在小鼠早期胚胎发育和种系中特异表达的转录因子OCT4

在小鼠早期胚胎发育和种系中特异表达的转录因子ＯＣＴ４童英尚克刚（北京大学生命科学学院,北京１００８７１）ＯＣＴ４：ａＴｒａｎｓｃｒｉｐｔｉｏｎＦａｃｔｏｒＥｘｐｒｅｓｅｄＳｐｅｃｉｆｉｃａｌｙｉｎＭｏｕｓｅＥａｒｌｙＤｅｖｅｌｏｐｍｅｎｔａｎｄＧｅｒ...     

A Spectrophotometric Analysis of Pigments in Apples

Methods of pigment extraction using traditional polar organic solvents (acetone or methanol) were compared to those employing a chloroform–methanol mixture. We found that, for spectrophotometric pigment analysis in the apple peel, the cuticular lipids must be preliminarily extracted from the samples with chloroform and MgO must be added during homogenization to prevent pigment degradation. The traditional extraction did not result in the complete extraction of intact pigments, and such extracts contained a considerable amount of light-absorbing impurities. The application of chloroform–methanol extraction allowed us to markedly reduce the content of such impurities and to increase the accuracy and sensitivity of the measurement of the content of chlorophylls and carotenoids. In addition, this extraction method proved useful for the analysis of phenolic substances (anthocyanins and flavonoids) in the water-methanol fraction of the extracts.     

Endogenous Gibberellins in the Xylem Exudate from Apple Trees

In the xylem exudate extracted from the current-year stems of apple (Malus domestica Borkh.), gibberellins A₁₅, A₁₇, A₁₈, A₁₉, A₂₃, A₄₄, and A₅₃ were identified, and 16,17-dihydro-17-hydroxy GA₁₉ was presumed from full-scan mass spectra and Kovats retention indices.     

Conditioning promotes regeneration and transformation in apple leaf explants

The conditioning of apple shoots for several days in an appropriate liquid medium enhances the regenerative capacity of leaf explants derived from the shoots, so that adventitious buds form in high frequency. The use of conditioning enables the transformation and rapid recovery of plants from otherwise recalcitrant cultivars without the need for an extended callus phase. Conditioning has a wide range of effects on the leaf cells, including increasing the density of the cytoplasm and the complexity of vacuoles, and increasing the porosity of the cell walls from of the order of 3.5 nm to 5.5 nm. The increased porosity may aid the insertion of T-DNA through the cell wall. Initial expression of introduced genes, as judged by the histochemical assay of the β-glucuronidase gene, occurs within 2 days of inoculation with Agrobacterium, usually in groups of 2–20 cells, termed foci. The foci are most commonly composed of an intensely expressing core cell with one or more surrounding layers of less intensely expressing cells. Explants from conditioned leaves contain at least three times as many foci as the control explants. It is concluded that conditioning of apple shoots promotes the recovery of transformed plants from leaf explants by two processes: increasing the number of cells containing and expressing the introduced genes, and by increasing the probability that cells will regenerate directly to shoots. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phacidiopycnis washingtonensis,Cause of a New Storage Rot of Apples in Northern Europe

Phacidiopycnis washingtonensis was identified by morphology and ITS sequence analysis as the cause of rubbery rot, a new storage disease of apples in northern Germany. Infected fruits had an unusually firm texture and pale appearance after storage in ultra‐low oxygen conditions, but turned dark brown to black in ambient atmosphere. Ultimately, the surface of rotted fruits became covered by black pycnidia producing cream‐coloured conidial exudates. Rubbery rot affected several apple varieties, including the commercially important ‘Jonagold’ and ‘Elstar’. Losses during storage were commonly below 1% but reached 5–10% in a few cases. Fruits of ‘Golden Hornet’ crab apple trees planted as pollinators in commercial orchards became heavily infected by P. washingtonensis in October. Conidia were released throughout the following season from infected fruit mummies, which remained attached to the crab apple tree.     

Investigation of Agrobacterium-mediated transformation of apple using green fluorescent protein: high transient expression and low stable transformation suggest that factors other than T-DNA transfer are rate-limiting

To investigate early events of Agrobacterium-mediated transformation of apple cultivars, a synthetic green fluorescent protein gene (SGFP) was used as a highly sensitive, vital reporter gene. Leaf explants from four apple cultivars (Delicious, Golden Delicious, Royal Gala and Greensleeves) were infected with Agrobacterium EHA101 harboring plasmid pDM96.0501. Fluorescence microscopy indicated that SGFP expression was first detected 48 h after infection and quantitative analysis revealed a high T-DNA transfer rate. Plant cells with stably incorporated T-DNA exhibited cell division and developed transgenic calli, followed by formation of transgenic shoots at low frequencies. The detection of SGFP expression with an epifluorescence stereomicroscope confirmed the effectiveness of SGFP as a reporter gene for detection of very early transformation events and for screening of putative transformants. The efficiency of the transformation and regeneration process decreased ca. 10000-fold from Agrobacterium infection to transgenic shoot regeneration, suggesting that factors other than Agrobacterium interaction and T-DNA transfer are rate-limiting steps in Agrobacterium-mediated transformation of apple.     

Controlling plant growth via the gibberellin biosynthesis system – I. Growth parameter alterations in apple seedlings

'York Imperial' apple seedlings ( Malus domestica Borkh.) were continuously supplied via the roots with paclobutrazol [(2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol)], a triazole GA biosynthesis inhibitor, at 0.68 μ M in a nutrient solution. In comparison to controls, seedlings treated with paclobutrazol for 66 days showed a 91% reduction in shoot length, a 66% reduction in leaf area but only a 17% reduction in leaf number. This effect could be reversed by GA₃ applied to the foliage at 71.4 μ M 0, 19 or 35 days after paclobutrazol was initially supplied and leaf area values for paclobutrazol-treated seedlings given both treatments did not differ significantly from controls. Plots of growth data indicate linearity of shoot longitudinal growth of GA₃-treated seedlings. Leaf area increase was non-linear after GA₃ treatment up to approximately 30 days, when the rate dropped. On a per shoot basis, leaf weight closely followed leaf area but on a per unit area basis, paclobutrazol-treated leaves were heavier than controls; GA₃ applications temporarily reversed this trend.     

Calcium ion transport across discs of the cortical flesh of apple fruit in relation to fruit development

Transport of Ca²⁺ through discs of apple fruit tissue was examined in tissue taken at different stages of fruit development. Transport rates decreased with fruit development when cation exchange was the predominant influence on transport (with 10⁻⁶ M ⁴⁵CaCl₂ as the source solution). This decrease was associated with a reduction in relative cell wall surface area, cation exchange capacity and cell wall yield that occurred during fruit growth. When diffusion was the major transport force, and when transport was influenced by solution infiltration of the tissue disc (10⁻² M ⁴⁵CaCl₂ in the source solution), transport rates increased during fruit growth. This increment was related to increases in air space of the tissue. Ca²⁺ transport through apple fruit tissue is influenced by the extent and nature of the cell wall, changing proportions of air space and Ca²⁺ concentration in the extracellular solution.     

高温对苹果花粉在花柱内萌发和生长的影响

以‘长富2号’红富士苹果为母本,‘红星’苹果为父本,研究35℃高温对苹果花粉在花柱内萌发和生长的影响。结果表明：温度影响花粉在柱头和花柱内的萌发及生长,与对照相比,高温促进花粉在柱头表面快速萌发,并加速花粉管在花柱内的伸长生长,但是在授粉72hN,高温处理下培养的花粉管形态出现花粉管变粗、弯曲并有瘤状小结的异化现象,同时,花柱发生褐变,并产生胼胝质,最终导致花粉管不能进入胚珠完成受精。     

Measurement of intracellular and extracellular free calcium in apple fruit cells using calcium-selective microelectrodes

Abstract. Intracellular and extracellular free calcium was measured in cortical cells from apple fruit ( Malus domestica Borkh cv. Cox's Orange Pippin) using calcium-selective microelectrodes. It was difficult to position microelectrodes in the cytoplasm, and thus most intracellular measurements reflected vacuolar concentrations of about 0.6mol m⁻³ free calcium. Extracellular free calcium was measured in wells (0.2mm³) that had been cut through the epidermis of the fruit, then filled with osmoticum. Concentrations of free calcium in the sampling well varied between 0.02 and 1.3 mol m⁻³, and were related to the calcium content of the tissue. Functioning of the plasma membrane, and perhaps the supply of calcium for intracellular needs during fruit ripening and storage, may require mol m⁻³ concentrations of extracellular free calcium. Thus, it is suggested that low levels of extracellular free calcium in calcium-deficient fruit may be partly responsible for development of deficiency disorders.     

Effect of thidiazuron on abscisic acid content in apple bud relative to dormancy

The effect of N -phenyl- N '-1,2,3,-thidiazol-5-ylurea (thidiazuron, Dropp, SN 49537) on abscisic acid (ABA) level in apple ( Malus domestica Borkh. cv. York Imperial) buds associated with bud break and bud development was determined. The data showed that increased ABA content in apple buds was associated with thidiazuron-induced bud break and bud development. ABA stimulated growth of apple buds that had emerged from dormancy by thidiazuron treatment. The ABA in apple buds was confirmed by GC-EIMS and GC-CIMS.     

Drought tolerance of apple rootstocks: Production and partitioning of dry matter

The drought tolerance of the commercial apple ( Malus domestica Borkh.) rootstocks M9, M26, M27 and MM111, and some new selections from the rootstock breeding programme at HRI-East Malling (AR69-7, AR295-6, AR360-19, AR486-1 and AR628-2), was assessed using potted, glasshouse-grown, unworked rootstocks. After an initial period of growth under well-watered conditions the amount of irrigation was gradually reduced, for some treatments, to simulate natural drying in the soil. At the end of a six-month growth period, the rootstocks were harvested and the production of dry matter and its partitioning to various plant parts determined. The rootstocks exhibited large differences in shoot and root dry matter, and root length but not all the rootstocks showed declines in root mass or length in response to the droughting treatment. The dwarfing rootstocks tended to have smaller amounts of both coarse (>2 mm diameter) and fine roots (<2 mm diameter), than the more vigorous rootstocks. Irrespective of rootstock or irrigation treatment there was a close linear relationship between coarse and fine root. There was also no change in the length/weight relationship for fine roots irrespective of rootstock or irrigation treatment, i.e. 42 m of fine root weighed 1 g dry weight. In some cases the amount of root produced could be directly correlated with the rootstock known potential to control scion vigour, but this was not true for all the rootstocks examined. The absence of this relationship was particularly evident in some of the new selections of rootstock. The possible causes for these differences, compared with commercially used rootstocks, is discussed in relation to the origin and parentage of the rootstock selections. Despite this lack of a root length/vigour relationship, the amount of dry matter partitioned to shoot growth reflected the rootstocks' known vigour. The different responses of these rootstocks to drought are discussed along with their implications for understanding the mechanisms by which rootstocks are thought to dwarf scion shoots.     

Abscisic acid in dormant apple seed tissues - a rapid purification scheme using pre-packed columns and GCMS-SIM quantitation

A rapid small scale procedure has been developed for quantitative abscisic acid (ABA) determination by gas chromatography - mass spectrometry with selected ion monitoping (GCMS-SIM). Extracts of apple seeds ( Malus domestica cv. Northern Spy) were passed first through 3 ml C₁₈ columns, and then through 3 ml silica gel columns. GCMS-SIM quantitation of ABA was done by adding an internal standard, hexadeuterated ABA. Ten sample extracts could be purified and analyzed by GCMS in 5 to 6 h, offering a quick and precise method to laboratories equipped with GC-MS instrumentation. The endosperm membrane and seed coat of apple seeds contained 2.5-3 times the ABA concentration found in the embryonic axis and cotyledons, supporting the hypothesis that it may be the ABA content of the membrane and seed coat that explains their inhibitory qualities with respect to seed germination.     

不同水分条件下盆栽苹果树蒸腾速率动态模拟

土壤水分是制约作物产量和品质的主要环境因素之一,估算不同水分条件下的蒸腾速率（r3对于作物的优质高产和节水灌溉等具有十分重要的意义。通过构建蒸腾．气孔．光合耦合模型可模拟出不同水分条件下苹果树的蒸腾动态,模型参数根据逐步干旱条件下盆栽‘富士’苹果树试验获取。结果表明,Tr主要由饱合水气压差和气孔导度（G。）驱动,同时气象因子和土壤水势对其有强烈的交互作用影响。Tr随土壤水势的下降而减小,当土壤水势低于-0．4MPa时减小幅度更加显著。晴天时,G。在一天中呈双峰曲线,而一呈单峰曲线,最大值出现在13：00左右,约为3．6mmol·m-Ls。根据该模型可计算出不同水分条件下1株盆栽苹果树（总叶片积为O．26m-2）全天的蒸腾总量,供水充足时为652．1g,严重干旱时（土壤水势为-1．5MPa）为85．4g。实测值和模拟值的比较表明,该耦合模型能够模拟出不同土壤水分条件下盆栽苹果树的蒸腾动态以及土壤的含水量。     

Changes in jasmonic acid concentration during early development of apple fruit

Apple fruits ( Malus domestica Borkh.) were harvested from 24 to 136 days after full bloom (DAFB) and endogenous jasmonic acid was analyzed by GC-MS. There were two isomers of jasmonic acid in apple fruit with a ratio of 37:63 ( cis:trans ). The cis:trans ratio remained relatively constant throughout this period of fruit development. The endogenous jasmonic acid concentration was 138 ng g⁻¹ fresh weight 24 DAFB and decreased as fruit developed. Changes in jasmonic acid concentration were coincident with those of respiration, ethylene production, and anthocyanin accumulation in patterns consistent with the reported responses to exogenous jasmonates. Possible roles for jasmonic acid during early fruit development are discussed.     

Calcium regulation of exogenous and endogenous 1-aminocylopropane-1-carboxylic acid bioconversion to ethylene

Ethylene production and overall levels of free and conjugated 1-aminocyclopropane-1-carboxylic acid (ACC) were studied in parenchymatous tissues, excised from clmacteric apples ( Malus domestica Borkh. cv. Granny Smith) and infiltrated with an incubation medium containing 0, 1, 10 or 100 m M Ca²⁺, with or without exogenous ACC (2 m M ). Irrespective of whether exogenous ACC was applied or not, ethylene production was inhibited to the same extent (40%) by an apoplastic effect of 100 m M Ca²⁺. In the absence of external ACC, the inhibition was associated with an increase in total endogenous ACC and may be related to a reduction in the rate of the last step of ethylene pathway. This suggests that the ethylene-forming enzyme (EFE) is localized in the plasma membrane. Low Ca²⁺ concentrations (1 m M ) enhanced basal ethylene synthesis due to influx of Ca²⁺ into the cytosol, while overall concentrations of ACC in the tissue decreased. However, 1 m M Ca²⁺ did not stimulate ACC-dependent ethylene formation. Thus, Ca²⁺ influx may stimulate the translocation of endogenous ACC from synthesis or storage compartment (s) to reactive site(s) of the plasma membrane. The concentration of 10 m M Ca²⁺ had no effect on basal ethylene production and appears to represent a balance point between the stimulating and inhibiting effects of 1 and 100 m M Ca²⁺, respectively, Furthermore, the charge-times of exogenous ACC observed with 0, 1 and 10 m M Ca²⁺ suggest that EFE is located on the inner side of the plasma membrane.     

Monitoring seasonal variation in apple fruit volatile emissions in situ using solid-phase microextraction

Emissions of volatiles from apple fruits (Malus domestica Borkh.) were monitored in situ over the course of a growing season (from early June to mid September) for two apple varieties, Golden Delicious and Maigold. Results indicate a characteristic time-course of volatile emissions as the sampling date was a statistically significant factor for nine of the 13 compounds considered. The amounts of volatiles collected were greatest early and late in the season. The temporal effect on emissions was generally much larger than the effect of variety, which was significant for only four of the 13 compounds considered. The possible sources of variation which are not explained by the statistical models are discussed, and it is considered that they are most likely related to differences in the emissions from individual fruits.