榆树转录组EST-SSR引物开发及遗传多样性分析

目前榆树已有的分子标记资源匮乏,无法满足榆树种质资源评价及分子标记辅助育种等相关研究需要。本研究对白榆叶片转录组数据进行EST-SSR标记的检测和开发,检测引物在不同榆树资源中的可用性,并对不同榆树资源的多样性进行分析。本研究共检测到8828个精确型和569个复合型SSR位点,SSR序列长度主要以10~22 bp的短序列为主。SSR重复单元比例最大的为A/T(3330,40.18%),其次为AG/CT(1211,14.61%)和AAG/CTT(568,6.85%)。随机挑选90对EST-SSR引物进行验证,有效扩增率为51.11%(46对),其中有63.04%的引物(29对)为高多态性引物,多态性信息含量PIC在0.054~0.683间变化,极大地丰富了榆树的SSR引物资源。聚类分析表明,绝大部分榆树无性系均按其起源聚类,从侧面也证明了本研究开发的EST-SSR引物的有效性。本研究开发的SSR引物可将绝大部分榆树资源进行区分,与传统分类学相吻合,为榆科植物的分类提供了分子依据。     

Amino Acid Composition of Elm Seeds

In the biosynthetic pathway of aromatic amino acids of Brevibacterium flavum, ratios of each biosynthetic flow at the chorismate branch point were calculated from the reaction velocities of anthranilate synthetase for tryptophan and chorismate mutase for phenylalanine and tyrosine at steady state concentrations of chorismate. When these aromatic amino acids were absent, the ratio was 61, showing an extremely preferential synthesis of tryptophan. The presence of tryptophan at 0.01 mM decreased the ratio to 0.07, showing a diversion of the preferential synthesis to phenylalanine and tyrosine. Complete recovery by glutamate of the ability to synthesize the Millon-positive substance in dialyzed cell extracts confirmed that tyrosine was synthesized via pretyrosine in this organism. Partially purified prephenate aminotransferase, the first enzyme in the tyrosine-specific branch, had a pH optimum of 8.0 and Km’s of 0.45 and 22 mM for prephenate and glutamate, respectively, and its activity was increased 15-fold by pyridoxal-5-phosphate. Neither its activity nor its synthesis was affected at all by the presence of the end product tyrosine or other aromatic amino acids. The ratio of each biosynthetic flow for tyrosine and phenylalanine at the prephenate branch point was calculated from the kinetic equations of prephenate aminotransferase and prephenate dehydratase, the first enzyme in the phenylalanine-specific branch. It showed that tyrosine was synthesized in preference to phenylalanine when phenylalanine and tyrosine were absent. Furthermore, this preferential synthesis was diverted to a balanced synthesis of phenylalanine and tyrosine through activation of prephenate dehydratase by the tyrosine thus synthesized. The feedback inhibition of prephenate dehydratase by phenylalanine was proposed to play a role in maintaining a balanced synthesis when supply of prephenate was decreased by feedback inhibition of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP*) synthetase, the common key enzyme. Overproduction of the end products in various regulatory mutants was also explained by these results.     

榆树叶片虫瘿形成过程中蛋白质组学分析

为探究榆树虫瘿叶片形成的分子机制,以不同时期榆瘿蚜取食诱导的榆树叶片为试材,利用iTRAQ技术分析榆树虫瘿叶片形成过程中蛋白表达丰度的差异。通过质谱鉴定,共得到2 689个蛋白,与KEEG数据库进行比对,发现2 145个蛋白被注释到126个不同的代谢通路中,和本研究相关的蛋白有12条,涉及代谢途径的蛋白数量最多,为813(37.9%)个。未被榆瘿蚜取食的叶片与榆瘿蚜取食诱导叶片形成虫瘿的前期、中期、后期相比,差异蛋白分别有418个、390个、244个,筛选出虫瘿发育的共有差异蛋白29个,这些蛋白在榆瘿蚜取食形成虫瘿的初始形成期和成长分化期持续发挥作用的有过氧化物酶、过氧化氢酶等氧化还原酶类;在前期持续发挥作用的有翻译控制肿瘤蛋白TCTP和肌动蛋白;在榆瘿蚜取食形成虫瘿的开裂期起到重要抗性作用的有溶质的抗坏血酸过氧化物酶、70 kD热激蛋白等。在榆树虫瘿叶片发展过程中,数个蛋白基因涉及了应激防御反应、氧化还原、免疫系统过程和光合作用等生理反应过程,建议进一步进行榆树虫瘿叶片形成的分子机制研究。     

Analysis of the Italian Dutch Elm Disease Fungal Population

Sixty‐two Ophiostoma ulmi sensu lato strains have been collected from symptomatic trees in seven areas of Central Italy. Isolates were compared with 10 reference strains, belonging to the species O. ulmi and to the two subspecies of O. novo‐ulmi, in order to establish the genetic variability within the Italian population of this fungal pathogen. The structure of the population has been analysed by means of morpho‐physiological features and of the direct amplification of minisatellite‐region DNA polymerase chain reaction (DAMD‐PCR) by using the M13 core sequence. The DNA profiles have been compared with taxonomic parameters (growth rate, culture aspect and fertility barriers). Taxa could thus be well separated. None of the isolates collected was recognized as O. ulmi. Isolates assigned to the two subspecies of O. novo‐ulmi (novo ulmi and americana) by means of the fertility test, showed short genetic distances with the respective reference strains and they constituted subgroups according to their geographical origin. The high level of variation detected indicates a postepidemic situation in Italy. Some inconsistency was found within the subspecies clusters. Several isolates, assigned to subspecies americana using fertility test, were in the novo‐ulmi cluster and vice versa. A possible explanation is that these isolates are americana–novo‐ulmi hybrids.     

Mechanisms Involved in Biological Control of Dutch Elm Disease

An effective suppression of Dutch elm disease symptom development was observed in elms resistant to the non-aggressive strain of Ophiostoma ulmi, but not to the aggressive strain, after a preventive treatment by this non-aggressive strain. Anequally effective isolate of Verticillium dabliae suppressed symptom development even in the very susceptible field elm (Ulmus carpinifolia). Induction of resistance is proposed for the major mechanism explaining these and earlier experiments. The primary stimuli initiating a resistance reaction are unknown, but the intermediates of the univalent reduction of oxygen may play a role. In an experiment the activities of two groups of enzymes involved in the catalytical scavenging of these radicals, superoxide dismutase and peroxidase, were monitored, but no correlation with induction of resistance was observed. Resistant elms showed the highest activities, probably reflecting a more effective defence against radicals than susceptible ones.     

Micropropagation and Regeneration of English Elm, Ulmus procera Salisbury

Rapidly elongating shoot tips from a clone of the English elm,Ulmus procera SR4, were taken in early summer and sterilizedby sodium hypochlorite treatment before transfer to three differentproliferation media. Proliferating shoot cultures readily establishedon Driver and Kuniyuki Walnut medium (DKW), but failed to establishon either Murashige and Skoog-based medium, or Woody Plant medium.On DKW medium 3–5 shoots were produced per 3 week subcultureperiod or up to 20 more shoots from the stem base callus, ifthis was subcultured separately. Excised leaves regeneratedshoots readily from the petiole region on standard DKW mediumafter 3–4 weeks, and this was unaffected by the antibioticcefotaxime, but prevented by concentrations of kanamycin above50 mg dm^–3. U. procera SR4, a well characterized clonaltree of known habit and high timber quality is, therefore, amenableto the procedures necessary for genetic manipulation. Key words: English elm, Ulmus procera, micropropagation, regeneration     

Identification of Elm Yellows Phytoplasma in Plum Trees in China

Plum plants (Prunus cerasifera Ehrh) with small and rolled leaves resembling symptoms of phytoplasma infection were observed during 2008 and 2009 in the ornamental garden of Northwest A&F University (Republic of China). Nested polymerase chain reaction (PCR) using a combination of phytoplasma‐specific universal primer pairs (R16F2m/R16R1m‐R16F2n/R16R2) amplified 16S rDNA with the expected size (1.2 kb) from all samples of symptomatic plum plants. Sequencing results and restriction fragment length polymorphism (RFLP) analysis of the 1248 bp R16F2n/R16R2 products showed that the phytoplasma belongs to group 16SrV. Phylogenetic analysis showed that the phytoplasma had a close relation to JWB phytoplasma. This is, we believe, the first report of elm yellows phytoplasma infecting plum plants in China.     

The spindle position checkpoint is coordinated by the Elm1 kinase

How dividing cells monitor the effective transmission of genomes during mitosis is poorly understood. Budding yeast use a signaling pathway known as the spindle position checkpoint (SPC) to ensure the arrival of one end of the mitotic spindle in the nascent daughter cell. An important question is how SPC activity is coordinated with mother-daughter polarity. We sought to identify factors at the bud neck, the junction between mother and bud, which contribute to checkpoint signaling. In this paper, we show that the protein kinase Elm1 is an obligate regulator of the SPC, and this function requires localization of Elm1 to the bud neck. Furthermore, we show that Elm1 promotes the activity of the checkpoint kinase Kin4. These findings reveal a novel function for Elm1 in the SPC and suggest how checkpoint activity may be linked to cellular organization.     

Microbial Decomposition of Elm and Oak Leaves in a Karst Aquifer

Dry Chinquapin oak (Quercus macrocarpa) and American elm (Ulmus americana) leaves were placed in four microcosms fed by groundwater springs to monitor changes in dry mass, ash-free dry mass, and microbial activity over a 35-day period. Oxygen microelectrodes were used to measure microbial activity and to estimate millimeter-scale heterogeneity in that activity. Oak leaves lost mass more slowly than elm leaves. Generally, there was a decrease in total dry weight over the first 14 days, after which total dry weight began to increase. However, there were consistent decreases in ash-free dry mass over the entire incubation period, suggesting that the material remaining after initial leaf decomposition trapped inorganic particles. Microbial activity was higher on elm leaves than on oak leaves, with peak activity occurring at 6 and 27 days, respectively. The level of oxygen saturation on the bottom surface of an elm leaf ranged between 0 and 75% within a 30-mm² area. This spatial heterogeneity in O₂ saturation disappeared when the water velocity increased from 0 to 6 cm s^-1. Our results suggest that as leaves enter the groundwater, they decompose and provide substrate for microorganisms. The rate of decomposition depends on leaf type, small-scale variations in microbial activity, water velocity, and the length of submersion time. During the initial stages of decomposition, anoxic microzones are formed that could potentially be important to the biogeochemistry of the otherwise oxic aquifer.     

Elm1 kinase activates the spindle position checkpoint kinase Kin4

Budding yeast asymmetric cell division relies upon the precise coordination of spindle orientation and cell cycle progression. The spindle position checkpoint (SPOC) is a surveillance mechanism that prevents cells with misoriented spindles from exiting mitosis. The cortical kinase Kin4 acts near the top of this network. How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established. Here, we show that the bud neck–associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4. Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment. These findings establish a novel function for Elm1 in the coordination of spindle positioning with cell cycle progression via its control of Kin4.     

Spatial Distribution and Site-Specific Spraying of Main Sucking Pests of Elm Trees

Elm trees are important landscape trees and sucking insects weaken the elm trees and produce large amounts of honeydew. The main objectives of this study were to identify main honeydew-producing pests of elm trees and do site-specific spraying against these pests. To map the spatial distribution of the sucking pests in the large scale, the study area was divided into 40?×?40 m grids and one tree was chosen randomly from each grid (a total of 55 trees). These trees were sampled twice a year in 2011 and 2012. Each sample was a 30-cm branch terminal. Eight samples were taken from each tree in four cardinal directions and two canopy levels. The number of sucking insects and leaves of each sample were counted and recorded. Spatial analysis of the data was carried out using geostatistics. Kriging was used for producing prediction maps. Insecticide application was restricted to the regions with populations higher than threshold. To identify within-tree distribution of the honeydew-producing pests, six and four elm trees were chosen in 2011 and 2012 respectively, and sampled weekly. These trees were sampled as described previously. European elm scale (EES), Gossyparia spuria (Modeer) and two species of aphids were the dominant honeydew-producing pests. The results revealed that the effects of direction, canopy level and their interactions on insect populations were not statistically significant (P?<?0.05). Site-specific spraying decreased the amount of insecticides used by ca. 20%, while satisfactory control of the sucking pests and honeydew excretion was obtained. Considering the environmental and economic benefits of site-specific spraying, it is worth doing more complementary works in this area.     

Ri-plasmid mediated transformation and regeneration of Ulmus procera (English Elm)

Infection of Ulmus procera (English elm) cloneSR4 internodal stem explants with Agrobacteriumtumefaciens C58 c1 pRiA4b resulted in callusdevelopment and extensive hairy root production. Shoots which regenerated from hairy roots, followingan extended culture period, were dwarf in stature,with reduced apical dominance and wrinkled leaves whencompared with wild type U. procera SR4. Shootswere rooted successfully and plants with extensiveroot systems have been transferred to soil. Thetransgenic status of regenerants was confirmed by PCRanalysis and DNA sequencing of pRiA4b TL- and TR- DNArolA (329 bp) and agropine synthase (490 bp)primed amplimers, which were 100% homologous to theexpected sequences. No vir D1 primed PCRproducts were obtained, indicating that the Agrobacterium was successfully removed. Thepotential of Ri plasmid mediated transformation forinducing altered elm xylem structure, restrictedspread of the Dutch elm disease fungus and inphytoremediation is discussed.     

Elm bark beetle boring and feeding deterrents from Phomopsis oblonga

The two groups of Phomopsis oblonga which invade the phloem of stressed elm trees have been investigated for the production in vitro of boring/feeding deterrents for elm bark beetles. The secondary metabolites of this fungus are compared with those of closely-related Phomopsis spp. associated with ash and sycamore. Active compounds isolated from P. oblonga include a novel norsesquiterpene γ-lactone, the tiglic esters of two novel 5,6-dihydro-5-hydroxy-2-pyrones, nectriapyrone, 4-hydroxyphenylethanol, 5-methylmellein, 2-furoic, orsellinic and 3-nitropropanoic acids and mellein-5-carboxylic acid; portensterol and thymine, from P. oblonga, were inactive. (+)-Mellein and furan-2,5-dicarboxylic acid were obtained as minor metabolites of the ash and sycamore Phomopsis strains, which also produced the norsesquiterpene γ-lactone and one of the tiglic esters.     

榆树叶片形成虫瘿过程中转录组学分析

榆瘿蚜取食侵染榆树叶片形成了榆树虫瘿,本研究采用新一代的高通量Illumina Hi Seq^TM 2000技术测序平台对榆瘿蚜取食刺激的榆树叶片进行转录组测序和功能注释,利用生物学方法对基因表达和功能进行研究。测序获得23.19 Gb碱基序列信息,通过对测序数据进行序列过滤、拼接和去冗余,共获得102 017个Unigenes,通过NR与BLAST等数据库比对,其中有37 899个(37.15%) Unigense被注释。利用KOG、GO、KEGG等数据库对榆树虫瘿叶片的Unigense进行比对,按功其能将匹配的Unigenes基因划分25大类;GO注释将信息归纳为基因的3大主类,57个亚类;以KEGG数据库为参考,将Unigene定位到110个不同的代谢通路,包括氧化应激防御、植物激素信号转导、碳水化合物以及次生物代谢等代谢相关的Unigenes通路。本研究通过二代高通量转录组测序技术研究榆瘿蚜侵染下榆树虫瘿的相关基因,为今后研究榆瘿蚜侵染榆树叶片形成虫瘿的分子机理提供了基础资料。     

Dendrosoter protuberans (Hymenoptera: Braconidae), An Important Elm Bark Beetle Parasitoid

The most favourable period for Dendrosoter protuberans development occurred when wasps were introduced 16 days after the beginning of Scolytus multistriatus development (the greatest number of emerged wasps per a single parasitoid female with a very positive sexual index and high percentage of parasitised elm bark beetle larvae). No significant differences were observed in parasitoid efficiency between the 11^th and the 21^st day. The most unfavourable period for elm bark beetle parasitisation was six days after the beginning of S. multistriatus development (a low reproduction of D. protuberans, a negative sexual index and a low number of destroyed S. multistriatus larvae). The reproduction of both the elm bark beetle and D. protuberans was greater in the first, than in the second generation. The developmental period of D. protuberans in the first generation was approximately two days longer than in the second generation. Furthermore, the development of females was 1-2 days longer than the development of males. D. protuberans developed most successfully on S. multistriatus (the most abundant parasitoid reproduction with the longest developmental period and a very positive sexual index that resulted in a high percentage of parasitised larvae) and then on S. ensifer, S. pygmaeus and Pteleobius kraatzi. On the other hand, S. scolytus larvae were the poorest host of all elm species - the reproduction of D. protuberans in this species was the scarcest with the shortest period of development and a negative sexual index, hence larval parasitisation was the lowest.     

The checkpoint kinase Hsl1p is activated by Elm1p-dependent phosphorylation

Saccharomyces cerevisiae cells growing in the outdoor environment must adapt to sudden changes in temperature and other variables. Many such changes trigger stress responses that delay bud emergence until the cells can adapt. In such circumstances, the morphogenesis checkpoint delays mitosis until a bud has been formed. Mitotic delay is due to the Wee1 family mitotic inhibitor Swe1p, whose degradation is linked to bud emergence by the checkpoint kinase Hsl1p. Hsl1p is concentrated at the mother-bud neck through association with septin filaments, and it was reported that Hsl1p activation involved relief of autoinhibition in response to septin interaction. Here we challenge the previous identification of an autoinhibitory domain and show instead that Hsl1p activation involves the phosphorylation of threonine 273, promoted by the septin-associated kinase Elm1p. We identified elm1 mutants in a screen for defects in Swe1p degradation and show that a phosphomimic T273E mutation in HSL1 bypasses the need for Elm1p in this pathway.     

Elm defence against herbivores and pathogens: morphological,chemical and molecular regulation aspects

Elms (Ulmus spp.) have long been appreciated for their environmental tolerance, landscape and ornamental value, and the quality of their wood. Although elm trees are extremely hardy against abiotic stresses such as wind and pollution, they are susceptible to attacks of biotic stressors. Over 100 phytopathogens and invertebrate pests are associated with elms: fungi, bacteria and insects like beetles and moths, and to a lesser extent aphids, mites, viruses and nematodes. While the biology of the pathogen and insect vector of the Dutch elm disease has been intensively studied, less attention has been paid so far to the defence mechanisms of elms to other biotic stressors. This review highlights knowledge of direct and indirect elm defences against biotic stressors focusing on morphological, chemical and gene regulation aspects. First, we report how morphological defence mechanisms via barrier formation and vessel occlusion prevent colonisation and spread of wood- and bark-inhabiting fungi and bacteria. Second, we outline how secondary metabolites such as terpenoids (volatile terpenoids, mansonones and triterpenoids) and phenolics (lignans, coumarins, flavonoids) in leaves and bark are involved in constitutive and induced chemical defence mechanisms of elms. Third, we address knowledge on how the molecular regulation of elm defence is orchestrated through the interaction of a huge variety of stress- and defence-related genes. We conclude by pointing to the gaps of knowledge on the chemical and molecular mechanisms of elm defence against pest insects and diseases. An in-depth understanding of defence mechanisms of elms will support the development of sustainable integrated management of pests and diseases attacking elms.