橡胶树死皮病的发生机理和假说

在阐述橡胶树死皮病的概念和已有关于死皮病因及其机理假说基础上,根据分子生物学新进展提出了死皮病发生的新现点.死皮病是可由生物、生理多种因子胁迫引发的、通过氧化跃变产生的信号分子转导,激发细胞程序性死亡机制而表现出的防卫反应.并论述了使该观点成立的依据和进一步研究的策略.该观点的提出,使关于死皮病的各种假说的合理现点达到统一.并对深入研究死皮病的分子机理具有深远的指导意义.     

产胶植物橡胶转移酶的研究进展

天然橡胶合成中,橡胶转移酶催化异戊二烯焦磷酸的多聚化过程,这一过程对天然橡胶的品质及产量至关重要。橡胶转移酶及其性质、橡胶生物合成分子机理及橡胶的分子量大小决定机制是亟待解决的重要科学问题。本文介绍了以巴西橡胶树为主的产胶植物橡胶转移酶的性质和生物学功能,对橡胶转移酶的分离与鉴定及其活性调节等研究进展进行了综述。     

橡胶草90年来主要研究成果及最新研究进展

天然橡胶是重要的国防战略物质,巴西橡胶树( Hevea brasiliensis (Willd. ex A. Juss.) Muell. Arg)是天然橡胶的唯一来源,天然橡胶商业化形式极为单一,潜在的供给不足问题亟待解决。因此,寻找可替代巴西橡胶树的产胶植物一直受到全世界高度重视。蒲公英属橡胶草( Taraxacum kok-saghyz Rodin)根部含有与橡胶树橡胶类似的天然橡胶分子,该植物主要分布在温带和寒带地区,具有易于机械化收获、生长周期短、遗传转化相对容易等特点,是最具开发潜力的产胶植物。本文对橡胶草90年(1931 - 2018)来的研究历史和主要成果进行了概括,对近10年取得的最新成果进行了深度分析,并预测橡胶草在未来天然橡胶产业中的作用,期望为开展橡胶草商业化生产和橡胶生物合成相关基础研究提供一定的参考。     

不同提取液对巴西橡胶树橡胶粒子蛋白质提取和分离的影响

橡胶粒子是巴西橡胶树（Hevea brasiliensis）乳管细胞内进行橡胶生物合成的亚细胞结构;对橡胶粒子的蛋白质组学研究,可为揭示天然橡胶生物合成机理提供线索。采用5种提取液分别提取橡胶粒子蛋白,并对橡胶粒子蛋白进行SDS—PAGE和16-BAC／SDS—PAGE电泳分离及MALDITOF／TOF串联质谱分析,证明不同提取液抽提的橡胶粒子蛋白具有不同组成,发现分子量较高的橡胶延伸因子家族蛋白更难从橡胶粒子上被洗脱和提取。通过检索橡胶树转录组数据库,鉴定了3个新的橡胶粒子蛋白,即醌氧化还原酶、含蓖麻毒素B链凝集素结构域蛋白及枯萎／脱水相关蛋白。本研究建立的橡胶粒子蛋白质提取和分离方法,为进一步鉴定低丰度和具有重要功能的橡胶粒子蛋白提供了参照体系。     

巴西橡胶成龄无性系茎段的试管微繁技术研究

巴西橡胶成龄无性系茎段组织培养在生产中有很大的潜在应用价值,但一直是橡胶树组织培养的难点,至今国内外鲜有成功的报道.本研究以在中国热带地区大面积推广种植的巴西橡胶树优良品种热研7-33-97不同生长时期成龄树茎段为外植体,进行无性系试管微繁技术研究.研究结果表明不同生长时期的茎段在自然条件中受污染程度不同,茎段真菌污染率为稳定期＞淡绿期＞古铜期,细菌污染率为淡绿期＞稳定期＞古铜期.不同生长时期的成龄树茎段需要通过不同的灭菌方法才能显著提高外植体的利用率,外植体灭菌方法显著影响橡胶成龄树茎段的组织培养效率;与稳定期相比,古铜期和淡绿期茎段在组织培养过程中诱导丛生芽萌芽快、萌芽多,是较优的外植体材料;古铜期和淡绿期茎段在激素配比为4.0～5.0 mg/L6-BA+0.5 mg/L GA3的条件下能很好的诱导抽出丛生芽;丛生芽在激素配比为2.0 mg/L 6-BA+0.5 mg/LNAA,1.0 mg/t 6-BA+1.0 mg/L KT+0.5 mg/L NAA或0.5 mg/L 6-BA+1.5 mg/L KT+0.5 mg/L NAA的条件下能很好的诱导丛生芽抽出健壮的芽条;丛生芽抽出的健壮芽条在激素配比为0.5 mg/L IBA+0.5 mg/L IAA的条件下能较好的抽根成苗.外植体生长时期、激素种类和浓度配比都是影响巴西橡胶成龄无性系茎段的试管微繁的重要因素.     

巴西橡胶树43 kD橡胶粒子膜蛋白基因的cDNA克隆及表达

对43 kD的橡胶粒子膜蛋白进行了分离纯化和其N端氨基酸序列分析,根据N端氨基酸序列,设计一简并引物,通过3'RACE(Rapid Amplification ofcDNA Ends)的方法,获得了43 kD的橡胶粒子膜蛋白的cDNA.该cDNA含有1 385个核苷酸,含有完整的阅读框架,编码381个氨基酸.在终止密码子下游,包含有一个239bp的3'非编码区.该cDNA由5个首尾相连的重复单元组成,每个单元编码76个氨基酸组成的泛素(ubiquitin)单体.编码43 kD橡胶粒子蛋白的基因具有多个拷贝,在胶乳、叶片和树皮都表达.     

巴西橡胶树的脂酰辅酶A还原酶cDNA克隆及其序列分析

从巴西橡胶树差减cDNA文库中筛选到一个与脂酰辅酶A还原酶同源性较高的基因片段,根据该基因片段序列信息,设计特异引物,采用RACE进行差异片段的5’和3’端的扩增,获得长度为1365bp的cDNA克隆R28（GenBank登陆号：AY461413）。序列分析表明,该基因包含1149bp的开放阅读框,5＇-UTR为96bp,3＇-UTR为128bp,编码382个氨基酸,推测其蛋白质的分子量为43．5kDa,等电点为8．97,有一个跨膜螺旋N（187至215位氨基酸）和1个由17个氨基酸组成的信号肽（1至17位氨基酸）。R28含有脂酰辅酶A还原酶的保守（NADP结合蛋白保守区）,推测该基因是一个脂酰辅酶A还原酶基因。     

一种快速、高效的橡胶树胶乳总RNA提取方法

胶乳是橡胶树（Hevea brasiliensis）乳管中特殊的细胞质,主要由橡胶粒子、黄色体、F-W粒子和普通细胞质成分构成,其中橡胶粒子占20％-40％,蛋白含量高达1％-2％。由于高比例橡胶粒子和蛋白质的干扰,目前使用的胶乳RNA提取方法都具有步骤繁琐、胶乳需求量大、操作技巧性强不易掌握等缺点。为快速、高效地获取高质量的胶乳RNA,我们在现有方法的基础上摸索出一套步骤简单、容易操作、快速、高效提取橡胶树胶乳总RNA的简易方法,获得了较好的实验效果。紫外分光光度计、RT-PCR和RACE分析结果表明,使用该方法提取的胶乳RNA质量完全能够满足相应的分子操作,但所需时间仅为目前常用方法的50％,RNA获得率提高了2-3倍,操作难度大大降低。     

Agrobacterium-mediated transformation and regeneration of guayule

In vitro grown shoot tissue of facultative apomictic lines of guayule (Parthenium argentatum Gray), a rubber producing desert shrub, were transformed by Agrobacterium-mediated DNA transfer and regenerated into complete plants. Guayule shoots of lines 11591, UC101 and UC104 were inoculated with A. tumefaciens strains LBA4404 or PC2760 harboring the binary vector pCGN1557. Axillary shoots were regenerated from transformed cells and rooted in vitro in the presence of kanamycin. Genetic transformation in all cases was verified by Southern blot analysis. Transgenic plants were grown to maturity in the greenhouse and, as predicted for apomictic species, all seed produced possessed kanamycin resistance. Because apomicts have limitations for gene transfer by normal sexual crosses, this method offers a new means of transferring genes into this species.Abbreviations BA benzyladenine - EDTA ethylene diamine tetraacetate - kan_R kanamycin resistance - MS salts salts of Murashige and Skoog medium (1962) - NAA naphthalene acetic acid - NPT-II neomycin phosphotransferase - SDS sodium dodecyl sulfate     

巴西橡胶树赤霉素信号转导途径HbRGA1结构和功能分析

DELLA蛋白是赤霉素激素信号负调控因子,具有抑制植物生长发育的作用。解析其家族成员结构与功能将有助于揭示橡胶树DELLA蛋白家族成员调控橡胶树生长发育的机制。本研究从橡胶树热研73397叶片中克隆HbRGA1的cDNA全长序列。该基因长为2136 bp,含1839 bp的ORF,编码613个氨基酸。HbRGA1蛋白序列包含DELLA和GRAS保守结构域,与杨树、木薯和橡胶树DELLA基因相似性高达82.5%。qRT-PCR分析发现HbRGA1在橡胶树叶片中表达量高,在树皮和胶乳中表达量极低。叶片中HbRGA1表达量受喷施赤霉素和脱落酸等诱导显著上调。本研究表明HbRGA1与橡胶树赤霉素等激素信号密切相关,为深入研究其在橡胶树生长发育中的结构和功能打下良好基础。     

New aspects of rubber biosynthesis

New aspects of rubber biosynthesis. Following a review of the site of rubber biosynthesis in Hevea brasiliensis and Parthenium argentalum, evidence is given for the initiation of polyisoprene molecules from (ranMerpenoid precursors including geranylgeranyl pyrophosphate. All franj-¹⁴C-geranylgeraniol has been isolated from incubations of H. brasiliensis latex serum with ¹⁴C-isopentenyl pyrophosphate. Gel-filtration chromatography of the serum yields very small rubber particles of high biosynthetic activity, and two proteinaceous fractions. One of these increases the biosynthesis of rubber and may contain the enzyme, isopentenyldiphosphate δ-isomerase, whilst the other appears to inhibit rubber formation. The nature and molecular weight of the rubber formed in vitro is discussed and a mechanism for the de novo formation of rubber particles is suggested.     

Determination of biotic aetiology of tapping panel dryness (TPD) syndrome of rubber tree (Hevea brasiliensis) by return-polyacrylamide gel electrophoresis (R-PAGE) technique

Tapping panel dryness (TPD) syndrome affecting rubber tree (Hevea brasiliensis) is known to reduce natural latex production. Its aetiology remains ambiguous despite long years of research. A low molecular weight RNA similar to viroid RNA was isolated from TPD-affected samples of rubber trees. In the present study, a modified return-polyacrylamide gel electrophoresis procedure was standardised. The viroid-like low molecular weight (LMW) RNA was found associated with leaf, bark and root tissues and rubber seedlings. The technique was employed to detect LMW RNA in different clones of rubber planted in different locations and in bud-grafted plants. The LMW RNA isolated from TPD-affected trees was found infectious on seedlings of tomato cv Pusa Ruby. The LMW RNA was reisolated from symptomatic tomato leaves but not from control plants. This is for the first time that a biotic agent, a viroid RNA, is found consistently associated with the syndrome. The technology developed can be useful to demonstrate the onset of TPD in untapped trees in the absence of other methods such as nucleic acid hybridisation.     

Growth and gas exchange responses of Hevea brasiliensis seedlings to inoculation with Glomus mosseae

 The beneficial effect of arbuscular mycorrhizal (AM) fungi on plant growth is well known, but the physiological processes involved are still discussed. The purpose of this study was to determine if Glomus mosseae affects the growth of Hevea brasiliensis seedlings and, if it is the case, if it could be associated with variations in leaf CO₂ and H₂O gas exchange. H. brasiliensis rubber trees were grown for 9 months in a medium containing either propagules of G. mosseae or sterilized inoculum. Plant size, root collar diameter and leaf area, as well as net CO₂ assimilation, stomatal conductance (g_s) and water-use efficiency of photosynthesis were evaluated during the first 5 stages of growth. At stage 2, a growth depression occurred in the mycorrhizal seedlings coincident with the first AM infections. Then, at stage 5, Glomus mosseae-inoculated plants had moderate colonization (47% of root length) and were taller than control plants with a larger root collar diameter and an enhanced leaf organogenesis. This enhanced growth was accompanied by increased photosynthesis, transpiration, and stomatal conductance. After 9 months, dry weights of shoots and roots of inoculated plants were greater than those of controls by 27 and 17%, respectively. Received: 10 May 1997 / Accepted: 9 September 1997     

橡胶树不同品种雌花分布和座果规律研究

以橡胶树（Hevea brasiliensis Muell.Arg.）RRIM600、GT1、RRII105、IAN873、RRIM712、PB217等6个品种为对象,对不同品种橡胶树雌花分布和座果位置进行了观察和统计。结果表明：不同橡胶树品种间,圆锥花序上雌花的个数、雌花在花序上的位置分布和结果侧枝上的花序数存在极显著差异。雌花和果实在圆锥花序上的分布规律相似,主要分布在圆锥花序的顶端和下部;结果花序主要分布在结果侧枝的下部,圆锥花序距离树干越远,则花序结果的几率越小。因此,在橡胶树杂交育种时,可以只对相关品种座果率高的结果侧枝相应位置上的花序及花序上相应的雌花进行授粉,而摘掉其余座果率低的花序和雌花,从而提高杂交育种效率和效果。     

Rubber particles from four different species, examined by transmission electron microscopy and electron-paramagnetic-resonance spin labeling, are found to consist of a homogeneous rubber core enclosed by a contiguous, monolayer biomembrane

The physical characteristics of rubber particles from the four rubber (cis-1,4-polyisoprene) producing species Euphorbia lactiflua Phil., Ficus elastica Roxb., Hevea brasiliensis Müll. Arg., and Parthenium argentatum Gray, were investigated using transmission electron microscopy (TEM) and electron-paramagnetic-resonance (EPR) spin labeling spectroscopy. Transmission electron microscopy showed the rubber particles to be composed of a spherical, homogeneous, core of rubber enclosed by a contiguous, electron-dense, single-track surface layer. The biochemical composition of the surface layer and its single-track TEM suggested that a monolayer biomembrane was the surface structure most compatible with the hydrophobic rubber core. The EPR spectra for a series of positional isomers of doxyl stearic acid, used to label the surface layer of the rubber particles, exhibited flexibility gradients and evidence for lipid-protein interactions for all four rubber particle types that is consistent with a biomembrane-like surface. The EPR spectra confirmed that the surface biomembrane is a monolayer. Thus, rubber particles appear similar to oil bodies in their basic architecture. The EPR spectra also provided information on protein location and degree of biomembrane penetration that correlated with the known properties of the rubber-particle-bound proteins. The monolayer biomembrane serves as an interface between the hydrophobic rubber interior and the aqueous cytosol and prevents aggregation of the particles. An unexpected observation for the probes in pure polyisoprene was evidence of an intrinsic flexibility gradient associated with the stearic acid molecule itself. Received: 22 May 1999 / Accepted: 21 June 1999     

橡胶树凝集因子hevein基因及其启动子序列的分离与分析

橡胶树 (HeveabrasiliensisMuell._Arg .)凝集因子hevein是引起橡胶粒子凝集的主要因素 ,它是胶乳中黄色体内主要的蛋白质 ,具有几丁结合的功能。通过PCR技术扩增并克隆了橡胶树hevein基因共 6 80bp的序列。继而通过步移法分离启动子区域 130 6bp的序列 ,序列含典型的TATA盒和CAAT盒以及ABA效应元件的同源序列。为证实该基因在乳管中特异表达 ,利用Northernblot分析hevein基因在胶乳和叶片中的表达 ,同时 ,分析乙烯和ABA处理后hevein基因的表达。结果表明 ,hevein基因主要在胶乳中表达 ,乙烯和ABA对基因的表达有诱导作用。     

Modelling the influence of assimilate availability on root growth and architecture

A model has been designed to simulate rubber seedling root development as related to assimilate availability. Each root of the system is defined both as an element of a network of axes, characterized by its order, position and connections and as an individual sink competing for assimilates. At each time step, the growth of each root is calculated as a function of its own growth potential and of assimilate availability calculated within the whole plant. The potential elongation rate of a root is estimated by its apical diameter, which reflects the size of the meristem. When a root is initiated, the apical diameter depends on root type, but it varies thereafter according to assimilate availability. Thus, the latter controls both current and potential elongation. The model was able to simulate periodicity in root development as related to shoot growth and to reproduce differences in sensitivity to assimilate availability related to root type. It thereby validated the hypothesis that root growth but also root system architecture depend on assimilate allocation and that apical diameter is a good indicator of root growth potential. Provided that specific calibration is done, this model may be used for other species.     

Synchronous flowering of the rubber tree (Hevea brasiliensis) induced by high solar radiation intensity

How tropical trees flower synchronously near the equator in the absence of significant day length variation or other meteorological cues has long been a puzzle. The rubber tree (Hevea brasiliensis) is used as a model to investigate this phenomenon. The annual cycle of solar radiation intensity is shown to correspond closely with the flowering of the rubber tree planted near the equator and in the subtropics. Unlike in temperate regions, where incoming solar radiation (insolation) is dependent on both day length and radiation intensity, insolation at the equator is due entirely to the latter. Insolation at the upper atmosphere peaks twice a year during the spring and autumn equinoxes, but the actual solar radiation that reaches the ground is attenuated to varying extents in different localities. The rubber tree shows one or two flowering seasons a year (with major and minor seasons in the latter) in accordance with the solar radiation intensity received. High solar radiation intensity, and in particular bright sunshine (as distinct from prolonged diffuse radiation), induces synchronous anthesis and blooming in Hevea around the time of the equinoxes. The same mechanism may be operational in other tropical tree species.     

Homologous Hevea brasiliensis REF (Hevb1) and SRPP (Hevb3) present different auto-assembling

HbREF and HbSRPP are two Hevea brasiliensis proteins present on rubber particles, and probably involved in the coagulation of latex. Their function is unclear, but we previously discovered that REF had amyloid properties, which could be of particular interest during the coagulation process. First, we confirmed that REF and SRPP, homologous and principal proteins in hevea latex, are not glycoproteins. In this work, we investigated various aspects of protein interactions: aggregation, auto-assembling, yeast and erythrocyte agglutination, co-interactions by various biochemical (PAGE, spectroscopy, microscopy), biophysical (DLS, ellipsometry) and structural (TEM, ATR-FTIR, PM-IRRAS) approaches. We demonstrated that both proteins are auto-assembling into different aggregative states: REF polymerizes as an amyloid rich in β-sheets and forms quickly large aggregates (> μm), whereas SRPP auto-assembles in solution into stable nanomultimers of a more globular nature. Both proteins are however able to interact together, and SRPP may inhibit the amyloidogenesis of REF. REF is also able to interact with the membranes of yeasts and erythrocytes, leading to their agglutination. In addition, we also showed that both REF and SRPP did not have antimicrobial activity, whereas their activity on membranes has been clearly evidenced. We may suspect that these aggregative properties, even though they are clearly different, may occur during coagulation, when the membrane is destabilized. The interaction of proteins with membranes could help in the colloidal stability of latex, whereas the protein–protein interactions would contribute to the coagulation process, by bringing rubber particles together or eventually disrupting the particle monomembranes.