTs) play important role in the process of antioxidant response to thermal stress. Although T-AOC changed slightly, SOD and GSTs could be induced by the accumulation of products from oxidative stress. As one important index of lipid peroxidation, malondialdehyde (MDA) kept at an almost normal level in a time-dependent manner, which suggest that the citrus red mites is capable of efficiently dealing with ROS induced by thermal stresses.
3 Evaluation and validation of reference genes for RT-qPCR in P. citri
Reference genes used for the quantification of mRNA expression may be affected by the experimental condition, so the stabilities of seven candidate reference genes (Pc5.8SrRNA, PcActin, PcEf-1α, PcGapdh, PcRpⅡ, PcSdha, and Pcα-Tubulin) in P. citri were systematically analyzed at different developmental stages and after thermal stresses using geNorm and NormFinder software, respectively. The stage of egg, larva, nymph, and adult (male and female) was used for different developmental stages experiment, and thermal stresses included cold shock (0,5 and 10℃) and heat shock (35,38 and 41℃), respectively. The treated time was 1 h, and 25℃served as a control. The aim of this study was to evaluate housekeeping genes of P. citri for their suitability as reference genes in quantitative real-time PCR (RT-qPCR), and provide appropriate reference genes to explore the gene expression patterns of the heat shock proteins (Hsps) in P. citri. According to the analysis by geNorm and NormFinder, it was found that Pc5.8SrRNA, PcActin, and PcSdha were less stabe whether at developmental stages or under thermal stresses. Whereas, PcRpⅡwas the most stable gene at different developmental stages, PcGapdh and Pcα-Tubulin also kept stable during 1 h cold shock. If multiple reference genes needed for validation target genes mRNA expression under above condition, the most appropriate genes number was 2, and the combination of PcEf-1α+PcRpⅡwas recommended at different developmental stages; for cold shock experiment, the most appropriate genes number was also 2, the combination of PcGapdh+Pcα-Tubulin was recommended; whereas for heat shock experiment, the most appropriate genes number was 3, and the combination of PcEf-1α+PcGapdh+ PcRpⅡwas recommended.
4 Molecular cloning and sequences analysis of Hsp90 and Hsp70 family genes in P. citri
4.1 Hsp90
The full-length cDNA of Hsp90 family gene PcHsp90 was cloned from P. citri using the combined techniques of reverse transcriptase-PCR (RT-PCR) with rapid amplification of cDNA ends (RACE). Its GenBank accession number was GQ495086. The complete cDNA of consisted of 2,763 nucleotides with an open reading frame (ORF) of 2,193 nucleotides which encode a protein of 730 amino acids residues.
Molecular characterizations of the putative proteins have been predicted by Protparam software. The deduced amino acid sequence included 5 typical motifs of the Hsp90 family:NKEIFLRELISNSSDALDKIR, LGTIARS, IGQFGVGFYSAYLVAD, IKLYVRRVFI, and GVVDSEDLPLNISRE. The amino acid sequences ended with "MEEVD" motif which was the characteristic of cytosolic Hsp90. A phylogenetic tree was constructed based on the amino acid sequence deduced from PcHsp90 with other 12 Hsp90 amino sequences from GenBank using software MEGA 4.1 with Neighbor-joining method. The result showed that PcHsp90 clustered with Hsp90 from Tetranychus cinnabarinus firstly, next with Hsp90 of Ixodes scapularis, and kept relatively longer hereditary distance with Hsp90s from insects. In addition, its 3-D structure model was constructed by SWISSS-MODEL. Similar to other Hsp90, three domains were found:NBD (Nuleotide binding domain), MD (Middle domain), and CTD (C-terminus domain).
4.2 Hsp70s
The full-length cDNA of three Hsp70 family genes PcHsp70-1, PcHsp70-2, and PcHsp70-3 were cloned from P. citri. GenBank accession numbers were GQ495083, GQ495084, and GQ495085, respectively. The complete cDNA of PcHsp70-1 consisted of 2,586 nucleotides with an ORF of 1,977 nucleotides which encoded a protein of 658 amino acids residues. The complete cDNA of PcHsp70-2 consists of 2.405 nucleotides with an ORF of 1,968 nucleotides, encoding a protein of 655 amino acids residues. The complete cDNA of PcHsp70-3 consists of 2,300 nucleotides with an ORF of 2,028 nucleotides, encoding a protein of 675 amino acids residues.
Molecular characterizations of the putative proteins have been predicted by Protparam software. The amino acids sequences were encoded by PcHsp70-1, PcHsp70-2, and PcHsp70-3. respectively, included three typical motifs of the Hsp70 family genes. The motifs for PcHsp70-1 amino acids sequence were as follows: IDLGTTYS. IFDLGGGTFDVSLL, and IVLVGGSTRIPKIQK. The motifs for PcHsp70-2 were IDLGTTYS, IFDLGGGTFDVSIL, and IVLVGGSTRIPKIQK. The motifs for PcHsp70-3 were IDLGTTYS. VFDLGGGTFDVSLL, and IVLVGGSTRIPKIQQ. The C-terminus motifs of PcHsp70-1 and PcHsp70-2 amino acid sequences both were "EEVD", which was the characteristic of cytosolic Hsp70. So PcHsp70-1 and PcHsp70-2 were located in cytosol. However, the C-terminus motif of PcHsp70-3 amino acid sequence was "KDEL", which was the typical characteristic of ER (endoplasmic reticulum) protein at C-terminus. Accordingly PcHsp70-3 was attributed to ER Hsp70. The percentages of similarity among the three amino acids sequences were as follows:87% between PcHsp70-l and PcHsp70-2,64.7% between PcHsp70-l and PcHsp70-3, and 65.8% between PcHsp70-2 and PcHsp70-3. To analyze the sequence homology and phylogenetic relationships, the complete deduced protein sequences of the three Hsp70s, together with other Hsp70 sequences from 27 other organisms obtained from GenBank, were used for the phylogenetic tree construction. A Neighbor-joining phylogenetic tree was constructed by MEGA 4.1 The tree consisted of three branches, including the Hsp70 family members of the different intracellular compartments (cytosol, endoplasmic reticulum, and mitochondria). As expected, PcHsp70-l and PcHsp70-2 belonged to cytosolic Hsp70s and the PcHsp70-3 was clustered with the endoplasmic reticulum Hsp70 cluster from other species. In addition, their 3-D structure models were constructed by SWISSS-MODEL. All of them included 2 domains, NBD (Nucleotide binding domain) and SBD (Substrate binding domain).
5 The mRNA expression profiles of Hsp90 and Hsp70 genes
5.1 Developmental expression profiles of the four Hsp genes
The developmental expression profiles of Hsp90 and Hsp70 genes from P. citri were analyzed using PcRpII as an internal control gene. The developmental stages included egg, larvae, nymph, male adult, and female adult. The results indicated that expression of PcHsp90 in egg, nymph, and adult was high. Expressions of PcHsp70-l and PcHsp70-3 genes changed slightly at all stages. However, the expression level of PcHsp70-3 distinctly increased with development. Based on the above results, it was concluded that PcHsp90 and PcHsp70-2 possibly have connection with growth and development of P. citri. It was necessary for PcHsp70-l and PcHsp70-3 to maintain basal physiological function of the mite.
5.2 Expression profiles under short-term low-temperature stress
The expression profiles of Hsp90 and Hsp70 genes from P. citri were analyzed with Pcα-Tubulin gene as an internal control gene under low-temperature (0,5, and 10℃) stress for 1 h. Temperature of 25℃served as a control. The mRNA expressions levels of the four genes were all decreased compared with the control. It may suggest that other Hsps or substances were synthesized during stress, thus resulting in the decrease of the 4 Hsps under short-term low-temperature stress.
5.3 Expression profiles under short-term high-temperature stress
The expression patterns of Hsp90 and Hsp70 genes from P. citri were analyzed with PcRpII as an internal control gene under high-temperature (35,38, and 41℃) stress for 1 h. Temperature of 25℃served as a control. The results showed that expressions of the 4 genes were all up-regulate after heat shock, but only the expressions of PcHsp90 and PcHsp70-2 significantly enhanced at 41℃compared to control. At 41℃, the expression of PcHsp90 and PcHsp70-2 was up to 6.75- and 28.37-fold compared with the control. It was concluded that all the four genes all can be induced under short-term high-temperature, but PcHsp90 and PcHsp70-2 had more important role in deal with heat stress.
In conclusion, PcHsp70-1 and PcHsp70-3 were necessary to maintain basal physiological function of P. citri, while wouldn't be induced under short-term thermal stress. PcHsp90 can be induced by heat shock and was also relative with basal physiological function; there was a close relationship between PcHsp70-2 and development of P. citri, especially it can be dramatically induced by heat shock.
6 Heterologous expression of P. citri Hsp70s in Escherichia coli
Applying the double digestion of FastDigest" BamHⅠand FastDigestR NotⅠrestriction enzymes with the DNA recombination technology, the expression vectors for PcHsp70-1,PcHsp70-2, and PcHsp70-3 based on pET 28a (+) vector in Escherichia coli was constructed. This provided the basis for Hsp70s expression in vitro, and made it possible for study the characteristics and physiological function of Hsp70s in future.
In conclusion, the current study made it clear that short-term high-temperature had negative effect on the survival and fecundity of P. citri. The antioxidant response of P. citri to thermal stress was subsequently determined. To explore the molecular mechanisms of P. citri response to thermal stress, one Hsp90 and 3 Hsp70 genes were cloned from P. citri. To validate the stability of 7 housekeeping gene, the best stable reference genes at developmental stages and under thermal stress were selected using geNorm and NormFinder software. Then the mRNA expression profiles of the four genes at different developmental stages and under thermal stress were analyzed, and the role of the Hsp90 and Hsp70 genes in response to thermal stress were clarified. Besides, the expression vectors for 3 Hsp70 genes based on pET 28a (+) vector in Escherichia coli were constructed for studying the Hsp70 characteristics in future. These results not only help us understanding the mechanisms of P. citri in response to thermal stress at biological, physiological and molecular levels, but also provided the evidence to understand the population dynamics of the mite.
引文
1. Vassiliou VA, Papadoulis G. 2009. First record of the citrus red mite Panonychus citri in Cyprus. Phytoparasitica 37:99-100.
2.徐南昌,郎国良,刘立峰.2003.柑橘全爪螨发生规律及防治措施.植保技术与推广23：22-23.
3.忻介六.1988.农业螨类学.北京：农业出版社.1-466p.
4.刘映红.1992.桔全爪螨对柑桔生理的影响.西南农业大学学报14：235-239.
5. Ye H, Zhao ZM, Zhu WB.1998. Life table of Panonychus citri (McGregor) with particular reference to temperature. Journal of Yunnan University 20:367-369.
6. Gotoh T, Ishikawa Y, Kitashima Y.2003. Life-history traits of the six Panonychus species from Japan (Acari:Tetranychidae). Experimental and Applied Acarology 29:241-252.
7. Kasap I.2009. The biology and fecundity of the citrus red mite Panonychus citri (McGregor) (Acari:Tetranychidae) at different temperatures under laboratory conditions. Turkish Journal of Agriculture and Forestry 33:593-600.
8. Altincicek B, Knorr E, Vilcinskas A.2008. Beetle immunity:Identification of immune-inducible genes from the model insect Tribolium castaneum. Developmental and Comparative Immunology 32:585-595.
9. Abad-Moyano R, Pina T, Dembilio O, Ferragut F, Urbaneja A.2009. Survey of natural enemies of spider mites (Acari:Tetranychidae) in citrus orchards in eastern Spain. Experimental and Applied Acarology 47:49-61.
10. Chen C, Zheng J, Xie J, Xie X, Mao R.2009. Pest management based on petroleum spray oil in navel orange orchard in Ganzhou, South China. Journal of Pest Science 82:155-162.
11. Zhang Z.2010. Review of a new guide to the identification, biology and control of citrus mites. Systematic and Applied Acarology 15:36-38.
12. Gotoh T, Yamaguchi K, Mori K.2004. Effect of temperature on life history of the predatory mite Amblyseius (Neoseiulus) californicus (Acari:Phytoseiidae). Experimental and Applied Acarology 32:15-30.
13. Chen ZY, Ran C, Zhang L, Dou W, Wang JJ.2009. Susceptibility and esterase activity in citrus red mite Panonychus citri (McGregor) (Acari:Tetranychidae) after selection with phoxim. International Journal of Acarology 35:33-40.
14.刘永华,蒋红波,袁明龙,樊钰虎,杨丽红,陈静,王进军.2010.柑橘全爪螨 对4种杀螨剂的抗性监测及增效作用.果树学报27：570-574.
15. Stanley SM, Parsons PA, Spence GE, Weber L.1980. Resistance of species of the Drosophila melanogaster subgroup to environmental extremes. Australian Journal of Zoology 28:413-421.
16. Stratman R, Markow T.1998. Resistance to thermal stress in desert Drosophila. Functional Ecology 12:965-970.
17. Salt R.1961. Principles of insect cold-hardiness. Annual Review of Entomology 6: 55-74.
18. Sinclair B.1999. Insect cold tolerance:how many kinds of frozen? European Journal of Entomology 96:157-164.
19. Addo-Bediako A, Chown S, Gaston K.2000. Thermal tolerance, climatic variability and latitude. Proceedings of the Royal Society of London Series B: Biological Sciences 267:739-745.
20. Hoffmann AA, Sorensen JG, Loeschcke V.2003b. Adaptation of Drosophila to temperature extremes:bringing together quantitative and molecular approaches. Journal of Thermal Biology 28:175-216.
21. Storey K.1997. Organic solutes in freezing tolerance. Comparative Biochemistry and Physiology, Part A:Physiology 117:319-326.
22. Joanisse DR, Storey KB.1996. Oxidative stress and antioxidants in overwintering larvae of cold-hardy goldenrod gall insects. The Journal of Experimental Biology 199:1483-1491.
23. Joanisse DR, Storey KB.1998. Oxidative stress and antioxidants in stress and recovery of cold-hardy insects. Insect Biochemistry and Molecular Biology 28: 23-30.
24. Lalouette L, Williams CM, Hervant F, Sinclair BJ, Renault D.2011. Metabolic rate and oxidative stress in insects exposed to low temperature thermal fluctuations. Comparative Biochemistry and Physiology, Part A 158:229-234.
25. Lindquist L.1986. The heat-shock response. Annual Review of Biochemistry 55: 1151-1191.
26. Ferrando R, Schuschereba S, Quong J, Bowman P.1995. Carbon dioxide laser induction of heat shock protein 70 synthesis:comparison with high temperature treatment. Lasers in Medical Science 10:207-212.
27. Livingstone D.2001. Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms. Marine Pollution Bulletin 42: 656-666.
28. Hernandez-Garcia D, Wood CD, Castro-Obregon S, Covarrubias L.2010. Reactive oxygen species:A radical role in development? Free Radical Biology and Medicine 49:130-143.
29. Fridovich I.2004. Mitochondria:are they the seat of senescence? Aging Cell 3: 13-16.
30. Marnett L, West J.2003. Endogenous generation of reactive oxidants and electrophiles and their reactions with DNA and protein. Journal of Clinical Investigation 111:583-593.
31. Dubovskii IM, Grizanova EV, Chertkova EA, Slepneva IA, Komarov DA, Vorontsova YL, Glupov VV.2010. Generation of reactive oxygen species and activity of antioxidants in hemolymph of the moth larvae Galleria mellonella (L.) (Lepidoptera:Piralidae) at development of the process of encapsulation. Journal of Evolutionary Biochemistry and Physiology 46:35-43.
32. Lopez-Martinez G, Elnitsky MA, Benoit JB, Lee Jr RE, Denlinger DL.2008. High resistance to oxidative damage in the Antarctic midge Belgica antarctica, and developmentally linked expression of genes encoding superoxide dismutase, catalase and heat shock proteins. Insect Biochemistry and Molecular Biology 38:796-804.
33. Sies H.1997. Oxidative stress:oxidants and antioxidants. Experimental Physiology 82:291.
34. Felton GW, Summers CB.1995. Antioxidant systems in insects. Archives of Insect Biochemistry and Physiology 29:187-197.
35. McCord JM, Fridovich I.1969. Superoxide dismutase. Journal of Biological Chemistry 244:6049.
36. McCord JM, Keele BB, Fridovich I.1971. An enzyme-based theory of obligate anaerobiosis:the physiological function of superoxide dismutase. Proceedings of the National Academy of Sciences of the United States of America 68:1024.
37. Misra HP, Fridovich I.1976. Superoxide dismutase and the oxygen enhancement of radiation lethality. Archives of Biochemistry and Biophysics 176:577-581.
38. Datkhile KD, Mukhopadhyaya R, Dongre TK, Nath BB.2009. Increased level of superoxide dismutase (SOD) activity in larvae of Chironomus ramosus (Diptera: Chironomidae) subjected to ionizing radiation. Comparative Biochemistry and Physiology, Part C 149:500-506.
39. Claravon-Mathews M, Summers CB, Felton GW.1997. Ascorbate peroxidase:a novel antioxidant enzyme in insects. Archives of Insect Biochemistry and Physiology 34:57-68.
40. Ahmad S.1992. Biochemical defence of pro-oxidant plant allelochemicals by herbivorous insects. Biochemical systematics and ecology 20:269-296.
41. Felton GW, Duffey SS.1991. Protective action of midgut catalase in Lepidopteran larvae against oxidative plant defenses. Journal of Chemical Ecology 17: 1715-1732.
42. Grubor-Lajsic G, Block W, Telesmanic M, Jovanovic A, Stevanovic D, Baca F. 1997. Effect of cold acclimation on the antioxidant defense system of two larval epidoptera (Noctuidae). Archives of Insect Biochemistry and Physiology 36:1-10.
43. Serrano-Pinto V, Acosta-Perez M, Luviano-Bazan D, Hurtado-Sil G, Batista CVF, Martinez-Barnetche J, Lanz-Mendoza H.2010. Differential expression of proteins in the midgut of Anopheles albimanus infected with Plasmodium berghei. Insect Biochemistry and Molecular Biology 40:752-758.
44. Michaud RM, Benoit JB, Lopez-Martinez G, Elnitsky MA, Lee Jr RE, Denlinger DL.2008. Metabolomics reveals unique and shared metabolic changes in response to heat shock, freezing and desiccation in the Antarctic midge,Belgica antarctica. Journal of Insect Physiology 54:645-655.
45. Corona M, Robinson GE.2006. Genes of the antioxidant system of the honey bee: annotation and phylogeny. Insect Molecular Biology 15:687-701.
46. Ritossa F.1962. A new puffing pattern induced by temperature shock and DNP in Drosophila. Cellular and Molecular Life Sciences18:571-573.
47. Tissieres A, Mitchell HK, Tracy UM.1974. Protein synthesis in salivary glands of Drosophila melanogaster:relation to chromosome puffs. Journal of Molecular Biology 84:389-392.
48. Kiang JG, Tsokos GC.1998. Heat shock protein 70 kDa:Molecular biology, biochemistry, and physiology. Pharmacology & Therapeutics 80:183-201.
49. Daugaard M, Rohde M, Jaattela M.2007. The heat shock protein 70 family:Highly homologous proteins with overlapping and distinct functions. FEBS Letters 581: 3702-3710.
50. Munro S, Pelham HRB.1987. A C-terminal signal prevents secretion of luminal ER proteins. Cell 48:899-907.
51. Guy C, Li Q.1998. The organization and evolution of the spinach stress 70 molecular chaperone gene family. The Plant Cell Online 10:539-556.
52. Feder ME, Hofmann GE.1999. Heat-shock proteins, molecular chaperones, and the stress response:Evolutionary and ecological physiology. Annual Review of Physiology 61:243-282.
53. Parsell DA, Lindquist S.1993. The function of heat-shock proteins in stress tolerance:degradation and reactivation of damaged proteins. Annual Review of Genetics 27:437-496.
54. Rinehart JP, Li A, Yocum GD, Robich RM, Hayward SAL, Denlinger DL.2007. Up-regulation of heat shock proteins is essentail for cold survival during insect diapause. Proceedings of the National Academy of Sciences of the United States of America 104:11130-11137.
55. Lindquist S, Craig EA.1988. The heat-shock proteins. Annual Review of Genetics 22:631-677.
56. Morimoto RI.1993. Cells in stress:transcriptional activation of heat shock genes. Science 259:1409-1410.
57. Carper SW, Duffy JJ, Gerner EW.1987. Heat shock proteins in thermotolerance and other cellular processes. Cancer Research 47:5249-5255.
58. Gupta SC, Sharma A, Mishra M, Mishra RK, Chowdhuri DK.2010. Heat shock proteins in toxicology:How close and how far? Life Sciences 86:377-384.
59. Kim KK, Kim R, Kim S.1998. Crystal structure of small heat-shock protein. Nature 394:595-599.
60. Boorstein WR, Ziegelhoffer T, Craig EA.1994. Molecular evolution of the HSP70 multigene family. Journal of Molecular Evolution 38:1-17.
61. Denlinger DL, Rinehart JP, Yocum GD.2001. Stress proteins:a role in insect diapause. In:Denlinger DL, Giebultowicz, J. and Saunders, D.S., eds, editor: Elsevier, Amsterdam.155-171 p.
62. Sorensen JG 2010. Application of heat shock protein expression for detecting natural adaptation and exposure to stress in natural populations. Current Zoology 56:703-713.
63.王秋香,李宗芸,谢艳,曹慧.2008.果蝇热激蛋白的研究进展.昆虫知识45：698-702.
64. Pearl LH, Prodromou C.2006. Structure and mechanism of the Hsp90 molecular chaperone machinery. Annual Review of Biochemistry 75:271-294.
65. Stebbins CE, Russo AA, Schneider C, Rosen N, Hartl FU, Pavletich NP.1997. Crystal structure of an Hsp90-geldanamycin complex:targeting of a protein chaperone by an antitumor agent. Cell 89:239-250.
66. Prodromou C, Roe SM, O'Brien R, Ladbury JE, Piper PW, Pearl LH.1997. Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone. Cell 90:65-75.
67. Fontana J, Fulton D, Chen Y, Fairchild TA, McCabe TJ, Fujita N, Tsuruo T, Sessa WC.2002. Domain mapping studies reveal that the M domain of hsp90 serves as a molecular scaffold to regulate Akt-dependent phosphorylation of endothelial nitric oxide synthase and NO release. Circulation Research 90: 866-873.
68. Sato S, Fujita N, Tsuruo T.2000. Modulation of Akt kinase activity by binding to Hsp90. Proceedings of the National Academy of Sciences of the United States of America 97:10832-10837.
69. Meyer P, Prodromou C, Hu B, Vaughan C, Roe SM, Panaretou B, Piper PW, Pearl LH.2003. Structural and functional analysis of the middle segment of hsp90: Implications for ATP hydrolysis and client protein and cochaperone interactions. Molecular Cell 11:647-658.
70. Panaretou B, Siligardi G, Meyer P, Maloney A, Sullivan JK, Singh S, Millson SH, Clarke PA, Naaby-Hansen S, Stein R.2002. Activation of the ATPase activity of hsp90 by the stress-regulated cochaperone ahal. Molecular Cell 10: 1307-1318.
71. Young JC, Obermann WMJ, Hartl FU.1998. Specific binding of tetratricopeptide repeat proteins to the C-terminal 12-kDa domain of hsp90. Journal of Biological Chemistry 273:18007-18010.
72. Wang TF, Chang JH, Wang C.1993. Identification of the peptide binding domain of hsc70.18-Kilodalton fragment located immediately after ATPase domain is sufficient for high affinity binding. Journal of Biological Chemistry 268: 26049-26051.
73. Zhong M, Kim SJ, Wu C.1999. Sensitivity of Drosophila heat shock transcription factor to low pH. Journal of Biological Chemistry 274:3135-3140.
74. Zeng XC, Bhasin S, Xufeng WU, Lee JG, Maffi S, Nichols CJ, Taylor JP, Greene LE, Eisenberg E.2004. Hsp70 dynamics in vivo:Effect of heat shock and protein aggregation. Journal of Cell Science 117:4991-5000.
75. Kroeger PE, Sarge K.D, Morimoto RI.1993. Mouse heat shock transcription factors 1 and 2 prefer a trimeric binding site but interact differently with the HSP70 heat shock element. Molecular and Cellular Biology 13:3370-3383.
76. Price BD, Calderwood SK.1991. Ca2+ is essential for multistep activation of the heat shock factor in permeabilized cells. Molecular and Cellular Biology 11: 3365-3368.
77. Wiederrecht G, Seto D, Parker CS.1988. Isolation of the gene encoding the S. cerevisiae heat shock transcription factor. Cell 54:841-853.
78. Nakai A, Tanabe M, Kawazoe Y, Inazawa J, Morimoto RI, Nagata K.1997. HSF4, a new member of the human heat shock factor family which lacks properties of a transcriptional activator. Molecular and Cellular Biology 17:469-481.
79. Morimoto RI, Santoro MG.1998. Stress-inducible responses and heat shock proteins:New pharmacologic targets for cytoprotection. Nature Biotechnology 16:833-838.
80. Pirkkala L, Nykanen P, Sistonen LEA.2001. Roles of the heat shock transcription factors in regulation of the heat shock response and beyond. The FASEB Journal 15:1118-1131.
81. Harrison CJ, Bohm AA, Nelson HC.1994. Crystal structure of the DNA binding domain of the heat shock transcription factor. Science 263:224-227.
82. Ananthan J, Goldberg AL, Voellmy R.1986. Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. Science 232: 522-524.
83. Audibert F.2003. Adjuvants for vaccines, a quest. International Immunopharmacology 3:1187-1193.
84. Li B, Liu HT, Sun DY, Zhou RG.2004. Ca2+ and calmodulin modulate DNA-binding activity of maize heat shock transcription factor in vitro. Plant and Cell Physiology 45:627-634.
85. Xuan Y, Zhou S, Wang L, Cheng Y, Zhao L.2010. Nitric oxide functions as a signal and acts upstream of AtCaM3 in thermotolerance in Arabidopsis seedlings. Plant Physiology 153:1895-1906.
86. Saidi Y, Finka A, Muriset M, Bromberg Z, Weiss YG, Maathuis FJM, Goloubinoff P.2009. The heat shock response in moss plants is regulated by specific calcium-permeable channels in the plasma membrane. The Plant Cell Online 21: 2829-2843.
87. Zhang W, Zhou RG, Gao YJ, Zheng SZ, Xu P, Zhang SQ, Sun DY.2009. Molecular and genetic evidence for the key role of AtCaM3 in heat-shock signal transduction in Arabidopsis. Plant Physiology 149:1773-1784.
88. Beckmann RP, Mizzen LE, Welch WJ.1990. Interaction of Hsp 70 with newly synthesized proteins:implications for protein folding and assembly. Science 248:850-854.
89. Agashe VR, Hartl FU.2000. Roles of molecular chaperones in cytoplasmic protein folding. Seminars in Cell &Developmental Biology 11:15-25.
90. Young JC, Agashe VR, Siegers K, Hartl FU.2004. Pathways of chaperone-mediated protein folding in the cytosol. Nature Reviews Molecular Cell Biology 5:781-791.
91. Gething MJ.1999. Role and regulation of the ER chaperone BiP. Seminars in Cell & Development Biology 10:465-472.
92. Welch WJ.1992. Mammalian stress response:cell physiology, structure/function of stress proteins, and implications for medicine and disease. Physiological Reviews 72:1063-1086.
93. Rossi S, Snyder MJ.2001. Competition for space among sessile marine invertebrates:changes in HSP70 expression in two Pacific cnidarians. The Biological Bulletin 201:385-393.
94. Hartl FU.1996. Molecular chaperones in cellular protein folding. Nature 381: 571-579.
95. Goloubinoff P, Rios PDL.2007. The mechanism of Hsp70 chaperones:(entropic) pulling the models together. Trends in Biochemical Sciences 32:372-380.
96. Sibly RM, Calow P.1989. A life-cycle theory of responses to stress. Biological Journal of the Linnean Society 37:101-116.
97. Moseley PL.1997. Heat shock proteins and heat adaptation of the whole organism. Journal of Applied Physiology 83:1413-1417.
98. Katschinski DM.2004. On heat and cells and proteins. News in Physiological Sciences 19:11-15.
99. Huang LH, Chen B, Kang L.2007. Impact of mild temperature hardening on thermo tolerance, fecundity, and Hsp gene expression in Liriomyza huidobrensis. Journal of Insect Physiology 53:1199-1205.
100. Morales M, Planello R, Martinez-Paz P, Herrero O, Cortes E, Martinez-Guitarte JL, Morcillo G.2011. Characterization of Hsp70 gene in Chironomus riparius: expression in response to endocrine disrupting pollutants as a marker of ecotoxicological stress. Comparative Biochemistry and Physiology, Part C 153: 150-158.
101. Butler CD, Trumble JT.2010. Predicting population dynamics of the parasitoid Cotesia marginiventris (Hymenoptera:Braconidae) resulting from novel interactions of temperature and selenium. Biocontrol Science and Technology 20:391-406.
102. Raisuddin S, Kwok KWH, Leung KMY, Schlenk D, Lee JS.2007. The copepod Tigriopus:A promising marine model organism for ecotoxicology and environmental genomics. Aquatic Toxicology 83:161-173.
103. Rinehart JP, Robich RM, Denlinger DL.2006. Enhanced cold and desiccation tolerance in diapausing adults of Culex pipiens, and a role for hsp70 in response to cold shock but not as a component of the diapause program. Journal of Medical Entomology 43:713-722.
104. Lopez-Martinez G, Benoit JB, Rinehart JP, Elnitsky MA, Lee RE, Denlinger DL. 2009. Dehydration, rehydration, and overhydration alter patterns of gene expression in the Antarctic midge, Belgica antarctica. Journal of Comparative Physiology 179:481-491.
105. Parsell DA, Taulien J, Lindquist S.1993. The role of heat-shock proteins in thermotolerance. Philosophical Transactions:Biological Sciences 339: 279-286.
106. Fulda S, Gorman A, Hori O, Samali A.2010. Cellular stress responses:cell survival and cell death. International Journal of Cell Biology: doi:10.1155/2010/214074.
107. Liang P, MacRae TH.1997. Molecular chaperones and the cytoskeleton. Journal of Cell Science 110:1431-1440.
108. Pai KS, Mahajan VB, Lau A, Cunningham DD.2001. Thrombin receptor signaling to cytoskeleton requires Hsp90. Journal of Biological Chemistry 276: 32642-32647.
109. Verrills NM, Liem NL, Liaw TYE, Hood BD, Lock RB, Kavallaris M.2006. Proteomic analysis reveals a novel role for the actin cytoskeleton in vincristine resistant childhood leukemia Can in vivo study. Proteomics 6:1681-1694.
110. Prokhnevsky AI, Peremyslov VV, Dolja VV.2005. Actin cytoskeleton is involved in targeting of a viral Hsp70 homolog to the cell periphery. Journal of Virology 79:14421-14428.
111. Bardella L, Comolli R.1994. Differential expression of c-jun, c-fos and hsp 70 mRNAs after folic acid and ischemia-reperfusion injury:effect of antioxidant treatment. Experimental Nephrology 2:158-165.
112. Khassaf M, McArdle A, Esanu C, Vasilaki A, McArdle F, Griffiths RD, Brodie DA, Jackson MJ.2003. Effect of vitamin C supplements on antioxidant defence and stress proteins in human lymphocytes and skeletal muscle. The Journal of Physiology 549:645-652.
113. Wallin RPA, Lundqvist A, More SH, von Bonin A, Kiessling R, Ljunggren HG. 2002. Heat-shock proteins as activators of the innate immune system. Trends in Immunology 23:130-135.
114. Zhou J, Wang WN, He WY, Zheng Y, Wang L, Xin Y, Liu Y, Wang AL.2010. Expression of HSP60 and HSP70 in white shrimp, Litopenaeus vannamei in response to bacterial challenge. Journal of Invertebrate Pathology 103: 170-178.
115. Garrido C, Gurbuxani S, Ravagnan L, Kroemer G.2001. Heat Shock Proteins: Endogenous Modulators of Apoptotic Cell Death. Biochemical and Biophysical Research Communications 286:433-442.
116. Bierkens JGEA.2000. Applications and pitfalls of stress-proteins in biomonitoring. Toxicology 153:61-72.
117. Rossner P.2003. Heat shock proteins hsp32 and hsp70 as biomarkers of an early response? In vitro induction of heat shock proteins after exposure of cell culture to carcinogenic compounds and their real mixtures. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 542:105-116.
118. Sturzenbaum S, Arts M, Kammenga J.2005. Molecular cloning and characterization of Cpn60 in the free-living nematode Plectus acuminatus. Cell Stress & Chaperones 10:79.
119. Ryan JA, Hightower LE.1996. Stress proteins as molecular biomarkers for environmental toxicology. Stress-Inducible Cellular Responses 77:411-424.
120. Varo 1, Serrano R, Pitarch E, Amat F, Lopez FJ, Navarro JC.2002. Bioaccumulation of chlorpyrifos through an experimental food chain:Study of protein HSP70 as biomarker of sublethal stress in fish. Archives of Environmental Contamination and Toxicology 42:229-235.
121. Washburn BS, Moreland JJ, Slaughter AM, Werner I, Hinton DE, Sanders BM. 2002. Effects of handling on heat shock protein expression in rainbow trout Oncorhynchus mykiss. Environmental Toxicology and Chemistry 21:557-560.
122. Mukhopadhyay I, Saxena DK, Chowdhuri DK.2003. Hazardous effects of effluent from the chrome plating industry:70 kDa heat shock protein expression as a marker of cellular damage in transgenic Drosophila melanogaster (hsp70-lacZ). Environmental Health Perspectives 111: 1926-1932.
123. Roh JY, Lee J, Choi J.2006. Assessment of stress-related gene expression in the heavy metal-exposed nematode Caenorhabditis elegans:A potential biomarker for metal-induced toxicity monitoring and environmental risk assessment. Environmental Toxicology and Chemistry 25:2946-2956.
124. Singh MP, Reddy MMK, Mathur N, Saxena DK, Chowdhuri DK.2009. Induction of hsp70, hsp60, hsp83 and hsp26 and oxidative stress markers in benzene, toluene and xylene exposed Drosophila melanogaster: Role of ROS generation. Toxicology and Applied Pharmacology 235:226-243.
125. Singh MP, Ravi Ram K, Mishra M, Shrivastava M, Saxena DK, Chowdhuri DK. 2010. Effects of co-exposure of benzene, toluene and xylene to Drosophila melanogaster:Alteration in hsp70, hsp60, hsp83, hsp26, ROS generation and oxidative stress markers. Chemosphere 79:577-587.
126. Chen B, Kang L.2002. Trends of occurrence and geographic variation of pea leafminer Liriomyza huidobrensis in China. Plant Quarantine 16:138-140.
127. Kang L, Chen B, Wei JN, Liu TX.2009. Roles of thermal adaptation and chemical ecology in Liriomyza distribution and control. Annual Review of Entomology 54:127-145.
128. Huang LH, Kang L.2007. Cloning and interspecific altered expression of heat shock protein genes in two leafminer species in response to thermal stress. Insect Molecular Biology 16:491-500.
129. Yu H, Wan FH.2009. Cloning and expression of heat shock protein genes in two whitefly species in response to thermal stress. Journal of Applied Entomology 133:602-614.
130. Gehring WJ, Wehner R.1995. Heat shock protein synthesis and thermotolerance in Cataglyphis, an ant from the Sahara desert. Proceedings of the National Academy of Sciences of the United States of America 92:2994-2998.
131. Rinehart JP, Hayward SAL, Elnitsky MA, Sandro LH, Lee RE, Denlinger DL 2006. Continuous up-regulation of heat shock proteins in larvae, but not adults, of a polar insect. Proceedings of the National Academy of Sciences of the United States of America 103:14223-14227.
132. Evgen'ev MB, Garbuz DG, Shilova VY, Zatsepina OG.2007. Molecular mechanisms underlying thermal adaptation of xeric animals. Journal of Biosciences 32:489-499.
133. Bhargav D, Pratap Singh M, Murthy RC, Mathur N, Misra D, Saxena DK, Kar Chowdhuri D.2008. Toxic potential of municipal solid waste leachates in transgenic Drosophila melanogaster (hsp70-lacZ):hsp70 as a marker of cellular damage. Ecotoxicology and Environmental Safety 69:233-245.
134. Feng HZ, Wang L, Liu YH, He L, Li M, Lu WC, Xue CH.2010. Molecular characterization and expression of a heat shock protein gene (HSP90) from the carmine spider mite, Tetranychus cinnabarinus (Boisduval). Journal of Insect Science 10:1-12.
135. Yoshimi T, Minowa K, Karouna-Renier NK, Watanabe C, Sugaya Y, Miura T. 2002. Activation of a stress-induced gene by insecticides in the midge, Chironomus yoshimatsui. Journal of Biochemical and Molecular Toxicology 16: 10-17.
136. Mason PJ, Hall LMC, Gausz J.1984. The expression of heat shock genes during normal development in Drosophila melanogaster (heat shock/abundant transcripts/developmental regulation). Molecular and General Genetics 194: 73-78.
137. Zhang QR, Denlinger DL.2010. Molecular characterization of heat shock protein 90,70 and 70 cognate cDNAs and their expression patterns during thermal stress and pupal diapause in the corn earworm. Journal of Insect Physiology 56: 138-150.
138. Rinehart JP, Robich RM, Denlinger DL.2010. Isolation of diapause-regulated genes from the flesh fly, Sarcophaga crassipalpis by suppressive subtractive hybridization. Journal of Insect Physiology 56:603-609.
139. Shim JK, Jung DO, Park JW, Kim DW, Ha DM, Lee KY.2006. Molecular cloning of the heat-shock cognate 70 (Hsc70) gene from the two-spotted spider mite, Tetranychus urticae, and its expression in response to heat shock and starvation. Comparative Biochemistry and Physiology, Part B 145:288-295.
140. Li M, Lu WC, Feng HZ, He L.2009. Molecular characterization and expression of three heat shock protein70 genes from the carmine spider mite, Tetranychus cinnabarinus (Boisduval). Insect Molecular Biology 18:183-194.
141.韩柏明,金大勇,吕龙石.2007.苹果全爪螨在不同温度下的实验种群生命表.昆虫知识44：226-228.
142. Luo J, Zhang XX, Zhai BP.2005. Effect of high temperature on the growth, survival and reproduction of a laboratory population of the rice stem borer Chilo suppressalis Walker. Acta Ecologica Sinica 25:931-936.
143. Dean GJ.1974. Effect of temperature on the cereal aphids Metopolophium dirhodum (Wlk.), Rhopalosiphum padi (L.) and Macrosiphum avenue (F.) (Hem., Aphididae). Bulletin of Entomological Research 63:401-409.
144.马春森,陈瑞鹿.1993.温度对小菜蛾(Plutella xylostella L.)发育和繁殖影响的研究.吉林农业科学3：44-49.
145. Williams KD, Helin AB, Posluszny J, Roberts SP, Feder ME.2003. Effect of heat shock, pretreatment and hsp70 copy number on wing development in Drosophila melanogaster. Molecular Ecology 12:1165-1177.
146.向玉勇,郭晓军,张帆,李子忠,罗晨.2007.温度和湿度对北京地区B型烟粉虱个体发育和种群繁殖的影响.华北农学报22：152-156.
147. Ma CS, Hau B, Poehling HM.2004. Effects of pattern and timing of high temperature exposure on reproduction of the rose grain aphid, Metopolophium dirhodum. Entomologia Experimentalis et Applicata 110:65-71.
148. Mironidis GK, Savopoulou-Soultani M.2010. Effects of heat shock on survival and reproduction of Helicoverpa armigera (Lepidoptera:Noctuidae) adults. Journal of Thermal Biology 35:59-69.
149.崔旭红,谢明,万方浩.2008.短时高温暴露对B型烟粉虱和温室白粉虱存活以及生殖适应性的影响.中国农业科学41：424-430.
150. Neven LG. 2000. Physiological responses of insects to heat. Postharvest Biology and Technology 21:103-111.
151.杜尧,马春森,赵清华,马罡,杨和平.2007.高温对昆虫影响的生理生化作用机理研究进展.生态学报27：1565-1572.
152.何恒果.2010.桔全爪螨对甲氰菊酯和阿维菌素的抗性及其酯酶基因的克隆与表达研究[博士学位论文].重庆：西南大学.23-24 p.
153.刘映红.1991.桔全爪螨种群与柑桔相互作用的研究[博士学位论文].重庆：西南农业大学.16 p.
154. Gerson U, Cohen E.1989. Resurgences of spider mites (Acari:Tetranychidae) induced by synthetic pyrethroids. Experimental and Applied Acarology 6: 29-46.
155.陈道茂,陈卫民.1990.二种拟除虫菊酯对桔全爪螨繁殖的影响.植物保护学报17：279-282.
156.沈慧敏,张新虎.2002.三氟氯氰菊酯和甲氰菊酯对二点叶螨生命活力及繁殖的影响.植物保护学报29：183-188.
157.李定旭,田娟,沈佐锐.2006.不同药剂对山楂叶螨的亚致死效应.植物保护学报33：187-192.
158. Green DR, Reed JC.1998. Mitochondria and apoptosis. Science 281:1309-1312.
159. Meng JY, Zhang CY, Zhu F, Wang XP, Lei CL.2009. Ultraviolet light-induced oxidative stress:effects on antioxidant response of Helicoverpa armigera adults. Journal of Insect Physiology 55:588-592.
160. Imlay JA.2003. Pathways of oxidative damage. Annual Review of Microbiology 57:395-418.
161. Dubovskiy IM, Martemyanov VV, Vorontsova YL, Rantala MJ, Gryzanova EV, Glupov VV.2008. Effect of bacterial infection on antioxidant activity and lipid peroxidation in the midgut of Galleria mellonella L. larvae (Lepidoptera, Pyralidae). Comparative Biochemistry and Physiology, Part C 148:1-5.
162. Wang Y, Oberley LW, Murhammer DW.2001. Antioxidant defense systems of two lepidopteran insect cell lines. Free Radical Biology & Medicine 30: 1254-1262.
163. Ghiselli A, Serafini M, Natella F, Scaccini C.2000. Total antioxidant capacity as a tool to assess redox status:critical view and experimental data. Free Radical Biology and Medicine 29:1106-1114.
164. Abele D, Heise K, Portner HO, Puntarulo S.2002. Temperature-dependence of mitochondrial function and production of reactive oxygen species in the intertidal mud clam Mya arenaria. Journal of Experimental Biology 205: 1831-1841.
165. Bradford MM.1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry 72:248-254.
166. Habig WH, Pabst MJ, Jakoby WB.1974. Glutathione S-transferase. The Journal of Bilogical Chemistry 249:7130-7139.
167. Worner SP.1988. Ecoclimatic assessment of potential establishment of exotic pests. Journal of Economic Entomology 81:973-983.
168. Bale JS, Masters GJ, Hodkinson ID, Awmack C, Bezemer TM, Brown VK, Butterfield J, Buse A, Coulson JC, Farrar J.2002. Herbivory in global climate change research:direct effects of rising temperature on insect herbivores. Global Change Biology 8:1-16.
169. Wang XH, Kang L.2005. Differences in egg thermotolerance between tropical and temperate populations of the migratory locust Locusta migratoria (Orthoptera:Acridiidae). Journal of Insect Physiology 51:1277-1285.
170. Park MS, Jo PG, Choi YK, An KW, Choi CY.2009. Characterization and mRNA expression of Mn-SOD and physiological responses to stresses in the Pacific oyster Crassostrea gigas. Marine Biology Research 5:453-463.
171. Abele D, Burlando B, Viarengo A, Portner HO.1998. Exposure to elevated temperatures and hydrogen peroxide elicits oxidative stress and antioxidant response in the Antarctic intertidal limpet Nacella concinna. Comparative Biochemistry and Physiology, Part B 120:425-435.
172. An MI, Choi CY.2010. Activity of antioxidant enzymes and physiological responses in ark shell, Scapharca broughtonii, exposed to thermal and osmotic stress:effects on hemolymph and biochemical parameters. Comparative Biochemistry and Physiology, Part B 155:34-42.
173. Ahmad S, Duval DL, Weinhold LC, Pardini RS.1991. Cabbage looper antioxidant enzymes:tissue specificity. Insect Biochemistry and Molecular Biology 21:563-572.
174. Ahmad S, Pardini RS.1990. Antioxidant defense of the cabbage looper, Trichoplusia ni:enzymatic responses to the superoxide-generating flavonoid, quercetin, and photodynamic furanocoumarin, xanthotoxin. Photochemistry and Photobiology 51:305-311.
175. Li D, Blasevich F, Theopold U, Schmidt O.2003. Possible function of two insect phospholipid-hydroperoxide glutathione peroxidases. Journal of Insect Physiology 49:1-9.
176. Missirlis F, Rahlfs S, Dimopoulos N, Bauer H, Becker K, Hilliker A, Phillips JP, J ckle H.2003. A putative glutathione peroxidase of Drosophila encodes a thioredoxin peroxidase that provides resistance against oxidative stress but fails to complement a lack of catalase activity. Biological Chemistry 384:463-472.
177. Kono Y. Shishido T.1992. Distribution of glutathione S-transferase activity in insect tissues. Applied Entomology and Zoology 27:391-397.
178. Somogyi A, Rosta K, Pusztai P, Tulassay Z, Nagy G.2007. Antioxidant measurements. Physiological Measurement 28:R41-R55.
179. Kaur G, Alamb MS, Athar M.2009. Cumene hydroperoxide debilitates macrophage physiology by inducing oxidative stress:possible protection by a-tocopherol. Chemico-Biological Interactions 179:94-102.
180. Mahmud SA, Hirasawa T, Hiroshi S.2010. Differential importance of trehalose accumulation in Saccharomyces cerevisiae in response to various environmental stresses. Journal of Bioscience and Bioengineering 109: 262-266.
181. Zhou J, Wang L, Xin Y, Wang WN, He WY, Wang AL, Liu Y.2010. Effect of temperature on antioxidant enzyme gene expression and stress protein response in white shrimp, Litopenaeus vannamei. Journal of Thermal Biology 35: 284-289.
182. Rael LT, Thomas GW, Craun ML, Curtis CG, Bar-Or R, Bar-Or D.2004. Lipid peroxidation and the thiobarbituric acid assay:standardization of the assay when using saturated and unsaturated fatty acids Journal of Biochemistry and Molecular Biology 37:749-752.
183. Del Rio D, Stewart AJ, Pellegrini N.2005. A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. Nutrition, Metabolism and Cardiovascular Diseases 15:316-328.
184. Michaud MR, Denlinger DL.2004. Molecular modalities of insect cold survival: current understanding and future trends. International Congress Series 1275: 32-46.
185. Sorensen JG, Kristensen TN, Loeschcke V.2003. The evolutionary and ecological role of heat shock proteins. Ecology Letters 6:1025-1037.
186. Csermely P, Schnaider T, Soti C, Prohaszka Z, Nardai G.1998. The 90-kDa molecular chaperone family:structure, function, and clinical applications.a comprehensive review. Pharmacology & Therapeutics 79:129-168.
187. Krishna P, Gloor G.2001. The Hsp90 family of proteins in Arabidopsis thaliana. Cell Stress & Chaperones 6:238-246.
188. Wang W, Vinocur B, Shoseyov O, Altman A.2004. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends in Plant Science 9:244-252.
189. Ellis RJ, Van der Vies SM.1991. Molecular chaperones. Annual Review of Biochemistry 60:321-347.
190. Bukau B, Horwich AL.1998. The Hsp70 and Hsp60 chaperone machines. Cell 92: 351-366.
191. Johnston JA, Ward CL, Kopito RR.1998. Aggresomes:a cellular response to misfolded proteins. Journal of Cell Biology 143:1883-1898.
192. Zampieri M, Ciccarone F, Guastafierro T, Bacalini MG, Calabrese R, Moreno-Villanueva M, Reale A, Chevanne M, Burkle A, Caiafa P.2010. Validation of suitable internal control genes for expression studies in aging. Mechanisms of Ageing and Development 131:89-95.
193. Huggett J, Dheda K, Bustin S, Zumla A.2005. Real-time RT-PCR normalisation; strategies and considerations. Genes and Immunity 6:279-284.
194. Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR.2005. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiology 139:5.
195. Radoni A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A.2004. Guideline to reference gene selection for quantitative real-time PCR. Biochemical and Biophysical Research Communications 313:856-862.
196. Lord JC, Hartzer K, Toutges M, Oppert B.2010. Evaluation of quantitative PCR reference genes for gene expression studies in Tribolium castaneum after fungal challenge. Journal of Microbiological Methods 80:219-221.
197. Argyropoulos D, Psallida C, Spyropoulos CG.2006. Generic normalization method for real-time PCR. FEBS Journal 273:770-777.
198. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F.2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3:research0034.0031-0034.0011.
199. Andersen CL, Jensen JL, rntoft TF.2004. Normalization of real-time quantitative reverse transcription-PCR data:a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Research 64:5245-5250.
200. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP.2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: bestKeeper-Excel-based tool using pair-wise correlations. Biotechnology Letters 26:509-515.
201. Ashihara W.1987. Infestation and reproduction of the citrus red mite, Panonychus citri (McGregor) (Acari:Tetranychidae) on leguminous plants. Applied Entomology and Zoology 22:512-518.
202. Gotoh T, Kubota M.1997. Population dynamics of the citrus red mite, Panonychus citri (McGregor) (Acari:Tetranychidae) in Japanese pear orchards. Experimental & Applied Acarology 21:343-356.
203. Jeppson LR, Keifer HH, Baker EW.1975. Mites injurious to economic plants. Berkeley, CA, USA:University of California Press.528 p.
204. Takafuji A, Fujimoto H.1986. Winter survival of the non-diapausing population of the citrus red mite, Panonychus citri (McGregor) (Acarina:Tetranychidae) on pear and citrus. Applied Entomology and Zoology 21:467-473.
205. Park H, Ahn IY, Lee HE.2007. Expression of heat shock protein 70 in the thermally stressed Antarctic clam Laternula elliptica. Cell Stress & Chaperones 12:275-282.
206. Crevel G, Bates H, Huikeshoven H, Cotterill S.2001. The Drosophila Dpit47 protein is a nuclear Hsp90 co-chaperone that interacts with DNA polymerase alpha. Journal of Cell Science 114:2015-2025.
207. Chen B, Zhong D, Monteiro A.2006. Comparative genomics and evolution of the HSP 90 family of genes across all kingdoms of organisms. BMC Genomics 7: 156-174.
208. Johnson JL, Brown C.2009. Plasticity of the Hsp90 chaperone machine in divergent eukaryotic organisms. Cell Stress and Chaperones 14:83-94.
209.李明,卢文才,冯宏祖,袁亮,工进军,何林.2008.朱砂叶螨热激蛋白HSP70基因TCHSP70-4的克隆与表达.昆虫学报51：1235-1243.
210. Knowlton AA, Salfity M.1996. Nuclear localization and the heat shock proteins. Journal of Biosciences 21:123-132.
211. Bukau B, Weissman J, Horwich A.2006. Molecular chaperones and protein quality control. Cell 125:443-451.
212. Demand J, Luders J, Hohfed J.1998. The carboxy-terminal domain of HSC70 provides binding sites for a district set of chaperone cofactors. Molecular and Cellular Biology 18:2023-2028.
213. Pelham HRB.1988. Evidence that luminal ER proteins are sorted from secreted proteins in a post-ER compartment. The EMBO Journal 7:913-918.
214. Cha JY, Jung MH, Ermawati N, Mukhamad S, Rho GJ, Han CD, Lee KH, Son D. 2009. Functional characterization of orchardgrass endoplasmic reticulum-resident Hsp90 (DgHsp90) as a chaperone and an ATPase. Plant Physiology and Biochemistry 47:859-866.
215. Haas IG.1994. BiP (GRP78), an essential hsp70 resident protein in the endoplasmic reticulum. Experientia 50:1012-1020.
216. Nicolai A, Senet P, Delarue P, Ripoll DR.2010. Human inducible Hsp70: structures, dynamics, and interdomain communication from all-atom molecular dynamics simulations. Journal of Chemical Theory and Computation 6: 2501-2519.
217. Sun W, Jin Y, He L, Lu WC, Li M.2010. Suitable reference gene selection for different strains and developmental stages of the carmine spider mite, Tetranychus cinnabarinus, using quantitative real-time PCR. Journal of Insect Science 10:1-12.
218.李钱峰,蒋美艳,于恒秀,辛世文,顾铭洪,刘巧泉.2008.水稻胚乳RNA定量RT-PCR分析中参照基因选择.扬州大学学报(农业与生命科学版)29：61-66.
219. Christians E, Michel E, Renard JP.1997. Developmental control of heat shock and chaperone gene expression Hsp70 genes and heat shock factors during preimplantation phase of mouse development. Cellular and Molecular Life Sciences 53:168-176.
220. Xu JJ, Shu JA, Zhang Q.2010. Expression of the Tribolium castaneum (Coleoptera:Tenebrionidae) hsp83 gene and its relation to oogenesis during ovarian maturation. Journal of Genetics and Genomics 37:513-522.
221. Kelty JD, Lee REJ.2001. Rapid cold-hardening of Drosophila melanogaster (Diptera:Drosophilidae) during ecologically based thermoperiodic cycles. Journal of Experimental Biology 204:1659¨C1666.
222. Wang HD, Kazemi-Esfarjani P, Benzer S.2004. Multiple-stress analysis for isolation of Drosophila longevity genes. Proceedings of the National Academy of Sciences of the United States of America 101:12610-12615.