甜瓜海藻糖-6-磷酸合成酶基因鉴定及表达

为了明确甜瓜海藻糖-6-磷酸合成酶基因(CmTPS)家族信息及对逆境信号的响应,该研究采用生物信息学方法,通过拟南芥TPS家族基因与甜瓜基因组数据库比对,从甜瓜基因组中共鉴定出7个海藻糖-6-磷酸合成酶基因,按照其在染色体上的位置分别命名为CmTPS1~7。系统进化分析结果显示,CmTPS4和CmTPS7为第1类,二者均含有16个内含子,推测其编码产物均具有海藻糖-6-磷酸合成酶(TPS)活性;其余5个CmTPS基因归为第2类,分别含有2~4个内含子;在这7个甜瓜TPS中,除CmTPS3只有TPS结构域外,其余CmTPS都含有TPS、TPP及UDP-forming结构域;蛋白序列比对结果显示,甜瓜TPS家族各成员间相似性较低(15.90%~57.31%);亚细胞定位预测表明,CmTPS1、CmTPS2和CmTPS6定位在细胞核内,其余4个CmTPS定位在细胞质内。qRTPCR表达分析表明,低温胁迫下甜瓜叶片可能以CmTPS4为主要的TPS编码基因;CmTPS基因家族对盐胁迫较为敏感,同时在ABA信号传递中起调控作用。这为进一步研究甜瓜TPS基因家族奠定了基础。     

昆虫海藻糖及其合成酶基因的特性与功能研究进展

海藻糖广泛存在于细菌、真菌、昆虫、无脊椎动物和植物等大量生物中。它不仅可以作为昆虫的能量来源,而且在抗逆等方面起着重要作用。海藻糖合成酶(Trehalose-6-phosphate synthase,TPS)是海藻糖合成过程中的一个关键酶。目前细菌、真菌和植物中都已经被发现和克隆,但其不存在于哺乳动物中。海藻糖是昆虫的"血糖",主要通过海藻糖合成酶和海藻糖-6-磷酸脂酶(Trehalose-6-phosphate phosphatase,TPP)在脂肪体中催化合成。TPS基因所编码的蛋白序列一般都包含两个保守的结构域:TPS和TPP,分别对应着酵母中的Ots A和Ots B基因。昆虫海藻糖合成酶的基因表达和酶活性的变化与昆虫的多项生理过程有着密切的关系,海藻糖合成酶有可能成为控制害虫的新靶标。     

垫状卷柏海藻糖-6-磷酸合成酶基因的克隆及功能分析

海藻糖-6-磷酸合成酶(Trehalose-6-phosphate synthse, TPS)是植物海藻糖合成途径的关键酶, 在旱生卷柏等复苏植物对逆境胁迫应答中起重要作用。文章以我国特有旱生植物垫状卷柏(Selaginella pulvinata)为材料, 采用同源扩增与RACE技术相结合的方法克隆了海藻糖-6-磷酸合成酶基因SpTPS1, cDNA全长3 223 bp, 包括一个2 790 bp的开放阅读框, 推导的氨基酸序列与模式物种的海藻糖-6-磷酸合成酶具有较高的序列相似性, 催化活性中心保守位点基本一致。酵母功能互补实验证明, 用SpTPS1基因开放阅读框转化的海藻糖合成酶基因突变(tps1△)酵母菌株, 可恢复在以葡萄糖作为唯一碳源培养基上的生长, 说明垫状卷柏海藻糖-6-磷酸合成酶基因SpTPS1的编码蛋白具有生物活性, 可应用于植物抗逆性的转基因改良。     

柑橘大实蝇滞育和非滞育蛹体内海藻糖含量的调控

【目的】通过比较柑橘大实蝇Bactrocera minax蛹滞育期与滞育前和滞育后以及滞育蛹与非滞育蛹体内海藻糖和葡萄糖含量的变化、海藻糖合成代谢途径中关键酶的活力变化以及关键酶基因的表达量变化,明确蛹滞育期间海藻糖合成代谢途径中关键酶对海藻糖含量的调控。【方法】利用分光光度法检测柑橘大实蝇滞育前(1日龄蛹)、滞育期(30,60和90日龄蛹)以及滞育后(120和150日龄蛹)蛹体内海藻糖与葡萄糖含量的变化,以及海藻糖合成代谢途径中的海藻糖-6-磷酸合成酶(TPS)、海藻糖-6-磷酸磷酸酯酶(TPP)和海藻糖酶(Tre)活力的变化;利用实时定量荧光PCR(qPCR)检测TPS,TPPB,TPPC-1,TPPC-2和Tre-1基因表达量的变化。向1日龄蛹体内注射20-羟基蜕皮酮(20E)作为处理(以注射10%乙醇为对照),分别于注射后1和30 d比较处理组与对照组蛹体内海藻糖与葡萄糖含量、关键酶活力以及上述基因表达量的差异。【结果】柑橘大实蝇蛹进入滞育后,海藻糖含量显著升高,葡萄糖含量无显著变化; TPS和TPP的酶活力以及TPS,TPPC-1和TPPC-2表达量在化蛹后逐渐升高,于滞育期达到最高水平,维持至羽化前显著下降;TPPB表达量在整个蛹期无显著差异; Tre酶活力以及Tre-1表达量在化蛹后逐渐升高,于滞育早期达到最高水平,随后显著下降,羽化前再次显著上升。注射20E后1 d,与对照组相比,处理组蛹体内海藻糖与葡萄糖含量、关键酶(TPS,TPP和Tre)活力以及TPS,TPPC-2和Tre-1表达量无显著变化,TPPB表达量显著下降,TPPC-1表达量显著上升;注射后30 d,与对照组滞育蛹相比,处理组非滞育蛹海藻糖含量显著上升,葡萄糖含量、TPS和Tre酶活力、TPS和Tre-1表达量显著下降,TPP酶活力以及TPPB和TPPC-2表达量无显著差异。【结论】柑橘大实蝇蛹体内海藻糖的含量在合成代谢途径中关键酶的调控下,随着滞育状态发生变化,表明海藻糖与滞育之间存在密切的关系,但其作用机理仍待进一步研究。     

干旱和低温胁迫影响烟草幼苗海藻糖代谢的差异比较

为了明确海藻糖代谢在干旱和低温胁迫下的响应及其差异,以及海藻糖在烟草耐旱和耐冷性中的作用,选用烟草品种云烟203幼苗为实验材料,通过对烟草幼苗进行干旱和4℃低温胁迫处理,研究叶片中海藻糖含量及代谢相关酶(海藻糖-6-磷酸合成酶TPS、海藻糖-6-磷酸磷酸酶TPP、海藻糖酶THase)活性及基因表达变化情况。结果表明,在干旱和低温胁迫下,海藻糖含量表现出先升高后降低趋势,均在处理2 d时海藻糖积累达到最大,且干旱胁迫下海藻糖含量高于低温胁迫。TPS、TPP活性在干旱和低温胁迫下先升高后降低,且干旱高于低温;THase活性在干旱和低温胁迫下均不断升高,低温下的THase活性高于干旱。TPS、TPP、THase基因在干旱下的表达量均高于0 d对照,低温下低于0 d对照。以上结果表明,干旱和低温胁迫均能促使烟草体内海藻糖的积累,干旱更能诱导相关基因的表达、相关酶活性及海藻糖含量的升高,表明海藻糖对干旱响应更敏感。     

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。     

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。     

结核分枝杆菌海藻糖磷酸磷酸酶诱导小鼠体液和细胞免疫应答

目的 研究结核分枝杆菌(M．tuberculosis)海藻糖磷酸磷酸酶(TPP)诱导小鼠体液和细胞免疫。方法 差速离心分离结核分枝杆菌H37Rv和卡介苗(BCG)的各细胞组分,通过Western杂交检测抗原TPP在结核分枝杆菌H37Rv和BCG中的亚细胞定位情况。分别用5×10~6CFU的BCG和50μg的TPP蛋白免疫C57BL/6小鼠,检测小鼠血清中抗TPP的IgG1和IgG2a抗体效价。取免疫小鼠的脾细胞,体外抗原刺激,用酶联免疫斑点试验(ELISPOT)检测γ干扰素(IFN-γ)分泌细胞。结果 TPP亚细胞定位于结核分枝杆菌H37Rv和BCG的胞壁和细胞膜组分。TPP蛋白免疫后小鼠产生的TPP特异性IgG1和IgG2a抗体效价明显高于BCG免疫小鼠,并且IgG2a的抗体效价高于IgG1。体外抗原刺激TPP蛋白和BCG免疫小鼠的脾细胞,都能诱导较高的IFN-γ分泌。结论 结核分枝杆菌细胞壁蛋白TPP能诱导小鼠Ⅰ型辅助性T细胞介导的免疫反应,可作为抗结核疫苗的候选抗原。     

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。     

Isolation and identification of a thermophilic strain producing trehalose synthase from geothermal water in China

A slightly thermophilic strain, CBS-01, producing trehalose synthase (TreS), was isolated from geothermal water in this study. According to the phenotypic characteristics and phylogenetic analysis of the 16s rRNA gene sequence, it was identified as Meiothermus ruber. The trehalose synthase gene of Meiothermus ruber CBS-01 was cloned by polymerase chain reaction and sequenced. The TreS gene consisted of 2,895 nucleotides, which specified a 964-amino-acid protein. This novel TreS catalyzed reversible interconversion of maltose and trehalose.     

Isolation and Identification of a Thermophilic Strain Producing Trehalose Synthase from Geothermal Water in China

A slightly thermophilic strain, CBS-01, producing trehalose synthase (TreS), was isolated from geothermal water in this study. According to the phenotypic characteristics and phylogenetic analysis of the 16s rRNA gene sequence, it was identified as Meiothermus ruber. The trehalose synthase gene of Meiothermus ruber CBS-01 was cloned by polymerase chain reaction and sequenced. The TreS gene consisted of 2,895 nucleotides, which specified a 964-amino-acid protein. This novel TreS catalyzed reversible interconversion of maltose and trehalose.     

Genetic modification of photosynthesis with E. coli genes for trehalose synthesis

Improvement in photosynthesis per unit leaf area has been difficult to alter by breeding or genetic modification. We report large changes in photosynthesis in Nicotiana tabacum transformed with E. coli genes for the trehalose pathway. Significantly, photosynthetic capacity (CO2 assimilation at varying light and CO2, and quantum yield of PSII electron transport) per unit leaf area and per leaf dry weight were increased in lines of N. tabacum transformed with the E. coli gene otsA, which encodes trehalose phosphate synthase. In contrast, transformation with otsB, which encodes trehalose phosphate phosphatase or Trec, encoding trehalose phosphate hydrolase, produced the opposite effect. Changes in CO2 assimilation per unit leaf area were closely related to the amount and activity of Rubisco, but not to the maximum activities of other Calvin cycle enzymes. Alterations in photosynthesis were associated with trehalose 6-phosphate content rather than trehalose. When growth parameters were determined, a greater photosynthetic capacity did not translate into greater relative growth rate or biomass. This was because photosynthetic capacity was negatively related to leaf area and leaf area ratio. In contrast, relative growth rate and biomass were positively related to leaf area. These results demonstrate a novel means of modifying Rubisco content and photosynthesis, and the complexities of regulation of photosynthesis at the whole plant level, with potential benefits to biomass production through improved leaf area.     

Contribution of trehalose biosynthetic pathway to drought stress tolerance of Capparis ovata Desf

Trehalose and the trehalose biosynthetic pathway are important contributors and regulators of stress responses in plants. Among recent findings for trehalose and its metabolism, the role of signalling in the regulation of growth and development and its potential for use as a storage energy source can be listed. The xerophytic plant Capparis ovata (caper) is well adapted to drought and high temperature stress in arid and semi‐arid regions of the Mediterranean. The contribution of trehalose and the trehalose biosynthetic pathway to drought stress responses and tolerance in C. ovata are not known. We investigated the effects of PEG‐mediated drought stress in caper plants and analysed physiological parameters and trehalose biosynthetic pathway components, trehalose‐6‐phosphate synthase (TPS), trehalose‐6‐phosphate phosphatase (TPP), trehalase activity, trehalose and proline content in drought stress‐treated and untreated plants. Our results indicated that trehalose and the trehalose biosynthetic pathway contributed to drought stress tolerance of C. ovata. Overall growth and leaf water status were not dramatically affected by drought, as both high relative growth rate and relative water content were recorded even after 14 days of drought stress. Trehalose accumulation increased in parallel to induced TPS and TPP activities and decreased trehalase activity in caper plants on day 14. Constitutive trehalose levels were 28.75 to 74.75 μg·g·FW^?1, and drought stress significantly induced trehalose accumulation (385.25 μg·g·FW^?1 on day 14) in leaves of caper. On day 14 of drought, proline levels were lower than on day 7. Under drought stress the discrepancy between trehalose and proline accumulation trends might result from the mode of action of these osmoprotectant molecules in C. ovata.     

啤酒酵母AS2.1416海藻糖合成酶基因tps1的cDNA克隆及序列分析

海藻糖 (Ｔｒｅｈａｌｏｓｅ,α ｇｌｕｃｏｐｙｒａｎｏｓｙｌ α 1,1 Ｄ ｇｌｕｃｏｐｙｒａ ｎｏｓｅ)是一种非还原性二糖 ,广泛存在于藻类、细菌、昆虫、无脊椎动物及酵母等许多生物体内。海藻糖除了作为一种储存性碳源外 ,业已被证明在许多逆境 ,诸如高温、高盐、干旱、重金属离子污染、冷冻、辐射等情况下 ,可以有效地保护生物的细胞膜、蛋白质及核酸[1～ 6] 。海藻糖合成酶为一多酶体系。在酵母细胞中 ,其合成分为两步进行。第一步 ,在 6 磷酸海藻糖合成酶 (Ｔｐｓ1)的作用下 ,由ＵＤＰ 葡萄糖和葡萄糖 6 磷酸合成海藻糖 6 磷酸 …     

Trehalose Biosynthesis in Response to Abiotic Stresses

Trehalose is a non‐reducing disaccharide that is present in diverse organisms ranging from bacteria and fungi to invertebrates, in which it serves as an energy source, osmolyte or protein/membrane protectant. The occurrence of trehalose and trehalose biosynthesis pathway in plants has been discovered recently. Multiple studies have revealed regulatory roles of trehalose‐6‐phosphate, a precursor of trehalose, in sugar metabolism, growth and development in plants. Trehalose levels are generally quite low in plants but may alter in response to environmental stresses. Transgenic plants overexpressing microbial trehalose biosynthesis genes have been shown to contain increased levels of trehalose and display drought, salt and cold tolerance. In‐silico expression profiling of all Arabidopsis trehalose‐6‐phosphate synthases (TPSs) and trehalose‐6‐phosphate phosphatases (TPPs) revealed that certain classes of TPS and TPP genes are differentially regulated in response to a variety of abiotic stresses. These studies point to the importance of trehalose biosynthesis in stress responses.     

Toluene-induced accumulation of trehalose by Pseudomonas sp. BCNU 106 through the expression of otsA and otsB homologues

AIM: The objective of this study was to investigate toluene-induced accumulation mechanism of trehalose in a toluene-tolerant bacterium Pseudomonas sp. BCNU 106. METHODS AND RESULTS: The accumulation of trehalose by a toluene-tolerant bacterium Pseudomonas sp. BCNU 106 was examined at various cultivation time by measuring the total intracellular trehalose content, trehalase activity and mRNA levels of the trehalose-biosynthetic genes. The pattern of trehalose accumulation corresponded to the mRNA expression pattern of the trehalose-biosynthetic genes with the maximum level at 12 h or 4 h of cultivation with 10% (v/v) toluene, respectively. The trehalose-biosynthetic genes were also cloned and sequenced. Furthermore, the effects of toluene addition on the intracellular osmotic pressure and pH were investigated. It was shown that homeostasis was maintained in the bacterial cells. CONCLUSIONS: In a toluene-tolerant bacterium Pseudomonas sp. BCNU 106, a significant amount of trehalose was accumulated through the toluene-induced expression of the trehalose-biosynthetic genes after the exposure to toluene. SIGNIFICANCE AND IMPACT OF THE STUDY: The accumulation of the high level of intracellular trehalose was preceded by the expression of otsA/B genes in toluene-tolerant bacteria, contributing to the elucidation of the tolerance mechanism.     

RNA interference of a trehalose-6-phosphate synthase gene reveals its roles in the biosynthesis of chitin and lipids in Heortia vitessoides(Lepidoptera:Crambidae)

Trehalose 6-phosphate synthase(TPS),an enzyme that hydrolyzes two glucose molecules to yield trchalose,plays a pivotal role in various physiological processes.In this study,we cloned the trehalose-6-phosphate synthase gene(HvTPS)and investigated its expression patterns in various tssues and d:velopmental stages in Heortia vitessoides Moore(Lepidoptera:Crambidac).HvTPS was highly expressed in the fat body and after pupation or before molting.We knocked down TPS in H.vitessoides by RNA interference and found that 3.0μg of dsHvTPS resulted in optimal interference at 24 h and 36 h post-injection and caused a sharp decline in the survival rate during the 5th instar larval-pupal stage and obviously abnormal or lethal phenotypes.Additionally.compared to the controls,TPS activity and trehalose contents were significantly lower and the glucose content was significantly higher 24 h or 36 h after injection with 3.0μg of dsHIvTPS.Furthermore,the silencing of HvTPS suppressed the cxpression of six key genecs in the chitin biosynthesis pathway and one key gene related to lipid catabolism.The expression levels of two genes associated with lipid biosynthesis were upregulated.These results strongly suggest that HvTPS is essential for the normal growth and development of H.vitessoides and provide a reference for further studies of the utility of key genes involved in chitin and lipid biosynthesis for controlling insect development.     

Platelet cryopreservation using a trehalose and phosphate formulation

Long-term storage of platelets is infeasible due to platelet activation at low temperatures. In an effort to address this problem, we evaluated the effectiveness of a formulation combining trehalose and phosphate in protecting platelet structure and function following cryopreservation. An annexin V binding assay was used to quantify the efficacy of the trehalose and phosphate formulation in suppressing platelet activation during cryopreservation. Of the platelets cryopreserved with the trehalose plus phosphate formulation, 23% +/- 1.2% were nonactivated, compared with 9.8% +/- 0.26% nonactivated following cryopreservation with only trehalose. The presence of both trehalose and phosphate in the cryopreservation medium is critical for cell survival and preincubation in trehalose plus phosphate solutions further enhances viability. The effectiveness of trehalose plus phosphate in preserving platelets in a nonactivated state is comparable to 6% dimethyl sulfoxide (Me(2)SO). Measurements of platelet metabolic activity using an alamarBlue assay also established that trehalose plus phosphate is superior to trehalose alone. Finally, platelets protected by the trehalose plus phosphate formulation exhibit similar aggregation response upon thrombin addition as fresh platelets, but an increase of cytosolic calcium concentration upon thrombin addition was not observed in the cryopreserved platelets. These results suggest that trehalose and phosphate protect several aspects of platelet structure and function during cryopreservation, including an intact plasma membrane, metabolic activity, and aggregation in response to thrombin, but not intracellular calcium release in response to thrombin.