巴西橡胶树的组织培养

介绍了上世纪70年代以来巴西橡胶树的组织培养,包括花药培养、未授粉胚珠和子房培养、茎尖及嫩茎培养、悬浮细胞和原生质体培养,以及体细胞植株的克隆增殖的研究进展.并探讨其存在的问题以及未来的发展方向.     

橡胶树EST-SSR标记的开发与应用

通过对橡胶树10 778条EST序列进行拼接, 共得到Unigene 3 090条, 从中发掘出分布于353条Unigene的430个EST-SSR位点, SSR发生频率为11.42%, 平均分布距离为3.93 kb。在橡胶树EST-SSR中, 二核苷酸和三核苷酸重复是主要的重复类型, 分别占63.49%、32.09%。TC/AG、CT/GA和CTT/GAA、AAG/TTC、AGA/TCT是二、三核苷酸的优势重复类型。利用PRIMER5.0设计引物148对, 随机挑选21对引物进行合成, 并用其中15对扩增较好的引物, 通过变性聚丙烯酰胺凝胶结合银染的方法对44个橡胶树无性系进行了遗传多样性检测, 构建了聚类分析树状图。研究结果表明, 从橡胶树EST中开发SSR标记是有效、可行的, 文章所开发的EST-SSR引物可用于橡胶树遗传分析研究。     

橡胶树种子对脱水的敏感性

橡胶树种子散落后能立即萌发,萌发率高（62．7％）。其在缓慢脱水的第一阶段和快速脱水的前2个阶段的萌发率均提高,此后,种子活力随着含水量的减少而下降。快速和缓慢脱水条件下的种子致死含水量分别为40．3％和48．9％。     

Effect of chilling on photosynthesis and antioxidant enzymes in Hevea brasiliensis Muell. Arg.

The aim of the present study was to assess the tolerance of Hevea brasiliensis to chilling temperatures since rubber production has been extended to sub-optimal environments. PB260 clone was used to analyze the responses of leaves chilled at 10°C during 96 h, as well as their recovery at 28°C. Some key parameters were used to evaluate photosynthetic apparatus functioning, membrane damage (electrolyte leakage) and oxidative stress. A short-term response versus a long-term one have been recorded, the time point of 24 h, when stomata closure was effective, being the border between the two responses. P _n decreased dramatically at 1 h, and Fv/Fm was slightly affected. NPQ reached its maximal level between 4 and 7 h. Lipid peroxidation and membrane lysis were observed between 48 and 96 h. Activities of antioxidant enzymes increased, along with the induction of antioxidant gene expression. Finally, the plants were capable to recover (net photosynthetic rate, photochemical efficiency, antioxidant enzymes activities) when placed back to 28°C showing that PB260 can withstand long-term chilling.     

橡胶树未成苗体细胞胚的茎芽分化及利用

以橡胶树未成苗体细胞胚为试材,选取未成苗双子叶胚、连体胚、多子叶胚、单子叶胚和子叶愈伤化胚作为外植体,研究不同体细胞胚类型的外植体分化茎芽的情况,并利用其进行幼态微型芽条的培育研究。结果表明:在植株诱导培养基中培养80d后,未成苗双子叶胚、连体胚、多子叶胚、单子叶胚、子叶愈伤化胚均能分化出芽,双子叶胚分化率最高(达90%),单子叶胚最低(20%),多子叶胚和连体胚最多可分化出芽3个,双子叶胚2个。幼态微型芽条增殖的最优培养基为MS+6-BA2mg·L-1或MS+6-BA2mg·L-1+KT1mg·L-1,而添加NAA对芽的伸长有抑制作用;适宜幼态微型芽条伸长培养的基本培养基为MS。     

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。     

巴西橡胶树HbMYB62转录因子基因的克隆和表达分析

R2R3-MYB类转录因子参与包括逆境胁迫反应等多种生物反应。为鉴定橡胶树中MYB转录因子的结构与功能,从橡胶树叶片中克隆HbMYB62全长的cDNA序列。该基因片段大小为1 013 bp,包含945 bp的ORF,编码314个氨基酸残基。其推导的氨基酸序列具有植物HTH_MYB的特异性结构域,并与拟南芥AtMYB62具有高度相似性。qRT-PCR发现HbMYB62主要在橡胶树花中表达,而在树皮、叶和乳胶中表达量较少。其叶片表达量在过氧化氢（H₂O₂）、脱落酸（ABA）、水杨酸（SA）等处理下显著上调。表明HbMYB62与橡胶树的抗逆反应和激素信号传导过程有关,为进一步研究其结构和功能打下基础。     

橡胶树(Hevea brasiliensis Muell.Arg.)幼茎木材的超微结构观察

橡胶树是中国重要的热带经济作物,橡胶种植的副产物橡胶木是我国木材供应的重要来源。我们以不同发育阶段的橡胶树幼茎木材为材料,借助扫描电子显微镜技术,对木质部细胞的超微结构进行了观察。结果表明,在橡胶树幼茎木材中,导管和木纤维细胞壁随着木质部发育成熟会发生明显的次生加厚,加厚方式主要为螺纹加厚;木质部各类型细胞均存在大量纹孔,纹孔排列方式主要有散生、网状、梯状和单串状等类型;在木质部发育过程中,木射线和部分薄壁细胞中会逐渐积累大量淀粉粒;木质部细胞内壁及其填充物表面存在不同类型的附着物。研究结果将为橡胶木材材性及其形成机制的研究提供一定理论参考。     

巴西橡胶树一个AP2／EREBP转录因子的克隆及表达分析

本文采用RACE技术从巴西橡胶树中鉴定出一个AP2／EREBP转录因子。该转录因子全长cDNANl225bp,最长开放阅读框699bp,预测编码蛋白包含232个氨基酸;序列比对分析发现,该转录因子具有一段保守的AP2／EREBP结构域,与拟南芥Soloist（At4g13040）具有较高的相似性,将该基因命名HbSoloist。半定量胙PcR分析结果表明,HbSoloist在巴西橡胶树的胶乳、叶片、树皮和花中均有表达。与健康橡胶树相比,死皮树胶乳HbSoloist表达量明显下降。研究发HbSoloist表达受乙烯利、茉莉酸和低温处理调控,表明HbSoloist基因可能在巴西橡胶树乙烯、茉莉酸和低温反应中发挥作用。     

巴西橡胶树6个PP2C家族基因成员分子信息学与抗旱功能分析

PP2C是一类丝氨酸/苏氨酸残基蛋白磷酸酶,在高等植物ABA信号途径中起着重要的作用。为阐明巴西橡胶树中PP2C基因的结构与功能,本研究通过生物信息学方法,从橡胶树转录组数据库中鉴定并获得6个PP2C家族基因,均含有PP2CD、F1和F2亚族。通过qRT-PCR技术对6个PP2C家族基因进行了干旱处理下的差异表达分析,发现6个基因都不同程度上响应橡胶树干旱胁迫。本研究为探究PP2C基因在橡胶树抗干旱反应机制提供了理论依据。     

Occurrence of Intraxylary Phloem in Hevea brasiliensis (Willd. ex A. Juss.) Muell. Arg.

The occurrence of intraxylary phloem in Hevea brasiliensis isreported. The phloem elements were observed as strands associatedwith the protoxylem group in the pericentral region. The natureand importance of such tissue in this species are discussed. Hevea brasiliensis, intraxylary phloem, laticifers, tapping     

Ethylene-Induced Increase in Glutamine Synthetase Activity and mRNA Levels in Hevea brasiliensis Latex Cells

Ethylene, used as a stimulant of latex production in Hevea brasiliensis, significantly activates the regenerating metabolism within the laticiferous cells. In this context, attention was focused on glutamine synthetase (GS; EC 6.3.1.2), a key enzyme in nitrogen metabolism. A specific and significant activation of the cytosolic glutamine synthetase (GScyt) in the laticiferous cells after ethylene treatment parallels the increase of latex yield. A marked accumulation of the corresponding mRNA was found, but in contrast, a slight and variable increase of the polypeptide level is at the limit of detection by western blotting. The GS response to ethylene might be mediated by ammonia that increases in latex cytosol following ethylene treatment. The physiological significance for such a regulation by ethylene of the GScyt is discussed in terms of the nitrogen requirement for protein synthesis associated with latex regeneration.     

Effects of Tapping, Wounding, and Growth Regulators on Turgor Pressure in Hevea brasiliensis Muell. Arg.

Various forms of wounding result in increases in hydrostaticpressure in the laticiferous phloem tissue of Hevea brasiliensis;regular tapping, a form of controlled wounding, probably causesa similar response. When a tree is ring-barked, there is a transientfall in turgor immediately above and below the ring, presumablyowing to loss of latex during cutting. This is followed by apressure increase which is particularly marked above the ring,suggesting the accumulation of metabolites. Isolation of an‘island’ of tissue, by cuts down to the wood, resultsin a steep fall in turgor within it, although a relatively slowrecovery may follow. Partially isolated panels show smallerfalls and a more rapid rise. It appears that a functional phloemconnexion permits a more rapid recovery of osmotic and turgorpressures following latex losses, both in the isolation experimentsand in normal tapping. Growth regulators such as 2,4,5-trichlorophenoxyaceticacid (2,4,5-T) increase latex yields by prolonging latex flowafter tapping. In untapped trees treatment with 2,4,5-T resultsin a slow and fairly small increase in turgor pressure, butthis effect may not be apparent if trees are regularly tapped.When the tapping cut is opened, there is a rapid fall in pressureimmediately under the cut. This loss in turgor spreads throughthe latex-vessel system as latex flows towards the cut, butrecovery is apparent near the cut even before flow ceases. Pressuregradients indicate a rapid formation of a localized resistanceto flow at or near the cut surface. This process appears toplay a major part in restricting flow. The increased yieldswhich result both from regular tapping and after 2,4,5-T treatmentappear to result from a delay in this sealing process. The mechanismsby which the barrier to flow is built up and delayed by 2,4,5-Ttreatment are not clearly understood.     

The Regulation of Invertase Activity in the Latex of Hevea brasiliensis Muell. Arg: THE EFFECTS OF GROWTH REGULATORS, BARK WOUNDING, AND LATEX TAPPING

Treatments which increase latex yield, e. g. bark scraping,latex tapping, and bark application of 2, 4-D or 2-chloroethylphosphonicacid (CEPA) were found to enhance the activity of latex invertase.In previously untapped trees, both the introduction of tappingand the application of 2, 4-D brought about an increase in thelevel of invertase. In regularly tapped trees, the amount oflatex invertase is several times higher than in untapped treesand evidence was obtained that its activity is regulated bythe variation of latex pH. The pH of latex of the clone investigated(PR 107) was shown to vary between 6.3 and 7.1 whereas the activityof invertase, as assayed directly in the latex, has a sharpoptimum at pH 7.5 and falls rapidly with the shift of pH tothe acid side. There was no increase in the content of latexinvertase when trees adapted to regular tapping were treatedwith 2, 4-D. The effect of auxin on actual invertase activitywas essentially mediated through related increase of latex pH.The CEPA and bark scraping were also shown to increase latexpH in tapped trees. The treatment of the bark of tapped trees with CEPA increasedthe level of latex sucrose, as did auxins. Bark scraping alsohad a slight stimulatory effect. The K_m of latex invertase asa function of pH was found to change in the same way as V_max,being highest at pH optimum.     

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。     

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。     

Structure of the Bark and Clonal Variability in Hevea brasiliensis Muell. Arg. (Willd. ex A. Juss.)

Detailed studies have been made on the structure of the barkof ten Hevea clones and the clonal variabilities with regardto the density and size of ray groups, density of laticifersper row per unit circumference of the tree, diameter of laticifersand the extent of connection between laticifers. Clonal variabilitywas highly significant with regard to the density of ray groups,ray height, ray width in the laticifer layer and the laticifercharacters. The influence of ray characters on the orientationof laticifers and thereby its quantity is discussed. The scopeof using anatomical parameters for clone identification is examined. Hevea brasiliensis, Para rubber tree, laticifers, bark (structure), anatomy, clonal variability, rubber     

一种提取橡胶树叶中总DNA的方法

橡胶树叶中富含多糖和多酚类物质,严重影响高质量基因组DNA的提取,文章针对这一特点提出了改良的CTAB法.这一方法可用于DNA的大量或小量提取,小量提取的产量为50μg·g-1(FW),大量提取可达到400μg·g-1(FW);可从橡胶树古铜期、变色期、稳定期等3个生长时期的叶中提取出高质量DNA,所得DNA可用于PCR分析和酶切分析等分子生物学研究.     

Field nursery inoculation of Hevea brasiliensis Muell. Arg. seedling rootstock with vesicular-arbuscular mycorrhizal (VAM) fungi

The growth response of Hevea brasiliensis to vesicular-arbuscular mycorrhizal (VAM) fungi inoculation was assessed in two field nursery sites containing indigenous mycorrhizal fungi (IMF). Seedling rootstocks were inoculated with mixed VAM-fungal species in a factorial combination with phosphorus (P) fertilizer application, and planted in randomised blocks on sandy (site 1) and clayey (site 2) soils. Plants were harvested after 26 weeks for measurements of shoot dry weight (DW), stem diameter, height, mycorrhizal root colonization and leaf nutrient contents. At site 1, VAM increased shoot DW, stem diameter and plant height only in treatments without P applied. Increases in shoot DW due to VAM were 70% greater than the uninoculated controls although this was reduced to 5% when P was applied. At site 2, VAM inoculation also increased shoot DW and stem diameter but the magnitude of the increases was smaller. Shoot DW response due to VAM was only 29%. At this second site, applying phosphate to uninoculated plants did not increase shoot yields further. Leaf concentrations of all nutrients were unaffected by VAM at both sites, except for copper (Cu) which was increased by VAM in treatments where P was not applied. However, leaf contents of P, potassium (K), magnesium (Mg) and Cu were increased by VAM at site 1, and of leaf nitrogen (N) and K at site 2. These experiments demonstrate that VAM-fungi could be introduced into field nursery sites to improve growth and P uptake by H. brasiliensis. The relevance of VAM-fungi to H. brasiliensis seedling rootstock development and the influence of IMF in determining field responses is discussed.