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.     

Effect of exogenous calcium on Agrobacterium tumefaciens-mediated gene transfer in Hevea brasiliensis (rubber tree) friable calli

The influence of CaCl₂ was investigated on Agrobacterium tumefaciens-mediated gene transfer in Hevea brasiliensis friable calli which are usually proliferated on maintenance medium (MM) containing 9 mM CaCl₂.Five A. tumefaciens strains (C58pMP90, C58pGV2260, AGL1, LBA4404 and EHA 105) and two binary vectors (pGIN and pCAMBIA2301) were tested and the strain EHA105pC2301 was selected to conduct further experiments. The calli were precultured on MM containing a range of CaCl₂ concentrations, then inoculated with Agrobacterium suspension. Transfer of friable calli from MM containing 9 mM CaCl₂ to calcium-free medium significantly enhanced the transient β-glucuronidase activity. Interestingly, the use of calcium-free Agrobacterium resuspension medium to inoculate friable calli again dramatically increased the transformation efficiency. Induction of Agrobacterium's virulence with acetosyringone remained an important factor to stimulate transformation. Received: 14 October 1999 / Revision received: 3 January 2000 / Accepted: 4 January 2000     

巴西橡胶树的分子生物学研究进展

巴西橡胶树是一种重要的热带经济作物,由于橡胶树体内橡胶含量多,且容易采收,所以橡胶树一直是天然橡胶的商业来源。相比于模式植物和粮食等经济作物来说,分子生物学研究显滞后。,而且橡胶树是多年生乔木,经济性状多集中于胶乳,因此研究难度大,研究也不多,本就巴西橡胶的分子生物学方面的研究进行综述。     

橡胶树死皮病胶乳C-乳清差异表达蛋白质的筛选与鉴定

橡胶树死皮病(Tapping Panel Dryness,TPD)在世界各橡胶种植园普遍发生,给橡胶种植业带来严重的危害。为了更好地了解和阐明死皮病发生、发展的分子机制,本研究应用双向凝胶电泳技术（2-DE）比较橡胶树死皮株与健康株胶乳C-乳清蛋白质组表达的差异。采用固相pH梯度双向凝胶电泳分离橡胶树死皮株与健康株C-乳清的总蛋白质,凝胶经考染显色后,用PDQuest7.40图像分析软件进行比较分析,识别差异表达的蛋白质。这些点经过胶内酶切后进行基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)分析获取肽质指纹图谱(PMF),Mascot软件搜索SWISS-PROT和NCBInr数据库鉴定蛋白质。结果：①橡胶树死皮株与健康株C-乳清凝胶的平均蛋白质点数分别为1075±35和1134±27,其平均匹配的点数分别为982±38和1008±22,组内图像匹配率达91.89﹪和88.72﹪。②橡胶树死皮株与健康株C-乳清组间的平均匹配蛋白点数为970±25。利用MALDI-TOF-MS质谱技术对40个差异明显的蛋白点进行分析鉴定,通过查询数据库鉴定了27个蛋白质。本研究建立了分辨率高且重复性较好的橡胶树死皮株与 健康株胶乳C-乳清的双向凝胶电泳图谱,并应用质谱技术鉴定了其中表达差异的蛋白质点,这些差异表达的蛋白质可能参与了死皮发生和发展的过程。     

Cloning and characterization of cDNA encoding farnesyl diphosphate synthase from rubber tree (Hevea brasiliensis)

Commercially used natural rubber (cis-1,4-polyisoprene) is a secondary metabolite of the rubber tree (Hevea brasiliensis). Previous studies have shown the involvement of a prenyl transferase in the final steps of natural rubber biosynthesis which includes polymerization of isopentenyl pyrophosphate into rubber. Using synthetic oligonucleotides corresponding to the partial amino acid sequences of this protein as probes to screen a laticifer-specific cDNA library, we have isolated a full-length cDNA which encodes a 47 kDa protein with strong homology to farnesyl diphosphate synthases from many species. The catalytic activity of this protein was confirmed by complementing the deletion yeast mutant. In Hevea, this gene is expressed in latex producing cells and in the epidermal region of the rubber plant suggesting a dual role for the protein in the biosyntheses of rubber and other isoprenoids. Although the expression level of this gene is not significantly affected by hormone treatment (e.g. ethylene), regeneration of latex due to tapping increases its expression level.     

巴西橡胶树SSR遗传图谱的构建

以热研88-13×IAN873的94个F1群体为试材, 利用简单序列重复(Simple sequence repeat, SSR)标记, 采用FsLinkageMAP 1.0软件, 构建了巴西橡胶树热研88-13×IAN873的遗传连锁图谱。从441对SSR引物中筛选出160对具有多态信息的引物, 在分离群体中共检测到206个多态性位点, 176个位点用于遗传图谱的构建; χ²检验结果显示, 有147个位点符合1:1分离比例, 有12个符合1:2:1分离比例, 有17个符合1:1:1:1的分离比例, 共有13个偏分离位点, 偏分离率低(7.38%); 91个SSR位点被分为18个连锁群, 覆盖橡胶树基因组1 937.06 cM, 每个连锁群包含2～16个位点, 标记间的平均距离为21.29 cM。     

Molecular cloning,characterization and expression of Mn-superoxide dismutase from the rubber tree (Hevea brasiliensis)

The gene and the RNA from Arabidopsis thaliana for the plastid-located glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) and their encoded product have been studied. The gene (designated ATS1) was isolated by screening a DASH genomic library for cross-hybridization with a radiolabeled probe prepared from cDNA for GPAT from squash. cDNA clones representing the mRNA were isolated by screening a ZAPII cDNA library for hybridization with a radiolabeled probe prepared from a DNA fragment of ATS1. The nucleotide sequences of the gene and the cDNA were determined, and the 5 end of the RNA was mapped by primer extension. Sequences similar to the TATA box, polyadenylation sequences and intron-splicing sequences were found at the expected locations. The pre-mRNA was 3288 nucleotides long and contained 5 and 3-untranslated sequences of 57 and 442 nucleotides, respectively. The coding sequence of 1377 nucleotides was interrupted by 11 introns of 1412 nucleotides in total and the 3-untranslated sequence contained another intron of 94 nucleotides. The open-reading frame encoded a polypeptide of 459 amino acid residues, the amino acid sequence of which was highly homologous to those of precursors to plastid-located GPATs from squash and pea. The enzymatic activity of a gene product that was over-produced in Escherichia coli confirmed the indentity of the gene.Abbreviations ACP acyl carrier protein - GPAT glycerol-3-phosphate acyltransferase - IPTG isopropyl--thiogalactopyranoside.     

A convenient and efficient protocol for isolating high-quality RNA from latex of Hevea brasiliensis (para rubber tree)

Isolating high-quality RNA from latex of H. brasiliensis is a prerequisite to elucidating the molecular mechanisms of rubber biosynthesis and its regulation. Here, an improved protocol was developed for latex collection, transportation, storage, and RNA isolation. Compared with existing ones, our protocol eliminated liquid nitrogen for latex collection and subsequent low-temperature (-70 degrees C) condition for latex storage, making it more convenient and feasible when latex was collected in remote sampling sites, and latex storage and RNA isolation were conducted in poorly-equipped laboratories. Different methods (UV absorbance scans, denaturing gel electrophoresis, autoradiograph monitoring of cDNA synthesis) were used to confirm the high quality of the RNA prepared with this protocol, whose usefulness was further verified by several practical applications, including construction of one high-quality cDNA library, cloning of the full-length cDNAs of 3 novel Hevea sucrose transporter genes, and semi-quantitative RT-PCR analysis of two rubber-biosynthesis essential genes and one sucrose transporter gene.     

Xylem embolism and stomatal regulation in two rubber clones (<Emphasis Type="Italic">Hevea brasiliensis</Emphasis> Muell. Arg.)

Vulnerability to water-stress-induced embolism of stems, petioles, and leaf midribs was evaluated for two rubber clones (RRIM600 and RRIT251). The xylem conduits were relatively vulnerable to cavitation with 50% of embolism measured for xylem pressures between –1 and –2 MPa. This feature can be related to the tropical-humid origin of the species. A distinct basipetal gradient of vulnerability was found, leaf midribs being the least vulnerable. Substantial variation in vulnerability to cavitation was found between the two clones only at the petiole level. A correlation was found between the stomatal behavior and the development of cavitation. Stomata were nearly closed when the xylem pressure reached the point of xylem dysfunction. Stomata may thus contribute to controlling the risk of cavitation. However, for one clone a poor correlation was found between stomatal regulation and petiole vulnerability. This was consistent with a high degree of embolism measured in the petioles after a soil drought event. Therefore, xylem cavitation might represent a promising criterion to evaluate the performance of rubber clones under drought conditions.     

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.     

橡胶树种质资源遗传多样性研究——Ⅰ．速生种质遗传多样性RAPD分析

应用RAPD技术对8份野生种质和12份栽培种质进行遗传多样性分析,筛选到18个具有多态性扩增的引物．共扩增出128条带。据Nei-Li相似系数将20份材料分别聚为野生种质和栽培种质两大类。5个野生种质聚为野生种质类群,12个栽培种质和3个野生种质聚为栽培种质类群。研究结果表明,RAPD技术用于橡胶树种质资源研究,能够为野生种质优良特性导入栽培种质提供分子水平的参考依据。