植物肌醇半乳糖苷合酶的生理功能和调控机制

植物肌醇半乳糖苷合酶（galactinol synthase, GolS）是高等植物棉子糖类寡糖合成途径中的关键酶,为棉子糖系列寡糖提供活化的半乳糖基,调控植物体内棉子糖（raffinose, RFO）系列寡糖的生物合成与积累。编码该酶的基因属于糖基转移酶（glycosyltransferases, GTs）GT8基因家族的亚家族。GolS参与合成的最终产物棉子糖家族低聚糖（raffinose family oligosaccharides,RFOs）是植物中重要的碳水化合物存在形式,在细胞内可溶性强,可作为脱水保护剂;还能发挥稳定膜结构的作用。同时,GolS催化合成的直接产物肌醇半乳糖苷（galactinol）和RFOs都能作为羟基自由基捕获分子参与活性氧的清除。因此,GolS参与的代谢途径在植物碳同化物的贮存与运输、生物和非生物逆境响应、种子的脱水效应等生命过程中均发挥了重要作用。GolS基因结构差异与表达模式不同,导致不同GolS基因参与的生物学功能具有很大的差异。研究植物中不同GolS基因的结构特征,组织特异性表达特性及它们响应不同生长发育阶段、环境变化的表达特性,对了解GolS参与的生物学功能具有重要意义。同时,在分子生物学水平上,深入了解调控植物GolS基因的分子调控机制,为通过遗传工程或分子辅助育种等手段,利用GolS改良农林作物的经济性状提供理论支持。本文针对近年来植物中GolS基因的生理功能和调控机制的研究进行了综述。     

大豆GmGolS1的克隆及转基因烟草耐高温性鉴定

棉子糖系列寡糖(RFOs,raffinose family oligosaccharides)是植物体内一种重要的渗透调节物质,肌醇半乳糖苷合成酶(G01S,galactinolsynthase)是RFOs合成过程中的关键酶.本研究从大豆中克隆了 GmGolS1基因.序列分析结果显示,GmGolS1基因位于大豆3号染色...     

植物中棉子糖系列寡糖代谢及其调控关键酶研究进展

棉子糖系列寡糖代谢与植物生长发育、逆境胁迫、种子耐贮性及脱水耐性等关系密切.棉子糖系列寡糖的合成从棉子糖的合成开始,由半乳糖苷肌醇上的半乳糖基的转移依次生成棉子糖、水苏糖、毛蕊花糖等.寡糖代谢是一个复杂的调控体系,其中肌醇-1-磷酸合成酶、肌醇半乳糖苷合成酶、蔗糖合成酶、棉子糖合成酶、水苏糖合成酶和毛蕊花糖合成酶等参与了棉子糖系列寡糖的生物合成过程.本文对植物中棉子糖系列寡糖的代谢及其重要调控酶的特性、功能及分子生物学研究进展进行综述.     

油菜（Brassica napus L.）种子脱水耐性获得过程中肌醇半乳糖苷合成酶活性与可溶性糖含量的变化

在这个研究中测量不同发育时期的油菜种子中可溶性糖含量与肌醇半乳糖苷合成酶（galactinol synthase,GOLS）活性,将二者的变化趋势与种子脱水耐性获得的过程相比较并对结果进行相关性分析。结果显示油菜种子脱水耐性获得过程中,葡萄糖和果糖含量均随着发育期的延长而下降,蔗糖则保持较高水平;肌醇含量下降而肌醇半乳糖苷含量上升;棉子糖系列寡糖（raffinose familyolig osaccharides,RFO）含量随着种子发育而上升,特别是水苏糖,在成熟种子中可以达到相当高的浓度。油菜种子发育中期,细胞内GOLS活性开始上升,至贮藏物积累完成时达到最大。GOLS活性变化与种子肌醇半乳糖苷积累速度、RFO含量及种子的脱水耐性呈一定的正相关关系。我们认为GOLS促使RFO积累,从而对种子脱水耐性的获得产生重要影响。     

拟南芥肌醇半乳糖苷合成酶与棉子糖合成酶的体外催化活性比较

[目的]基因克隆及原核表达纯化后比较拟南芥的2个肌醇半乳糖苷合成酶及2个棉子糖合成酶的体外催化活性,为微生物法或酶法合成棉子糖尊定基础。[方法]RT-PCR克隆拟南芥的肌醇半乳糖苷合成酶(GolS1及GolS3)与棉子糖合成酶(RafS1及RafS5)的基因,分别构建原核表达菌株,诱导表达纯化获得酶,电泳检测及蛋白定量后进行体外酶催化反应,HPLC分析产物。[结果]克隆到GolS1与GolS3及RafS1与RafS5的基因,原核表纯化获得纯酶,以反应体系中目标产物生成速率衡量,GolS1与GolS3催化速率分别为0.51和0.28mmol/(mg·min),RafS1与RafS5的催化速率分别为0.45和0.21mmol/(mg·min)。[结论]拟南芥的肌醇半乳糖苷合成酶(GolS1及GolS3)与棉子糖合成酶(RafS1及RafS5)基因经异源表达后具有良好酶活,其中GolS1酶活是GolS3的1.82倍,RafS1酶活是RafS5的2.14倍。     

摘除雌花对甜瓜成熟叶片中糖及相关酶活性的影响

甜瓜有果株的成熟叶片中蔗糖、葡萄糖、果糖含量与无果株的无显著差异,水苏糖与棉子糖含量略低于无果株,肌醇半乳糖苷(合成水苏糖的前体)含量显著低于无果株,蔗糖磷酸合成酶(SPS)和肌醇半乳糖苷合成酶活性与无果株的无显著差异,水苏糖合成酶活性显著高于无果株。     

基于RNA-Seq解析小桐子棉子糖代谢途径在干旱适应中的作用

作为新兴的能源植物,小桐子所具备的耐贫瘠、种子含油量高、油质优良等特性使其受到广泛关注。本研究对小桐子幼苗进行10%PEG模拟干旱锻炼与空气干旱胁迫处理,以叶片样品进行RNA-Seq文库构建并基于Illumina Hiseq2000平台进行高通量测序,对差异表达基因进行鉴定和通路富集分析,发现糖代谢通路得到了显著富集。其中,肌醇半乳糖苷合成酶(inositol galactoside synthase,GS)和棉子糖合成酶(raffinose synthase,RS)作为棉子糖系列寡糖合成途径的2类关键酶,其家族成员的不同编码序列在干旱胁迫下呈现不同的表达模式。实时荧光定量(RT-q PCR)验证了两个酶基因家族关键成员对干旱处理的诱导响应,暗示了棉子糖合成途径参与了干旱锻炼和胁迫耐受性的形成,为进一步揭示小桐子抗旱性形成的分子机制及其基因工程改良奠定了坚实的理论基础。     

大豆GolS基因家族鉴定及盐旱胁迫下的表达分析

肌醇半乳糖苷合成酶（galactinol synthase,GolS）是棉子糖家族寡糖（raffinose family oligosaccharides,RFOs）生物合成途径中的关键酶,在植物对非生物胁迫的反应中发挥重要作用。然而,关于大豆（Glycine max）GolS基因家族成员的分子结构特征还未见研究报道。本研究在全基因组水平上鉴定了6个大豆GolS基因家族成员,并对其理化性质、染色体定位、进化关系、基因结构、保守基序、二级结构、三级结构、组织特异性表达模式以及盐和干旱胁迫下的表达量进行了分析。结果表明：6个大豆GolS基因不均匀地分布在4条染色体上,6个大豆GolS蛋白的等电点为5.45-6.08,分子量变化范围为37 567.07-38 817.59 Da,氨基酸数量为324-339 aa;亚细胞定位预测结果发现4个蛋白定位在叶绿体上,2个蛋白定位在细胞质。系统进化树分析表明,大豆GolS基因家族成员在进化树中呈现出两两紧邻的现象,在进化上较为保守。6个基因成员含有的外显子数目为3或4。二级结构和三级结构预测表明,该家族所有成员蛋白质的空间结构主要由α螺旋和无规则卷曲结构组成,有较少的β转角结构和延伸链结构。组织特异性表达分析表明,6个GmGolS家族成员在种子、根、根毛、花、茎、豆荚、根瘤和叶中均有不同程度表达。基于qRT-PCR的表达分析显示,盐旱处理后所有GmGolS基因成员表现出不同程度的上调表达,表明这些基因可能与植物的耐盐抗旱响应有关。本研究结果为后续开展大豆GolS基因的功能解析奠定了基础。     

泡核桃肌醇半乳糖苷合成酶基因克隆及表达分析

肌醇半乳糖苷合成酶是植物中棉子糖系列寡糖合成中的关键酶,在植物抗性生理中扮演重要作用。本研究利用RT-PCR技术从泡核桃中成功克隆获得一个受冷害诱导的肌醇半乳糖苷合成酶基因(Js GS1),其含有1 026 bp的开放阅读框,编码341个氨基酸,登录号为:KX657831。该基因推断的蛋白与核桃(Juglans regia)GS蛋白的相似性为89%;系统进化树分析显示其与核桃、麻疯树(Jatropha curcas)、蓖麻(Ricinus communis)形成一个分支。半定量PCR显示:低温4℃处理3 h后有微弱表达,6 h后大量表达。这说明Js GS1是典型的受冷害诱导的GS基因。本研究为揭示泡核桃抗寒机理以及肌醇半乳糖苷合成酶在冷害胁迫下的作用提供帮助,并为利用基因工程手段培育抗寒新品种提供理论依据。     

Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana

Raffinose family oligosaccharides (RFO) accumulating during seed development are thought to play a role in the desiccation tolerance of seeds. However, the functions of RFO in desiccation tolerance have not been elucidated. Here we examine the functions of RFO in Arabidopsis thaliana plants under drought- and cold-stress conditions, based on the analyses of function and expression of genes involved in RFO biosynthesis. Sugar analysis showed that drought-, high salinity- and cold-treated Arabidopsis plants accumulate a large amount of raffinose and galactinol, but not stachyose. Raffinose and galactinol were not detected in unstressed plants. This suggests that raffinose and galactinol are involved in tolerance to drought, high salinity and cold stresses. Galactinol synthase (GolS) catalyses the first step in the biosynthesis of RFO from UDP-galactose. We identified three stress-responsive GolS genes (AtGolS1, 2 and 3) among seven Arabidopsis GolS genes. AtGolS1 and 2 were induced by drought and high-salinity stresses, but not by cold stress. By contrast, AtGolS3 was induced by cold stress but not by drought or salt stress. All the GST fusion proteins of GST-AtGolS1, 2 and 3 expressed in Escherichia coli had galactinol synthase activities. Overexpression of AtGolS2 in transgenic Arabidopsis caused an increase in endogenous galactinol and raffinose, and showed reduced transpiration from leaves to improve drought tolerance. These results show that stress-inducible galactinol synthase plays a key role in the accumulation of galactinol and raffinose under abiotic stress conditions, and that galactinol and raffinose may function as osmoprotectants in drought-stress tolerance of plants.     

Physiological aspects of raffinose family oligosaccharides in plants: protection against abiotic stress

Abiotic stresses resulting from water deficit, high salinity or periods of drought adversely affect plant growth and development and represent major selective forces during plant evolution. The raffinose family oligosaccharides (RFOs) are synthesised from sucrose by the subsequent addition of activated galactinol moieties donated by galactinol. RFOs are characterised as compatible solutes involved in stress tolerance defence mechanisms, although evidence also suggests that they act as antioxidants, are part of carbon partitioning strategies and may serve as signals in response to stress. The key enzyme and regulatory point in RFO biosynthesis is galactinol synthase (GolS), and an increase of GolS in expression and activity is often associated with abiotic stress. It has also been shown that different GolS isoforms are expressed in response to different types of abiotic stress, suggesting that the timing and accumulation of RFOs are controlled for each abiotic stress. However, the accumulation of RFOs in response to stress is not universal and other functional roles have been suggested for RFOs, such as being part of a carbon storage mechanism. Transgenic Arabidopsis plants with increased galactinol and raffinose concentrations had better ROS scavenging capacity, while many sugars have been shown in vitro to have antioxidant activity, suggesting that RFOs may also act as antioxidants. The RFO pathway also interacts with other carbohydrate pathways, such as that of O‐methyl inositol (OMI), which shows that the functional relevance of RFOs must not be seen in isolation to overall carbon re‐allocation during stress responses.     

Galactinol synthase across evolutionary diverse taxa: Functional preference for higher plants?

Galactinol synthase (GolS), a GT8 family glycosyltransferase, synthesizes galactinol and raffinose series of oligosaccharides (RFOs). Identification and analysis of conserved domains in GTs among evolutionarily diverse taxa, structure prediction by homology modeling and determination of substrate binding pocket followed by phylogenetic analysis of GolS sequences establish presence of functional GolS predominantly in higher plants, fungi having the closest possible ancestral sequences. Evolutionary preference for a functional GolS expression in higher plants might have arisen in response to the need for galactinol and RFO synthesis to combat abiotic stress, in contrast to other organisms lacking functional GolS for such functions.     

Allocation of raffinose family oligosaccharides to transport and storage pools in Ajuga reptans: the roles of two distinct galactinol synthases

Raffinose family oligosaccharides (RFOs) are important phloem transport and storage carbohydrates for many plants. Ajuga reptans, a frost-hardy evergreen labiate, ideally combines these two physiological roles and served as our model plant to study the regulation and importance of RFO metabolism. Galactinol is the galactosyl donor for the synthesis of raffinose (RFO-trisaccharide) and stachyose (RFO-tetrasaccharide), and its synthesis by galactinol synthase (GolS) is the first committed step of the RFO biosynthetic pathway. Two cDNAs encoding two distinct GolS were isolated from A. reptans source and sink leaves, designated GolS-1 and GolS-2, respectively. Warm- and cold-grown sink and source leaves were compared, revealing both isoforms to be cold-inducible and GolS-1 to be source leaf-specific; GolS-1 expression correlated positively with GolS activity. Conversely, GolS-2 expression was comparatively much lower and its contribution to the total extractable GolS activity is most probably only minor. These observations, together with results from phloem exudation and leaf shading experiments suggest that GolS-1 is mainly involved in the synthesis of storage RFOs and GolS-2 in the synthesis of transport RFOs. Furthermore, in situ hybridization studies showed GolS-1 to be primarily expressed in the mesophyll, the site of RFO storage, and GolS-2 in the phloem-associated intermediary cells known for their role in RFO phloem loading. A model depicting the spatial compartmentation of the two GolS isoforms is proposed.     

Expression of a GALACTINOL SYNTHASE gene is positively associated with desiccation tolerance of Brassica napus seeds during development

Desiccation tolerance of seeds is positively correlated with raffinose family oligosaccharides (RFOs). However, RFOs’ role in desiccation tolerance is still a matter of controversy. The aim of this work was to monitor the accumulation of RFO during acquisition of desiccation tolerance in rapeseed (Brassica napus L.). Rapeseeds become desiccation tolerant at 21-24 d after flowering (DAF), and the time was coincident with an accumulation of raffinose and stachyose. A gene encoding galactinol synthase (GolS; EC2.4.1.123), involved in RFO biosynthesis, was cloned and functionally characterized. Enzymatic properties of recombinant galactinol synthase were also determined. Accumulation of BnGOLS-1 mRNA in developing rapeseeds was concomitant with dry weight deposition and the acquisition of desiccation tolerance, and was concurrent with the formation of raffinose and stachyose. The physiological implications of BnGOLS-1 expression patterns in developing seeds are discussed in light of the hypothesized role of RFOs in seed desiccation tolerance.