Activity of Gibberellins A1 to A0 in the Avena First-leaf Bioassay, and Location after Chromatography

Activity curves are determined for gibberellins A₁ to A₀ bythe Avena first-leaf bioassay method. Gibberellins A¹, A₄ andA₅ can be detected at 10^-11 or 10^-10 g/ml and give optimum activityof approximately 230 per cent elongation (water controls = 100per cent). Gibberellins A²A³, and A⁹ can be detected at 10^-3g/mland give optimum activity of approximately 200 per cent. GibberellinsA₆ and A₇ can be detected at 10^-5g/ml; GA₇ gives optimum activityof around 190 per cent. All the gibberellins except GA₈ canbe detected by this bioassay method after chromatography inn-butanol: 1.5 N ammonia (3: 1) and benzene: acetic acid: water(4: 2: 1) when applied to the paper at concentrations from O.Ito µg. The sensitivity of the method is compared withthat of other gibberellin bioassay methods.     

Cytokinins in Gladiolus (Gladiolus grandiflorus) Corms

Ten cytokinin-like substances, termed X₁, X₂, X₃, X_4a, X_4b,X_5a, X_5b, X₆, X₇, and X₈, active in the soya bean hypocotyltest, were detected in gladiolus corms. The factors X_4a, X_5aand X₄ were tentatively identified as zeatin (Z), isopentenyladenosine (iPA) and isopentenyl adenine (iP), respectively,based on their behaviour in Sephadex LH-20 column chromatography,paper chromatography and high pressure liquid chromatography.Factor X₃, which behaved like zeatin riboside (ZR) in the abovesystems could be ZR and/or dihydrozeatin riboside (DHZR). Thebehaviour in Sephadex and ion-exchange column chromatographysuggested that X₁ and X₂ may be cytokinin glucosides and X₅a cytokinin nucleotide or a cytokinin conjugate similar to lupinicacid. The total cytokinin content and the concentration of Z,ZR/DHZR and iPA were higher in non-dormant than in dormant corms.The concentrations of X₁ and X₂ were higher in dormant corms. Gladiolus grandiflorus, gladiolus, dormancy, cytokinins, Sephadex column chromatography, high pressure liquid chromatography     

Identification and Semi-Quantification of Gibberellins from the Pollen and Anthers of Zea mays by Immunoassay and GC/MS

Radioimmunoassays and enzyme-linked immunosorbent assays formethyl esters of gibberellins A₁, A₃, A₄, and A₇ were establishedusing an antiserum specific for GA₁-Me. The antiserum was characterizedby high titer and specificity for such C₁₉-GAs with 3ß-hydroxylgroup as GA₁, GA₃, GA₄ and GA₇. Combination of this antiserumand HPLC enabled us to identify and quantify GA, and GA₄ fromthe pollen of Zea mays with a high degree of reliability. Similarly,identification and quantification of GA₉ and GA₂₀ were alsomade possible by use of an antiserum specific for GA₂₀-Me. Combineduse of immunoassays and GC/MS enabled us to identify nine GAsfrom the pollen and four from the anthers of Zea mays. The identificationof non-13-hydroxylated GAs, such as GA₄ and GA₉, in additionto 13-hydroxylated GAs from the pollen and the anthers suggeststhat the early-non-hydroxylation pathway, as well as the early-13-hydrox-ylationpathway, operates in the male reproductive organs of Zea mays,and that the organ-specific biosynthesis and/or localizationof GAs in Zea mays is similar to that in Oryza saliva. (Received May 7, 1990; Accepted August 20, 1990)     

Interactions of the Growth Retardants Daminozide and Piproctanyl Bromide, and Gibberellins A1, A3, A4+7, A5 and A13 in Stem Extension and Inflorescence Development in Chrysanthemum morifolium Ramat

Interactions between the growth retardants daminozide (a substitutedsuccinamic acid) or piproctanyl bromide (a quaternary ammonium,piperidinium compound), and a subsequent application of a singledose (40 µg) of either gibberellin A₁, A₃, A₄₊₇ or A₁₂,showed that, in Chrysanthemum morifolium Ramat. cv. Bright GoldenAnne, a strong interdependence exists between elongation ofthe lateral shoot and the rate of development of its terminalinflorescence. A₁, A₃, and A₄₊₇ were highly active in overcoming the restrictionson both internode extension and the rate of flower-bud developmentimposed by either retardant, suggesting that these two retardanteffects are caused by a deficiency of active gibberellins (GN).In the absence of retardant, A₁, A₃, and A₄₊₇ markedly increasedstem elongation, and flowering occurred earlier than in plantsreceiving neither retardant nor GN. A₁₃ the only 20-carbon GNtested, was much less active, while A₅ had a relatively greatereffect on the time of flowering than on shoot elongation. Thus,it is not necessarily the rate of stem extension which determinesthe rate of inflorescence development. The response to different amounts of A₁, A₃ or A₁₃ (1, 5, 10,20, or 50 µg per shoot) neither suggest that different‘threshold’ levels of a particular GN are requiredto induce increases in either stem elongation or in the rateat which inflorescences develop, nor did a change in the dosegiven lead to any consistent differential effect on these twoprocesses. Chrysanthemum morifolium Ramat., stem extension, inflorescence development, growth retardants, gibberellins     

Effect of GA3, GA4$7, GA5 and GA9 on the sex expression of Luffa acutangula var. H-2

Gibberellins A₃, A_4$7, A₅, and A₉ increase the number of malebuds and flowers, but inhibit the opening of female flowersand shift their position to a higher node in Luffa acutangula.GA₃ is the most effective followed by GA_4$7, GA₅ and GA₉. (Received November 19, 1971; )     

A Modified Micro-Drop Bioassay Using Dwarf Rice for Detection of Femtomol Quantities of Gibberellins

The sensitivity of the micro-drop assay with dwarf rice (Oryzasativa L., cv. Tan-ginbozu and cv. Waito-Q to gibberellins (GAs)was increased conspicuously by the use of assay plants thathas been treated with uniconazole (S-3307), an inhibitor ofthe biosynthesis of GAs. The Tan-ginbozu plants treated withS-3307 responded to 10 fmol/plant of GA₃ (ca. 3.5 pg/plant)and to 30 fmol/plant of gibberellins A₁, A₄, A₇, A₁₉ and A₂₀.Waito-C plants treated with S-3307 responded to 10 fmol of GA₃and to 30 fmol/plant of gibberellins A₁, A₄ and A₇. GibberellinsA₉, A₁₉ and A₂₀ had much less of an effect on the treated Waito-Cplants than did gibberellins A₁, A₃, A₄ and A₇. Furthermore,treatment with S-3307 counteracted the inhibition of growthof both cultivars by abscisic acid. Thus, the modified micro-dropassay should prove very useful for the detection of minute amountsof GAs in plant extracts. (Received October 3, 1988; Accepted March 29, 1989)     

Photosynthetic Characteristics of C3-C4 Intermediate Flaveria Species II. Kinetic Properties of Phosphoenolpyruvate Carboxylase from C3, C4 and C3-C4 Intermediate Species

The kinetic properties of phosphoenolpyruvate (PEP) carboxylasehave been studied among several Flaveria species: the C₃ speciesF. cronquistii, the C₃–C₄ species F. pubescens and F.linearis, and the C₄ species F. trinervia. At either pH 7 or8, the maximum activities (in µmol.mg Chl^–1.h^–1)for F. pubescens and linearis (187–513) were intermediateto those of the C₃ species (12–19) and the C₄ species(2,182–2,627). The response curves of velocity versusPEP concentration were hyperbolic for the C₃ and C₃–C₄species at either pH 7 or 8 while they were sigmoidal for theC₄ species at pH 7 and hyperbolic at pH 8. The K_m values forPEP determined from reciprocal plots were lowest in the C₃ species,and of intermediate value in the C₃–C₄ species comparedto the K' values of the C₄ species determined from Hill plotsat either pH 7 or 8. Glucose-6-phosphate (G6P) decreased theK_m values for PEP at both pH 7 and 8 in the C₃ and C₃–C₄species. In the C₄ species, G6P decreased the K' values at pH8 but increased the K' values at pH 7. In all cases, G6P hadits effect by influencing the activity at limiting PEP concentrationswith little or no effect on the maximum activity. At pH 8 andlimiting concentrations of PEP the degree of stimulation ofthe activity by G6P was greatest in the C₄ species, intermediatein F. linearis, a C₃–C₄ species, and lowest in the C₃species. In several respects, the PEP carboxylases of the C₃–C₄Flaveria species have properties intermediate to those of theC₃ and C₄ species. (Received April 30, 1983; Accepted August 22, 1983)     

Comparative Potency of Nine Gibberellins

Gibberellins A₁ to A₉ have been compared, each at several doselevels, in bioassays based on extension of stems of dwarf gardenpea (Pisum sativum), dwarf bean (Phaseolus vulgaris) and Lunariaannua, of hypocotyls of cucumber (Cucumis sativus) and lettuce(Lactuca sativa), and of leaf sheaths of three dwarf mutants(d–1, d–3, d–5) of maize (Zea mays). GibberellinsA₃ (gibberellic acid) and A₇ are of high potency in most bioassays.A₈ is of negligible potency in all and is probably not a functionalhormone. The other gibberellins show a more or less marked tendencyto specificity. The plants used as bioassay material also differin the specificity of their response. Some, for example, maizedwarfs d–3 and d–5 and lettuce, respond well tomost gibberellins; others, for example, cucumber, respond onlyto a few; extreme specificity is shown by Lunaria annua which,in the unvernalized condition, responds by stem elongation onlyto gibberellin A₇. Dose/response curves of the various gibberellinsare usually parallel, but certain exceptions to this have beenfound. Possible explanations of specificity are discussed inrelation to the results obtained, and it is concluded that insufficientevidence is available to make it possible to draw any validconclusions. Current definitions of gibberellins, whether basedon chemical structure or biological activity, are unsatisfactory.     

Induction of HIV-1 resistance: cell susceptibility to infection is an inverse function of globotriaosyl ceramide levels

To examine the role of the glycosphingolipid (GSL), globotriaosylceramide(Gb₃, CD77, p^k blood group antigen) in HIV-1 infection, we havepharmacologically modulated Gb₃ metabolism in an X4 HIV-1 infectablemonocytic cell line (THP-1) that naturally expresses Gb₃ andin a Gb₃-expressing glioblastoma cell line (U87) transfectedto express both CD4 and CCR5 to permit R5 HIV-1 infection. THP-1and U87 cells were treated with either a competitive inhibitorof -galactosidase A, 1-deoxygalactonojirimycin (DGJ) to induceGb₃ accumulation, or a glucosylceramide synthase inhibitor,phenyl-2-palmitylamino-3-pyrrolidino-1-propanol (P4) to depletecells of Gb₃. HIV susceptibility was determined via measurementof p24^gag antigen production by ELISA. In addition, total cellularGb₃ content was determined using thin layer chromatography followedby Verotoxin1 overlay binding. The cell surface expression ofGb₃ was verified by FACS analysis. We found that DGJ significantlydecreased THP-1 and U87 cell susceptibility to HIV-1_IIIB andHIV-1_BaL infection, respectively, at a concentration of approximately100 µM. In contrast, P4 (2 µM) substantiallyincreased cellular susceptibility to HIV-1 infection. Totalcellular GSL analysis verified increased Gb₃ expression in cellstreated with DGJ and considerable reduction of Gb₃ in P4-treatedcells as compared to controls. These results show a reciprocalrelationship between Gb₃ expression and infection with eitherX4 HIV-1_IIIB or R5 HIV-1_Ba-L. These results support previousstudies that Gb₃ provides resistance to HIV infection. VariableGb₃ expression may provide a natural HIV resistance factor inthe general population, and pharmacological manipulation ofGb₃ levels may provide an approach to induction of HIV resistance.     

ERRATA

Please replace the paragraph Culture of material in Materialand method on page 574 (Fujii, Shimmen and Tazawa), Vol. 19,No. 4 with the following corrected paragraph. Materials and methods Culture of material Spirogyra sp. used for the experiments was collected in theriver Kamogawa in Kyoto. Cylindrical cells composing the filamentwere 55 µm in diameter and 100–200 µm in length.Each cell usually had one spiral ribbon-like chloroplast. Thealga was cultured in slightly modified Reichardt's medium (27),1000 ml of which contained: 200 mg KNO₃, 20 mg K₂HPO₄, 10mgH₃BO₃, 6.6 mg FeSO₄?7H₂O, 25 mg Na₂EDTA (ethylenediamine tetraaceticacid disodium salt), 200 mg NaHCO₃, 50 mg CaSO₄?2H₂O, 10mg MgSO₄?7H₂O,0.5 mg ZnSO₄?7H₂O, 5mg MnCl₂?4H₂O, 24 µg Na₂MoO₄, 2 µgCoCl₂?6H₂O, 500 mg Tris. The pH was adjusted to 7.4 with HCl.The alga was cultured in a Petri dish at 20?1?C under a 16 hr–8hr light-dark regime. The light intensity was about 2000 lux.Under such conditions, the cells divided once a day fairly synchronously. Experimental solutions Artificial pond water (APW) containing 0.1 mM each of KCl, NaCland CaCl₂     

Integrative Neurophysiology of the Lobster Cardiac Ganglion

The synchronized bursts of impulses produced by the nine neuronsof the isolated Homarus cardiac ganglion are usually initiatedby Cell 7. Activity in all other cells commences with very shortlatency thereafter. Impulses in most cells originate in triggerzones located 1–2 mm from the cell body, but the firstseveral impulses in Cells 8 and 9 frequently originate in distaltrigger zones some distance from the somata. Large cells fireat a high initial frequency, dropping rapidly to a low frequencyplateau. Small cells exhibit a more tonic behavior and fireat intermediate rates. More anterior small cells tend to firefaster than more posterior ones. The major synaptic interactionsare the impulse-mediated excitatory ones from small cells tolarge cells, and possibly to more anterior small cells. Thereare weak interactions from large cells back onto small cells,and very specific interactions from Cells 1 and 2 onto 3_A, 4_A,5_A, and 3_B 4_B 5_B respectively. The large discrete EPSPs generatedin large cells by small cell impulses appear to be the explanationfor "discrete positioning" in large-cell firing patterns. Inthis situation, large-cell impulses only fire at discrete timesduring the burst, regardless of the actual large-cell pattern. The overall view is of a two-layered neural system in whichthe small cells possess an endogenous oscillatory driver potential,synchronized by synaptic and electrotonic interactions, anddriving a train of impulses in each cell. This activates excitatorysynapses on the large cells, which combined with a triggereddriver potential in each large cell, produces synchronized trainsof motor impulses which activate the heart muscle, causing theheartbeat.     

ATP activates DNA synthesis by acting on P2X receptors in human osteoblast-like MG-63 cells

In human osteoblast-like MG-63cells, extracellular ATP increased [³H]thymidineincorporation and cell proliferation and synergistically enhancedplatelet-derived growth factor- or insulin-like growth factor I-induced[³H]thymidine incorporation. ATP-induced[³H]thymidine incorporation was mimicked by thenonhydrolyzable ATP analogs adenosine5'-O-(3-thiotriphosphate) and adenosine 5'-adenylylimidodiphosphate and was inhibited by the P2purinoceptor antagonist suramin, suggesting involvement of P2purinoceptors. The P2Y receptor agonist UTP and UDP and a P2Y receptorantagonist reactive blue 2 did not affect [³H]thymidineincorporation, whereas the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4-disulfonic acid inhibited ATP-induced[³H]thymidine incorporation, suggesting that ATP-inducedDNA synthesis was mediated by P2X receptors. RT-PCR analysis revealedthat MG-63 cells expressed P2X₄, P2X₅,P2X₆, and P2X₇, but not P2X₁,P2X₂, and P2X₃, receptors. In fura 2-loadedcells, not only ATP, but also UTP, increased intracellularCa²⁺ concentration, and inhibitors for severalCa²⁺-activated protein kinases had no effect on ATP-inducedDNA synthesis, suggesting that an increase in intracellularCa²⁺ concentration is not indispensable for ATP-induced DNAsynthesis. ATP increased mitogen-activated protein kinase activity in aCa²⁺-independent manner and synergistically enhancedplatelet-derived growth factor- or insulin-like growth factor I-inducedkinase activity. Furthermore, the mitogen-activated protein kinasekinase inhibitor PD-98059 totally abolished ATP-induced DNA synthesis. We conclude that ATP increases DNA synthesis and enhances the proliferative effects of growth factors through P2X receptors byactivating a mitogen-activated protein kinase pathway.

     

A2B receptor activation promotes glycogen synthesis in astrocytes through modulation of gene expression

Adenosinehas been proposed as a key factor regulating the metabolic balancebetween energy supply and demand in the central nervous system. Becauseastrocytes represent an important cellular element in the control ofbrain energy metabolism, we investigated whether adenosine could inducelong-term changes of glycogen levels in primary cultures of mousecortical astrocytes. We observed that adenosine increased glycogencontent, up to 300%, in a time- (maximum at 8 h) andconcentration-dependent manner with an EC₅₀ of 9.69 µM.Pharmacological experiments using the broad-spectrum agonist5'-(N-ethylcarboxamido)adenosine (NECA) and specificagonists for the A₁, A_2A, and A₃receptors [N⁶-cyclopentyladenosine (CPA),CGS-21680, and IB-MECA, respectively] suggest that the effect ofadenosine is mediated through activation of the low-affinityA_2B adenosine receptor subtype. Interestingly, adenosineinduces in parallel the expression of the protein targeting to glycogen(PTG), one of the protein phosphatase-1 glycogen-targeting subunitsthat has been implicated in the control of glycogen levels in varioustissues. These results indicate that adenosine can exert long-termcontrol over glycogen levels in astrocytes and might therefore play asignificant role in physiological and/or pathological processesinvolving long-term modulation of brain energy metabolism.

     

Betacyanin-Decolorizing Enzymes from Phytolacca americana

Betacyanin-decolorizing enzymes (BDEs) in Phytolacca americanawere partially purified and characterized. Acidic enzymes, BDE₁and BDE₂, and basic enzymes, BDE₃ and BDE₄, were partially purifiedby column chromatography on DEAE-Sepharose CL-6B and CM-SepharoseCL-6B. These enzymes degraded betanin to its aglycon (betanidin)and then to a product with a peak of absorbance at 485 nm. Theoptimum pH values were 5.0 for BDE₁, 3.5 for BDE₂, and 5.5 forBDE₃ and BDE₄. BDE activity was detected in extracts of theleaf, stem, seed, and root. The highest specific activity ofBDE₂ was found in the red epidermis of the stem. The activityof BDE₂ was inhibited by NH₄NO₃, KNO₃, KC1, and NaCl, but theactivities of the other BDEs were not inhibited. These resultssuggest that BDEs other than BDE₂ control the degradation ofbetacyanin in the intracellular space. (Received March 27, 1989; Accepted December 8, 1989)     

Gibberellin-Induced Flowering in Cordyline (Agavaceae)

Normal, terminal inflorescences of Cordyline terminalis, a woodymonocotyledon, appeared 4–6 weeks after apical buds weretreated with gibberellin A₃ (GA₃) or GA₄₊₇. There was no responseto GA₁₃. Large plants, newly rooted cuttings, and seedlingsall responded, although there were clonal differences. Floweringwas induced under natural day lengths throughout the year. Untreatedcontrol plants in all experiments remained vegetative. Dracaenaspp. did not respond to the three gibberellins.     

UBER DIE WIRKUNG DES GIBBERELLINS AUF DIE BLUTENBILDUNG VON PHARBITIS NILCHOIS

Die Blütenbildung der Pharbitis-Pñanzenwird durchBehandlung der Keimlingen mit Gibberellin vor der Dunkelperiodegefördert, und die Zufuhr auf dem Vegetationspunkt istebenso wirksam wie die auf Kotyledonen. Die Wirkung tritt bei der Zufuhr unmittelbar vor der Dunkelperiodebis zu 2 Stunden nach Anfang derselben am stärksten auf,ist aber sogar bei der Zufuhr unmittelbar nach der Dunkelperiodedeutlich bemerkbar. Die Blütenanlage kann in ununterbrochener Beleuchtung mitschwachem Licht ausgebildet werden; die kritische Intensitätwird durch Gibberellin deutlich gesteigert. Die Blühhemmung von Indolylessigsäure wird durch Gibberellinteilweise aufgehoben. Die Wirkungsreihe der einzelnen Gibberelline in der Strek-kungsförderungist A_s = A₁> A₄ = A₂. In derselben Reihe steht die Blühförderungdurch niedrige Konzentration (1 mg/l), bei höherer Konzentration(100mg/l)ist sie in der Reihe A₂=A₄>A₃ = A3. Mittlere Konzentration(100mg/l)zeigt eine Übergangsreihe zwischen den beiden. (Received April 21, 1961; )     

Metabolism and Translocation of Gibberellins in Seedlings of Pharbitis nil. (I) Effect of Photoperiod on Stem Elongation and Endogenous Gibberellins in Cotyledons and Their Phloem Exudates

Gibberellin A₁, (GA₁), GA₁₉, and GA₂₀ in phloem exudates andcotyledons of seedlings of Pharbitis nil cv. Violet, grown underdifferent photoperiodic conditions, were qualitatively and semi-quantitativelyanalyzed by a combination of high performance-liquid chromatography(HPLC) and radioimmunoassays (RIA). The levels of GA₁₉ and GA₂₀were higher in cotyledons from plants grown under dark treatment(DT) conditons of 16 h-light/8 h-dark for 6 days followed by8 h-light/16 h-dark for 3 days than in those grown under continuouslight (CL) for 9 days. This relationship was also observed forthe GAs in phloem exudates, although the levels were much lowerthan in the cotyledons. When GAs were applied to the cotyledons,elongation of the epicotyl was promoted more by GA₂₀ than byGA₁ or GA₁₉, especially under the CL treatment. The relativeeffect of GA₁ and GA₂₀ on the epicotyl elongation was reversedwhen these GAs were applied to epicotyls pre-treated with prohexadione,an inhibitor of 2-oxoglutarate-dependent dioxygenases. ³Present address: Frontier Research Program, The Institute ofPhysical and Chemical Research (RIKEN), 2-1 Hirosawa, Wakoshi,Saitama, 351-01 Japan ⁴Present address: Laboratory of Horticulture, Faculty of Agriculture,Nagoya University, Nagoya, 464-01 Japan     

An Analysis of Growth of Oil Palms under Nursery Conditions: II. The Effect of Spacing and Season on Growth

Three experiments on the growth of watered nursery oil palmsare described, the results of which provide estimates of seasonalvariation in net assimilation rate (E_A) and relative growth-rate(R_w) in the tropics (6° 33' N.). The range of values obtained for E_A and R_w is similar to thatfound with seedlings and during early growth in the nursery(E_A = o.I8–o.32 g/dm²/week, R_w= o.84–I.70 per cent/day)and there is very little effect of season on E_A; such variationas exists appears to be related to solar radiation. A spacing experiment indicated that E_A is independent of leafarea index (L) when L is below about 2.2, but that above thislevel E_A decreases with increasing L, falling to zero at L =5.4. The crop growth-rate (C) is maximal when L is between 2.5and 3, the maximum value observed was o.62 g/dm²/week (equivalentto 3.22 x10⁴ kg/ha/annum). These results are compared with other estimates of growth andassimilation rates of seedling, nursery and adult oil palms,and are discussed in relation to the efficiency of energy fixation,and apparent growth-rates.     

REGULATION OF BUD REST IN TUBERS OF POTATO SOLANUM TUBEROSUM L.: I. EFFECT OF GROWTH SUBSTANCES ON EXCISED POTATO BUDS

Excised plugs containing buds from potato tubers were treatedwith 5-µliter droplets of a number of growth-regulatingsubstances. Gibberellin A₃ stimulated sprouting over a widerange of concentrations. Gibberellins A₃, A₄, A₅, and A₇ stimulatedsprouting, and A₆, A₈, and A₉ either had no effect or slightlyinhibited. Extracts of gibberellinlike substances from potatopeelings promoted sprouting. NAA and IAA both promoted sproutingslightly at low concentrations (4 x 10^–8M) but inhibitedsprouting at 4 x 10^–5M. Leaching of plugs resulted indelayed sprouting, and gibberellin restored total sproutingpotential. Plug size influenced rate of sprouting: small plugs(8 mm in diameter) sprouted faster than large (23 mm) plugs,and gibberellin stimulated sprouting slightly faster in thelarger than in the 8 mm plugs. None of the presumed componentsof ß, including cinnamic, chlorogenic, and caffeicacids, and coumarin, convincingly inhibited sprouting; in fact,they stimulated sprouting at almost all concentrations tested.5- Fluorouracil (5-FU) inhibited sprouting only slightly; andgibberellin completely or partially promoted sprouting in plugspreviously treated with 5-FU. Failure of 5-FU to inhibit sproutingwas considered to be the result of slow penetration of the inhibitorinto the potato bud. The potato "eye" bioassay is deficient in certain aspects, especiallyin view of the inconsistent rates of sprouting between experimentsand of nonspecificity. The results of this study, however, donot obviate the use of potato buds as a bioassay for inhibitorsof sprouting. ¹This research was supported in part by United States PublicHealth Service Grant EF-61. ²Present address: Department of Botany, Hebrew University, Jerusalem,Israel. ³Present address: U.S.D.A.-A.R.S., Department of Agronomy, Universityof California, Davis, California, U.S.A.     

洛氏脊漠甲抗冻蛋白基因的克隆、表达和功能检测

为了研究抗冻蛋白基因是否在新疆荒漠昆虫洛氏脊漠甲 Pterocoma loczyi 中存在,利用 RT-PCR 技术克隆获得了洛氏脊漠甲抗冻蛋白的 cDNA 片段,命名为 Plafp743。测序结果表明洛氏脊漠甲 Plafp743 所编码的蛋白质由 94 个氨基酸组成,蛋白序列呈现规则结构 CTX₁X₂X₃X₄CX₅X₆X₇X₈X ₉。利用原核表达载体构建重组质粒 pGEX-4T-1-Plafp743,转化 Escherichia coli BL21 进行融合蛋白表达,SDS-PAGE 结果表明抗冻蛋白PLAFP基因以可溶性融合蛋白形式表达,相对分子质量 36 kD。用 Glutathione Sepharose 4B 亲和柱对表达蛋白进行纯化后,通过赤翅甲 Dendroides canadensis 抗冻蛋白的小鼠抗血清进行 Western blot 分析,结果表明纯化的 GST-PLAFP 融合蛋白与赤翅甲抗血清能够发生特异性的免疫反应。大肠杆菌的低温抗冻保护实验结果证明,30 μg/mL 的 GST-PLAFP 在-6℃ 对细菌具有显著的保护作用,且随着抗冻蛋白浓度的增加,抗冻保护作用的效 果也随之增加。本研究首次报道了从荒漠昆虫洛氏脊漠甲扩增得到抗冻蛋白基因,提示抗冻蛋白可能是荒漠昆虫普遍采取的过冬生存策略,为进一步开发利用昆虫抗冻蛋白奠定了基础。