抗除草剂草甘膦EPSPs基因在小麦中的转化

通过基因枪法,用抗除草剂草甘膦的ＥＰＳＰｓ基因转化小麦京花１号的幼穗约１０００个,及幼胚约８００个,经草甘膦选择后分别获得３８株和４株再生植株。这些再生植株经ＰＣＲ和（或）Ｓｏｕｔｈｅｒｎ杂交证明,其中有部分再生植株的基因组中稳定整合了外源ＥＰＳＰｓ基因,并且转化体中部分是可育的。首次用实验证明,抗除草剂草甘膦的ＥＰＳＰｓ基因作为单子叶禾谷类作物小麦基因转化的选择标记是行之有效的。     

小麦TaPSG719基因启动子的分离及序列分析

TaPSG719基因是从小麦中分离的花粉特异性表达基因,其功能未知.克隆和分析该基因的启动子有助于研究该基因的功能,解析小麦花器官的发育调控机制.本研究根据已报道的TaPSG719基因cDNA序列为基础设计引物,经过两次反向PCR获得了该基因起始密码子上游1776 bp的调控序列.应用PLACE和PlantCARE数据库系统对该序列进行分析研究,发现其具有启动子的基本元件TATA-box和CAAT-box、两种花粉特异性调控元件AGAAA和GTGA及光反应和激素响应元件.     

Free Polyamines in Senescing Wheat(Triticum aestivum L.)

The free polyamine content of flag leaves, peduncles, rachis,glumes, and grains of wheat (Triticum aestivum L., cv. Castell)plants, ripening under field conditions, has been investigatedduring three consecutive growing seasons. Putrescine was quantitativelythe most important of all polyamines detected in these organs.Concentrations were highest in the grains, glumes and flag leaves.No correlation was found between polyamine content and the onsetof senescence of flag leaves and other organs. Excised primaryleaves, however, showed a decrease in polyamine content in thedark and also in light/dark cycles, but in the latter case onlyafter an initial increase. Sink removal of otherwise intactwheat plants caused an accumulation of putrescine in flag leavesat the later stages of senescence, whereas removal of all otherleaves was without any significant effect. Putrescine was alsorecovered in phloem-exudate samples collected throughout theperiod of grain development. In both grains and glumes, peakconcentrations of polyamines were found early during seed development. Key words: Triticum aestivum, polyamines, ripening, senescence     

Agrobacterium-Mediated Gene Delivery in Various Tissues and Genotypes of Wheat (Triticum aestivum L.)

Agrobacterium-mediated wheat transformation has been attempted employing various bacterial strains, different tissues and explants, and different methods of co-cultivation. Of the various tissues/explants employed, i.e., leaf base, actively growing callus cells, mature seeds and seedlings punctured at the mesocotyl region, the mature seeds emerged as the most suited material for Agrobacterium-mediated gene delivery. Strain A281 of A. tumefaciens was found to be most effective followed by strains A348 and GV2260. Although the GUS activity in tissues/explants co-cultivated with strain GV2260 was relatively low, it indicated an efficient splicing of the intron (inserted in the coding region of the uidA gene). Various genotypes, namely, HD2329, Arjun and CPAN1676 were also screened for susceptibility against Agrobacterium strains and NPTII activity could be demonstrated in all of them, with minor variations.     

小麦幼苗期水分胁迫所诱导基因表达谱的初步分析

利用抑制差减杂交(Suppression Subtractive Hybridization,SSH)和高密度点阵膜技术研究小麦2叶幼苗期水分胁迫诱导表达基因。通过筛选具有1530个克隆的SSH文库,获得181个阳性克隆。序列同源性比较和功能查询结果发现,83．2％的水分胁迫诱导表达基因分别与不同逆境胁迫条件下表达的基因具有较高的同源性,这些基因在生物体内的功能都是直接或间接对细胞遭受逆境胁迫起保护作用。其中17个EST未找到同源性较高的匹配序列,已经在GenBank注册。用反向Northern、RT-PCR和Northern进一步检验所获得的功能已知EST,初步建立了小麦幼苗期水分胁迫诱导的基因表达谱。     

A Study of Floret Development in Wheat (Triticum aestivum L.)

Plants of wheat (Triticum aestlvum L.) cv. Aotea were grownat high or low nitrogen levels and in a natural photoperiodor continuous light. Starting 17–21 days from the double-ridgestage, eight plants from each treatment were sampled every 3days until anthesis, and the two basal, the sixth, and the terminalspikelets were sectioned longitudinally. A developmental scorewas assigned to each floret and rates of development calculated.Continuous light hastened development but reduced the numberof spikelets per ear, while high nitrogen delayed developmentbut increased spikelet numbers. The number of florets initiatedin each spikelet varied within narrow limits, but grain settingdepended strongly on spikelet position and on treatment. Althoughflorets were initiated in acropetal succession, the rate ofdevelopment tended to increase up to floret 4 but then declinedmarkedly. As a result grain setting was confined to basal floretpositions, although the two basal spikelets developed so slowlythat they contributed relatively little to grain yield. Distalflorets degenerated almost simultaneously at or before ear emergence,but those in intermediate positions continued to develop untilafter fertilization in the lower florets. It is argued thatthe spikelet is an integrated system in which correlative mechanismsplay a part throughout the development of the florets.     

Colchicine-induced Paracrystals in Root Cells of Wheat (Triticum aestivum L.)

Tubulin conformations other than microtubules in the meristematiccells of wheat roots grown in the presence of 2 mM colchicinesolution were investigated by immunofluorescence and electronmicroscopy. In the affected cells microtubules disappeared andwere replaced by tubulin fluorescent strands that occurred inthe cortical cytoplasm. With increasing time of exposure tocolchicine the tubulin strands became better organized and occurredalso in the subcortical cytoplasm and finally they were restrictedto the area around the nucleus. In prophase and preprophasecells thick strands occupied the cortical cytoplasmic zone wherein normal cells a preprophase microtubule band (PMB) was expectedto be assembled. In the colchicine-treated cells electron microscopy revealedan accumulation of paracrystalline aggregates, which initiallyoccurred along the cell wall and later deeper in the cytoplasm,in the perinuclear regions and the cytoplasmic invaginationsof the nucleus. In transverse planes the paracrystalline strandsappear to consist of hexagonal subunits in a 'honeycomb' arrangement,while in longitudinal and oblique sections they exhibit variableimages. Since their distribution coincides with that of thetubulin strands visualized by immunofluorescence, they are consideredto be the same structure. Therefore, the paracrystals consistof, or at least contain, tubulin. They are most likely to bepolymers of tubulin-colchicine complexes.Copyright 1995, 1999Academic Press Wheat roots, colchicine, immunofluorescence, electron microscopy, tubulin paracrystals, Triticum aestivum L     

Floret Initiation and Development in Spring Wheat (Triticum aestivum L.)

The number and developmental stages of florets were determinedin each spikelet of the spike in the main shoots of spring wheat.Samples were taken frequently from plants grown in a phytotronand in a nitrogen application field-test. Ten stages of development,from floret initiation until anthesis, were recognized and described. Inter-spikelet variation in the total number of initiated floretswas rather small. However, the number of florets at advancedstages of development, as well as the number of grains, washighest in the central spikelets in which florets initiatedfirst. Floret initiation did not proceed beyond spike emergence,whereafter the distal florets and the spikelet apex degenerated.Grain-set was restricted to florets which had developed at leastto the stage of visible anther lobes at spike emergence. Thenumber of these florets was increased significantly by nitrogenapplication. Wheat, Triticum aestivum L., spikelet, floret, grain set, nitrogen     

Phenotypic Analysis of a Dwarf Wheat (Triticum aestivum L.) with Altered Phytochrome-Mediated Growth Responses

The influence of the amount of red light relative to far red light (red/far red ratio) on leaf-sheath elongation, leaf length, tillering, assimilate partitioning to shoots and roots, and chlorophyll content in a dwarf wheat (Triticum aestivum L. cv Tibet Dwarf) and seven other dwarf and standard cultivars was determined. All cultivars tested showed far red-stimulated leaf and leaf-sheath elongation except Tibet Dwarf. Tibet Dwarf was also unresponsive to a brief end-of-day exposure to far red light, although the other cultivars exhibited increased leaf-sheath and leaf length. In these cultivars, the effects of an end-of-day 5-min far red exposure were reversible by a subsequent 5-min red light exposure. Shoot/root ratios were higher and tillering was suppressed by increased far red irradiance in all cultivars except Tibet Dwarf. In addition, Tibet Dwarf was less responsive to dark-induced leaf and leaf-sheath elongation (etiolation) and retained chlorophyll in dark-adapted leaves longer than control cultivars. Tibet Dwarf did not differ from the other cultivars in leaf chlorophyll content. Western blot analysis, using an antibody against phytochrome A, showed that dark-grown Tibet Dwarf shoots contained at least twice the amount of detectable phytochrome A protein present in the other wheat cultivars. These results, plus the short, thick-stemmed, dark-pigmented phenotype of Tibet Dwarf suggest the possibility of a mutation in the phytochrome/signal transduction pathway.     

不同处理条件下小麦胚芽鞘长的遗传分析

以小麦双单倍体(DH)群体(旱选10号×鲁麦14)的150个株系及其亲本为材料,对不同处理条件下的胚芽鞘长、幼苗株高和幼苗生物学产量进行遗传分析,以探讨胚芽鞘长、幼苗株高及幼苗生物学产量的遗传基础及其相关性.结果表明:(1)随着播种深度的增加,胚芽鞘长、幼苗株高显著增加,16.1%PEG-6000处理条件下的幼苗株高显著低于对照,干旱胁迫条件下的幼苗生物学产量显著低于正常供水对照;(2)胚芽鞘长、幼苗株高、幼苗生物学产量的遗传力分别为92.5%、90.3%和78.1%;(3)胚芽鞘长、幼苗株高及幼苗生物学产量均受多基因控制,决定各性状的基因估计数目分别为7、8和14对;(4)根据对性状偏度和峰度系数的估算结果,控制这3个性状的多基因之间均有互补作用;胚芽鞘长与幼苗株高、生物学产量及成熟期株高之间呈显著正相关.     

Expression of Phosphoinositide-Specific Phospholipase C Isoforms in Native Endothelial Cells

Phospholipase C (PLC) comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-β, γ, δ, ε, ζ and η) based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs) remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA), pulmonary (PA) and middle cerebral arteries (MCA). mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA), δ4 (only expressed in MCA), η1 (expressed in all but MA) and ζ (not detected in any vascular beds tested). The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (β3, γ2 and δ1) in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found in the native endothelium.     

Polyphosphoinositide Phospholipase C in Plasma Membranes of Wheat (Triticum aestivum L.) : Orientation of Active Site and Activation by Ca and Mg

Polyphosphoinositide-specific phospholipase C activity was present in plasma membranes isolated from different tissues of several higher plants. Phospholipase C activities against added phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP₂) were further characterized in plasma membrane fractions isolated from shoots and roots of dark-grown wheat (Triticum aestivum L. cv Drabant) seedlings. In right-side-out (70-80% apoplastic side out) plasma membrane vesicles, the activities were increased 3 to 5 times upon addition of 0.01 to 0.025% (w/v) sodium deoxycholate, whereas in fractions enriched in inside-out (70-80% cytoplasmic side out) vesicles, the activities were only slightly increased by detergent. Furthermore, the activities of inside-out vesicles in the absence of detergent were very close to those of right-side-out vesicles in the presence of optimal detergent concentration. This verifies the general assumption that polyphosphoinositide phospholipase C activity is located at the cytoplasmic surface of the plasma membrane. PIP and PIP₂ phospholipase C was dependent on Ca²⁺ with maximum activity at 10 to 100 μm free Ca²⁺ and half-maximal activation at 0.1 to 1 μm free Ca²⁺. In the presence of 10 μm Ca²⁺, 1 to 2 mm MgCl₂ or MgSO₄ further stimulated the enzyme activity. The other divalent chloride salts tested (1.5 mm Ba²⁺, Co²⁺, Cu²⁺, Mn²⁺, Ni²⁺, and Zn²⁺) inhibited the enzyme activity. The stimulatory effect by Mg²⁺ was observed also when 35 mm NaCl was included. Thus, the PIP and PIP₂ phospholipase C exhibited maximum in vitro activity at physiologically relevant ion concentrations. The plant plasma membrane also possessed a phospholipase C activity against phosphatidylinositol that was 40 times lower than that observed with PIP or PIP₂ as substrate. The phosphatidylinositol phospholipase C activity was dependent on Ca²⁺, with maximum activity at 1 mm CaCl₂, and could not be further stimulated by Mg²⁺.     

In Vitro Synthesis of Wheat (Triticum aestivum L.) Storage Proteins

Free and membrane-associated polysomes were isolated in approximately equal amounts from endosperm of wheat kernels harvested 20 days after anthesis. The presence of heparin in the homogenizing buffer minimized polysome degradation. Ribonucleic acid from the isolated polysomes, when translated in vitro in a wheat germ system, yielded products ranging in size from about 12,000 to about 80,000 daltons, including at least two polypeptides that co-migrated with seed extract proteins in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The nature of the translation products of free and membrane-associated RNA are distinctly different, with membrane-associated RNA yielding a higher proportion of polypeptides in the size range of 30,000 to 37,000 daltons. Analysis of membrane-associated 3′-terminal polyadenylyl-containing RNA in vitro translation products, by solubility in 70% ethanol and by immunoprecipitation, indicates that the 33,000- to 37,000-dalton polypeptides contain gliadins, and the analysis provides evidence that these proteins are synthesized in association with membranous cell organelles. Gliadin polypeptides synthesized in vitro are larger than authentic gliadins and probably are precursors which, in vivo, undergo modification to yield the smaller final products.     

小麦雌性育性的主基因+多基因混合遗传分析

选用普通小麦中3种不同生态型育性正常品种与雌性不育系XND126杂交构建3个F2组合, 连续两年对3组合P₁、P₂、F₁和F₂雌性育性进行调查, 利用植物数量性状主基因+多基因混合遗传模型的4世代联合分析方法分析了小麦雌性育性的遗传。结果表明: 小麦雌性育性受两对主效基因+多基因联合控制, 两对主效基因之间存在互作效应。     

小麦抗倒性研究进展

倒伏是严重影响小麦子粒产量和品质的一个重要因素。本文系统阐述了小麦茎秆形态和结构特性、茎秆化学成分与抗倒伏关系以及抗倒性的遗传和分子标记等方面的最新研究进展。株高、基部节间长度与抗倒性呈负相关;而基部节间粗度、秆壁厚、单位长度干重与抗倒性呈正相关。茎秆机械组织细胞层数、厚度,维管束数目、面积以及髓腔大小与抗倒性密切相关。茎秆化学成分中纤维素、木质素以及碳水化合物含量和硅、钾元素含量与抗倒性呈正相关。小麦抗倒性呈数量性状遗传特征,除受多对主基因控制外,可能还受微效修饰基因作用。采用分子标记技术已将抗倒性以及与抗倒性相关的茎秆形态性状进行了QTL定位。     

Effect of Water Deficit on Sporogenesis in Wheat (Triticum aestivum L.)

Plants of Triticum aestivum L., cv. Gabo, were grown in a glasshousefor 4 weeks and then transferred to a controlled environmentwith 20±1 °C temperature and 16 h photoperiod. Theywere subjected to water deficit by withholding the water supplyduring various stages of floral development, including thoseimmediately before meiosis and all stages until just after anthesis. The proportion of apparently normal florets which produced grainwas reduced when water deficit occurred during and immediatelyafter meiosis in the generative tissues. The effect of thisreduced grain set on total grain yield was partially compensatedby an increase in the weight of the remaining grains. Cross-pollinationbetween stressed and well-watered plants showed that grain setwas reduced as a direct consequence of the induction of malesterility by water stress, whereas female fertility was unaffected.A large proportion of the anthers on water-stressed plants weresmall and shrivelled, did not dehisce normally and containedpollen which was devoid of normal cytoplasmic constituents andshowed no staining reaction with triphenyl tetrazolium chloride.This effect on male fertility was not a result of desiccationof the sporogenous tissue, but an indirect outcome of the decreasein water potential elsewhere in the plant. Water stress, Triticum aestivum L., wheat, pollen, sporogenesis, grain set, male sterility     

Intracellular Localization of Peptide Hydrolases in Wheat (Triticum aestivum L.) Leaves

Protoplasts from 8- to 9-day-old wheat (Triticum aestivum L.) leaves were used to isolate organelles which were examined for their contents of peptide hydrolase enzymes and, in the case of vacuoles, other acid hydrolases. High yields of intact chloroplasts were obtained using both equilibrium density gradient centrifugation and velocity sedimentation centrifugation on sucrose-sorbitol gradients. Aminopeptidase activity was found to be distributed, in approximately equal proportions, between the chloroplasts and cytoplasm. Leucyltyrosine dipeptidase was mainly found in the cytoplasm, although about 27% was associated with the chloroplasts. Vacuoles shown to be free from Cellulysin contamination contained all of the protoplast carboxypeptidase and hemoglobin-degrading activities. The acid hydrolases, phosphodiesterase, acid phosphatase, α-mannosidase, and β-N-acetylglucosamidase were found in the vacuole to varying degrees, but no β-glucosidase was localized in the vacuole.     

Gibberellic Acid Regulates Cell Wall Extensibility in Wheat (Triticum aestivum L.)

Mutations (Rht genes) blocking sensitivity to gibberellic acid (GA) were used to examine phytohormone mediated cell wall expansion in wheat (Triticum aestivum L.). Irreversible extensibility of immature leaf segments, as determined by stress/strain (instron) measurements, declined with Rht gene dose. Exogenous GA₃ significantly increased wall extensibility in the nonmutant controls but had no effect on the near-isogenic GA-insensitive genotypes. Furthermore, ancymidol, an inhibitor of gibberellin biosynthesis, diminished wall extensibility in the nonmutant control. Extensibility of immature segments was highly correlated with mature leaf sheath length (R = +0.95). The results indicate that wall yielding properties of expanding wheat leaves are associated with leaf cell expansion potential and that GA is involved in the determination of those properties.