葱蝇海藻糖-6-磷酸合成酶基因的克隆、序列分析及滞育相关表达

海藻糖-6-磷酸合成酶（trehalose-6-phosphate synthase, TPS）是昆虫海藻糖合成途径中的关键酶之一。本研究通过对葱蝇Delia antiqua海藻糖-6-磷酸合成酶基因的克隆、 序列分析及滞育相关表达的分析, 旨在证明该基因在能源合成以及抵御高温和低温环境方面发挥重要作用, 为进一步弄清葱蝇滞育分子机制提供理论依据。根据葱蝇抑制消减杂交文库中的EST序列信息, 设计特异性引物, 并通过RACE技术克隆了葱蝇海藻糖-6-磷酸合成酶基因全长cDNA, 命名为DaTPS1 （GenBank登录号： JX681124）, 其全长为2 904 bp, 开放阅读框2 448 bp, 编码815个氨基酸, 推测其相对分子质量为91.2 kD, 等电点为5.96。生物信息学分析表明, 该基因编码的氨基酸序列具有两个保守结构域, 与其他物种TPS具有较高的同源性, 其中和黑腹果蝇Drosophila melanogaster亲缘关系最近, 氨基酸序列一致性为92.1%; 其蛋白质三维结构有15条大的α螺旋和11股反向平行的β链折叠。RT-PCR分析表明, DaTPS1在葱蝇非滞育、 夏滞育和冬滞育期蛹中都有表达, 但是非滞育期各时期表达量基本没有变化, 而在夏滞育和冬滞育蛹的滞育前期表达量较高, 滞育保持期表达量较低, 滞育期后期表达量又有所升高。推断在葱蝇蛹夏滞育和冬滞育期前期, TPS1开始催化合成较多的海藻糖以提高滞育期抵御不良环境的能力, 滞育保持期蛹的新陈代谢降低, 所需能量较少, 所以TPS1处于低水平表达状态, 而滞育期结束后, 蛹生长发育逐渐恢复, 所需能量有所增加, TPS1的表达量再次升高。本研究对揭示昆虫TPS在能量代谢通路中的作用及昆虫滞育的分子机理具有一定的科学意义。     

德国小蠊两个海藻糖合成酶基因的克隆、组织分布及温度诱导表达分析

【目的】海藻糖合成酶（trehalose-6-phosphate synthase, TPS）是参与昆虫血糖-海藻糖合成的关键酶。本研究旨在克隆德国小蠊 Blattella germanica TPS基因,研究TPS基因在德国小蠊不同组织中的表达模式及在不同温度处理下的表达情况。【方法】通过RACE技术克隆德国小蠊TPS基因全长序列,利用荧光定量PCR的方法检测TPS基因在德国小蠊5龄幼虫不同组织中的表达模式及在高温(40℃和46℃处理30 min)及低温(0℃和10℃处理1 h)逆境下的表达量变化。【结果】从德国小蠊中克隆获得2个TPS基因,分别命名为 BgTPS1 (GenBank登录号：KR050213) 和 BgTPS2 (GenBank登录号：KR050214)。其中,BgTPS1基因cDNA序列全长2 987 bp,开放阅读框 (ORF) 2 502 bp,编码833个氨基酸;BgTPS2基因cDNA序列全长3 212 bp,开放阅读框2 469 bp,编码822个氨基酸。BgTPS1和BgTPS2基因都主要在5龄幼虫脂肪体中表达,且BgTPS2基因的表达量为BgTPS1基因表达量的3.9倍。在两种不同极端温度诱导下,BgTPS1和BgTPS2基因mRNA均上调表达。其中,BgTPS2 的表达量始终显著高于 BgTPS1。在0℃时,BgTPS1和BgTPS2的表达量最高。【结论】德国小蠊5龄幼虫中存在2个TPS基因。两个TPS基因均在脂肪体中高表达,且BgTPS2基因的表达量显著高于BgTPS1基因;低温和高温诱导下均能促进两个基因的表达量上升。该结果为进一步明确昆虫海藻糖的合成途径及其在昆虫对温度逆境的反应中的作用研究奠定了基础。     

野桑蚕酚氧化酶原基因cDNA的分子克隆及其特征

酚氧化酶在昆虫的免疫防御机制中起着重要作用。利用RT-PCR和RACE方法,克隆了野桑蚕酚氧化酶原基因,获得了其cDNA序列。该序列长2 134 bp,含有一个2 082 bp的完整开放阅读框,编码一个由693个氨基酸残基组成的蛋白质。推导的氨基酸序列与其他鳞翅目昆虫PPO2基因相应氨基酸序列有较高的同源性,该序列具有它们的PPO基因所共有的典型特征。组织特异性表达分析表明了该基因在野桑蚕5龄幼虫的血细胞、体壁、头部、精巢、卵巢、脂肪体和中肠等组织及其不同的发育阶段均有表达。这些结果为进一步研究野桑蚕酚氧化酶原基因的功能提供了分子基础。     

Cloning and characterization of genes encoding trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2) from Zygosaccharomyces rouxii

In many organisms, trehalose protects against several environmental stresses, such as heat, desiccation, and salt, probably by stabilizing protein structures and lipid membranes. Trehalose synthesis in yeast is mediated by a complex of trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2). In this study, genes encoding TPS1 and TPS2 were isolated from Zygosaccharomyces rouxii (designated ZrTPS1 and ZrTPS2, respectively). They were functionally identified by their complementation of the tps1 and tps2 yeast deletion mutants, which are unable to grow on glucose medium and with heat, respectively. Full-length ZrTPS1 cDNA is composed of 1476 nucleotides encoding a protein of 492 amino acids with a molecular mass of 56 kDa. ZrTPS2 cDNA consists of 2843 nucleotides with an open reading frame of 2700 bp, which encodes a polypeptide of 900 amino acids with a molecular mass of 104 kDa. The amino acid sequence encoded by ZrTPS1 has relatively high homology with TPS1 of Saccharomyces cerevisiae and Schizosaccharomyces pombe, compared with TPS2. Western blot analysis showed that the antibody against S. cerevisiae TPS1 recognizes ZrTPS1. Under normal growth conditions, ZrTPS1 and ZrTPS2 were highly and constitutively expressed, unlike S. cerevisiae TPS1 and TPS2. Salt stress and heat stress reduced the expression of the ZrTPS1 and ZrTPS2 genes, respectively.     

Purification of juvenile hormone esterase and molecular cloning of the cDNA from Manduca sexta.

Juvenile hormone esterase (JHE) is a highly specific enzyme important for regulating the onset of metamorphosis in lepidopteran insects. After affinity chromatography of the hemolymph proteins of Manduca sexta, the pure JHE protein was digested with Lys-C and the resultant peptides were purified by microbore HPLC. Two peptides were selected for sequencing. Based upon these amino acid sequences, degenerate RT-PCR was performed in order to amplify a partial cDNA sequence from mRNA from the fat body of M. sexta. A 1512bp partial cDNA was generated and found to be highly homologous to the JHE from Heliothis virescens. 5' and 3' RACE were performed to obtain the full length cDNA sequence. The cDNA has a total length of 2220bp, with a 1749bp coding region. The deduced protein sequence contains 573 amino acids.     

Characterization of a trehalose-6-phosphate synthase gene from Spodoptera exigua and its function identification through RNA interference

Trehalose is an important disaccharide and a key regulation factor for the development of many organisms, including plants, bacteria, fungi and insects. In order to study the trehalose synthesis pathway, a cDNA for a trehalose-6-phosphate synthase from Spodoptera exigua (SeTPS) was cloned which contained an open reading frame of 2481 nucleotides encoding a protein of 826 amino acids with a predicted molecular weight of 92.65 kDa. The SeTPS genome has 12 exons and 11 introns. Northern blot and RT-PCR analyses showed that SeTPS mRNA was expressed in the fat body and in the ovary. Competitive RT-PCR revealed that SeTPS mRNA was expressed in the fat body at different developmental stages and was present at a high level in day 1 S. exigua pupae. The concentrations of trehalose and glucose in the hemolymph were determined by HPLC and showed that they varied at different developmental stages and were negatively correlated to each other. The survival rates of the insects injected with dsRNA corresponding to SeTPS gene reached 53.95%, 49.06%, 34.86% and 33.24% for 36, 48, 60 and 204 h post-injection respectively which were significantly lower than those of the insects in three control groups. These findings provide new data on the tissue distribution, expression patterns and potential function of the trehalose-6-phosphate synthase gene.     

异色瓢虫海藻糖合成酶基因的克隆及低温诱导表达分析

海藻糖是昆虫的血糖, 在昆虫体内主要通过海藻糖合成酶（trehalose-6-phosphate synthase, TPS）催化合成。本研究通过同源克隆和cDNA末端快速扩增（rapid-amplification of cDNA ends, RACE） 技术, 从异色瓢虫Harmonia axyridis中克隆得到了TPS基因的cDNA全长序列, 命名为HaTPS（GenBank登录号： FJ501960）, 全长2 949 bp, 包含3′非翻译区为505 bp, 5′非翻译区为26 bp, 开放阅读框长2 418 bp, 共编码805个氨基酸。软件分析显示该基因编码蛋白的分子量为90.58 kD, 等电点为7.01, 包含两个糖基化位点, 无信号肽和跨膜结构。同源比对分析发现, 昆虫中TPS基因高度保守, 包含两个保守的结构域。同时, 采用实时荧光定量PCR技术对异色瓢虫HaTPS在不同发育阶段、 低温诱导条件下的表达量进行了研究。结果表明： HaTPS在预蛹期的表达量最高; 在短时低温诱导条件下, HaTPS的表达量随着温度的降低而显著升高, 在降温和升温处理条件下, HaTPS的表达量呈现先升高后下降的表达趋势。结果表明, TPS基因在昆虫抗逆中起到了重要的调节作用。昆虫经过低温诱导, 其TPS基因的调控能力得到提升。     

柞蚕蛹解除滞育过程中海藻糖合成酶基因的表达变化

【目的】克隆柞蚕Antheraea pernyi海藻糖合成酶(trehalose-6-phosphate synthase,TPS)基因,并对其进行组织表达分析,探讨该基因在柞蚕滞育蛹解除滞育过程中的表达规律,为阐明柞蚕滞育期间碳水化合物代谢规律与蛹滞育解除的关系提供数据支持。【方法】利用PCR及3'RACE技术从柞蚕幼虫脂肪体组织中克隆得到TPS基因,并进行生物信息学分析;RT-PCR检测该基因在柞蚕幼虫各组织中的表达分布,进一步采用Real-time PCR分析柞蚕滞育蛹解除滞育过程中该基因在脂肪体组织和血淋巴中的表达量变化。【结果】克隆获得柞蚕海藻糖合成酶基因并命名为ApTPS。其开放阅读框长2 487 bp,编码828个氨基酸,蛋白预测分子量为93.19 k D,等电点(p I)4.61;无信号肽,无跨膜区。蛋白质亚细胞定位预测该蛋白定位于细胞质中;蛋白质结构域分析表明,ApTPS有两个保守功能区:TPS(第22-497位氨基酸)和TPP(第532-772位氨基酸)。组织特异性分析表明,ApTPS基因在柞蚕幼虫脂肪体中表达量最高;柞蚕解除滞育过程中,ApTPS在脂肪体和血淋巴中的表达量均有所升高,且显著高于对照组(P0.05),但血淋巴中表达量的升高滞后于脂肪体。【结论】结果提示ApTPS参与了柞蚕蛹滞育中碳水化合物代谢调控并在其中发挥重要作用,与柞蚕蛹滞育解除关系密切。     

Aggregation dependent enhancement of trehalose-6-phosphate synthase activity in Saccharomyces cerevisiae

Trehalose-6-phosphate synthase (TPS) is one of the key subunits of the trehalose synthase complex, responsible for synthesis of trehalose in Saccharomyces cerevisiae. Different laboratories have tried to purify TPS, but have been unable to separate it from the complex. During the present study, active TPS has been isolated from the trehalose synthase complex as a free 59 kDa protein. A 158 fold purification was achieved with over 84% recovery of active TPS. N-terminal sequence confirmed the 59 kDa protein to be TPS. It was revealed to be a highly hydrophobic protein by amino acid analysis data. Activity of TPS was identified to be governed by association-dissociation of protein components. TPS activity of the isolated enzyme was highly unstable due to dissociation of the protein from the complex. Aggregation of active molecules was also seen to enhance as well as stabilize enzyme activity. This aggregation was concentration dependent and activity was seen to be enhanced by increasing the number of active molecules and fell with dilution. The association of the active complex was also found to be governed by ionic interactions.     

Aggregation dependent enhancement of trehalose-6-phosphate synthase activity in Saccharomyces cerevisiae

Trehalose-6-phosphate synthase (TPS) is one of the key subunits of the trehalose synthase complex, responsible for synthesis of trehalose in Saccharomyces cerevisiae. Different laboratories have tried to purify TPS, but have been unable to separate it from the complex. During the present study, active TPS has been isolated from the trehalose synthase complex as a free 59kDa protein. A 158 fold purification was achieved with over 84% recovery of active TPS. N-terminal sequence confirmed the 59kDa protein to be TPS. It was revealed to be a highly hydrophobic protein by amino acid analysis data. Activity of TPS was identified to be governed by association–dissociation of protein components. TPS activity of the isolated enzyme was highly unstable due to dissociation of the protein from the complex. Aggregation of active molecules was also seen to enhance as well as stabilize enzyme activity. This aggregation was concentration dependent and activity was seen to be enhanced by increasing the number of active molecules and fell with dilution. The association of the active complex was also found to be governed by ionic interactions.     

草地贪夜蛾海藻糖合成酶基因的克隆、时空表达谱及对温度胁迫的响应

【目的】本研究旨在通过克隆草地贪夜蛾Spodoptera frugiperda的海藻糖合成酶(trehalose-6-phosphate synthase)基因SfTPS,分析其在草地贪夜蛾不同发育阶段、不同组织中的表达水平及不同温度胁迫时5龄幼虫中的相对表达量,为进一步探究TPS在草地贪夜蛾生长发育及抗逆应激反应中的功能奠定基础。【方法】运用RT-PCR技术克隆草地贪夜蛾SfTPS的全长编码区,并进行生物信息学分析。运用RT-qPCR技术检测SfTPS在草地贪夜蛾不同发育阶段(卵、1-6龄幼虫、蛹和成虫)、5龄幼虫不同组织(体壁、中肠和脂肪体)中和经短期(2, 4和8 h)高(35℃)低温(10℃)胁迫后5龄幼虫中的表达变化。【结果】克隆获得2 571 bp的草地贪夜蛾TPS cDNA序列,命名为SfTPS(GenBank登录号： MT920672),全长开放阅读框(ORF)长2 481 bp,编码的826个氨基酸具有TPS和TPP两个保守结构域。同源比对和系统进化分析表明,昆虫TPS蛋白具有较高的保守性,SfTPS与斜纹夜蛾S. litura的TPS亲缘关系最近,序列一致性达到99.15%。SfTPS中α-螺旋、β-折叠和无规则卷曲占比分别为38.14%, 12.23%和48.55%;SfTPS的三级结构为同源二聚体。RT-qPCR结果表明,SfTPS在草地贪夜蛾卵期和1-5龄幼虫期低表达,在6龄幼虫期、蛹期和成虫期高表达,且草地贪夜蛾变态前后SfTPS的表达量变化较大。组织分布结果显示,SfTPS在5龄幼虫脂肪体中表达量最高。草地贪夜蛾5龄幼虫经2~8 h低温(10℃)和高温(35℃)胁迫后,SfTPS的相对表达量显著高于对照(25℃),分别为对照的4.43~9.34和2.50~6.03倍。【结论】SfTPS基因在草地贪夜蛾生长发育过程及抵御高低温度胁迫中可能具有重要作用。     

Preface

The vector of Chagas' disease, Rhodnius prolixus, feeds exclusively on blood. The blood meals are slowly digested, and these insects wait some weeks before the next meal. During the life of an insect, energy‐requiring processes such as moulting, adult gonadal and reproductive growth, vitellogenesis, muscular activity, and fasting, lead to increased metabolism. Carbohydrates are a major source of energy and their mobilization is important. We determined the amounts of glycogen, trehalose, and glucose present in the fat body and/or hemolymph of adult males of R. prolixus and recorded the processes of accumulation and mobilization of these carbohydrates. We also tested our hypothesis that these processes are under endocrine control. The amount of glycogen in the fat body progressively increased until the fourth day after feeding (from 9.3±2.2 to 77. 3±7.5 µg/fat body), then declined to values around 36.3±4.9 µg/fat body on the fifteenth day after the blood meal. Glycogen synthesis was eliminated in decapitated insects and head‐transplanted insects synthesized glycogen. The amount of trehalose in the fat body increased until the sixth day after feeding (from 16. 6±1.7 to 40. 6±5.3 nmol/fat body), decreased abruptly, and stabilized between days 7 and 15 at values ranging around 15–19 nmol/fat body. Decapitated insects did not synthesize trehalose after feeding, and this effect was reversed in head‐transplanted insects. The concentration of trehalose in the hemolymph increased after the blood meal until the third day (from 0.07±0.01 to 0.75±0.05 mM) and at the fourth day it decreased until the ninth day (0.21±0.01 mM), when it increased again until the fourteenth day (0.79±0.06 mM) after the blood meal, and then declined again. In decapitated insects, trehalose concentrations did not increase soon after the blood meal and at the third day it was very low, but on the fourteenth day it was close to the control values. The concentration of glucose in the hemolymph of untreated insects remained low and constant (0.18±0.01 mM) during the 15 days after feeding, but in decapitated insects it progressively increased until the fifteenth day (2.00±0.10 mM). We recorded the highest trehalase activity in midgut, which was maximal at the eighth day after feeding (2,830±320 nmol of glucose/organ/h). We infer that in Rhodnius prolixus, the metabolism of glycogen, glucose, and trehalose are controlled by factors from the brain, according to physiological demands at different days after the blood meal. © 2009 Wiley Periodicals, Inc.     

Characterization of a serine carboxypeptidase in the salivary glands and fat body of the orange wheat blossom midge, Sitodiplosis mosellana (Diptera: Cecidomyiidae)

A full-length cDNA encoding a serine carboxypeptidase (designated SmSCP-1) was recovered from an ongoing salivary gland EST project of the wheat midge. The deduced 461-amino acid sequence had a putative signal sequence at the amino terminus, indicating it was a secreted protein. The protein shared homology with serine carboxypeptidases from other insects, mammals, plants, and yeasts. SmSCP-1 mRNA was expressed in all stages of development and detected in salivary gland and fat body tissues but not in midgut tissue. Expression analysis and quantitative real-time PCR assays in male and female wheat midges and the fat body tissue of adult midges revealed that SmSCP-1 was up-regulated nearly four-fold in the female midges compared to males and nearly two-fold in female fat body compared to male fat body. The wheat midge serine carboxypeptidase (SmSCP-1) most likely has a dual function. As a secreted digestive enzyme, it could play a role in mobilizing host-plant seed reserves for feeding larvae and as expressed in fat body could function as an exopeptidase in degradation of vitellogenin and/or in post-translational processing of other enzymes.     

The adipokinetic hormones in the fall armyworm, Spodoptera frugiperda: cDNA cloning, quantitative real time RT-PCR analysis, and gene specific localization

Small neuropeptides of the adipokinetic/red pigment-concentrating hormone (AKH/RPCH) family regulate energy metabolism in insects. Within lepidopterans, the nonapeptide Manduca sexta AKH (Manse-AKH) represents a widely occurring AKH, whereas the decapeptide Helze-HrTH (at first isolated from Helicoverpa zea) seems to be restricted to moths. Here we report the identification of the Manse-AKH-like Spofr-AKH 1 and the Helze-HrTH-like Spofr-AKH 2 prohormone precursors from the fall armyworm, Spodoptera frugiperda. Moreover, by PCR screening of a random primer cDNA library and by RACE, three 668, 835 and 1008 bp cDNA sequences were obtained, which encode putative translation products of 67-74 amino acids, each containing one copy of a peptide sequence that in its processed form has the sequence of QLTFSSGW-amide (Spofr-AKH 3). Another cDNA sequence of 634 bp encodes a putative translation product of 40 amino acids, potentially leading to one copy of an elongated, non-amidated Helze-HrTH (pQLTFSSGWGNCTS-OH; Spofr-AKH 4). Q-RT-PCR analysis showed that the Spofr-AKH mRNAs are expressed in 2d-old female brain/corpora cardiaca complexes, but also in ovaries, midgut, fat body, accessory glands and muscle tissues. Expression was also found in the ovaries of 4d-old females. Whole-mount in situ RT-PCR analysis with ovaries from 2d-old females showed that the Spofr-AKH 2 and Spofr-AKH 4 were mainly localized in the germarium (phase 3), whereas the Spofr-AKH 1, and the three mRNA isoforms of Spofr-AKH 3 were localized at the end of the vitellarium and in the fully developed oocytes (phase 1 and 2). The results suggest that Spofr-AKH genes play a role in the regulation of oocyte maturation in S. frugiperda.     

Cloning and truncation modification of trehalose-6-phosphate synthase gene from Selaginella pulvinata

A homologous sequence was amplified from resurrection plant Selaginella pulvinta by RACE technique, proved to be the full-length cDNA of trehalose-6-phosphate synthase gene by homologous alignment and yeast complementation assay, and nominated as SpTPS1 gene. The open reading frame of this gene was truncated 225 bp at the 5′-end, resulting the N-terminal truncation modification of 75 amino acids for its encoding protein. The TPS1 deletion mutant strain YSH290 of the brewer's yeast transformed by the truncated gene SpTPS1Δ and its original full-length version restored growth on the medium with glucose as a sole carbon source and displayed growth curves with no significant difference, indicating their encoding proteins functioning as TPS enzyme. The TPS activity of the mutant strain transformed by the truncated gene SpTPS1Δ was about six fold higher than that transformed by its original version, reasoning that the extra N-terminal extension of the full-length amino acid sequence acts as an inhibitory domain to trehalose synthesis. However, the trehalose accumulation of the mutant strain transformed by the truncated gene SpTPS1Δ was only 8% higher than that transformed by its original version. This result is explained by the feedback balance of trehalose content coordinated by the comparative activities between trehalose synthase and trehalase. The truncated gene SpTPS1Δ is suggested to be used in transgenic operation, together with the inhibition of trehalase activity by the application of validamycin A or genetic deficiency of the endogenous trehalase gene, for the enhancement of trehalose accumulation and improvement of abiotic tolerance in transgenic plants.