黄瓜子叶扩张与细胞分裂素的关系

BA(benzyl adenine)专一性地促进离体黄瓜子叶的扩张。为了研究 BA 的作用机理,我们采用间接 EIISA 和 HPLC 的方法测定了子叶扩张过程中内源玉米素(Z)和玉米素核苷(ZR)含量的变化。离体黄瓜(Cucumis sativus,津研4号)子叶用10mg/l 的 BA 培养,72小时之后,处理子叶鲜重的增加比对照高70%。Z+ZR 在 BA 处理的子叶中有大量的积累。结果表明 BA 可能诱发了黄瓜子叶中的细胞分裂素生物合成和代谢的某些基因。     

拟南芥中4种细胞分裂素的高效液相色谱法测定

应用高效液相色谱法同时测定拟南芥中4种细胞分裂素组分玉米素(Z)、玉米素核苷(ZR)、6-r,r-二甲基烯丙基氨基嘌呤(2ip)和6-r,r-二甲基烯丙基氨基嘌呤核苷(2ipr)含量。结果表明,采用反相色谱柱Waters C₁₈柱(4.6×250 mm,5 μm),在35℃以乙腈和三乙胺缓冲液为流动相梯度洗脱,流速1 ml/min,在270 nm处能准确检测出拟南芥中4种细胞分裂素组分的含量,检测限达0.001 μg/ml。     

黄瓜子叶扩张与细胞分裂素的关系

BA(benzyl adenine)专一性地促进离体黄瓜子叶的扩张。为了研究BA的作用机理,我们采用间接EIISA和HPLC的方法测定了子叶扩张过程中内源玉米素 (Z) 和玉米素核苷(ZR)含量的变化。离体黄瓜(Cucumis sativus,津研4号)子叶用100mg／1的BA培养,72小时之后,处理子叶鲜重的增加比对照高70％。Z+ZR在BA处理的子叶中有大量的积累。结果表明BA可能诱发了黄瓜子叶中的细胞分裂素生物合成和代谢的某些基因。     

肌苷酶电极生物传感器

为了构建肌苷酶电极生物传感器,以固定化核苷磷酸化酶(EC 2.4.2.1)、黄嘌呤氧化酶(EC 1.2.3.2)与过氧化氢电极组成电流型酶电极生物传感器,用于检测肌苷片中的肌苷,其输出电流可达500nA.结果发现,肌苷测定的线性范围为1-268 mg/L,精度:RSD小于0.14%,响应时间:60 s,使用寿命大于25 d,实际测定肌苷片中肌苷含量回收率:100.8%.由此表明:采用双酶电极法测定肌苷片中的肌苷含量,由于酶促反应专一性高、样品不需分离直接进样分析、处理条件温和、反应时间短暂因而结果较为可靠.     

抗玉米素核苷的单克隆抗体制备及内源玉米素核苷的酶联免疫吸附测定

通过二次成功的细胞融合,共获得三株分泌抗玉米素核苷(ZR)单克隆抗体的稳定杂交瘤系F_2E_6、F_3G_6和F_1B_9。其中F_2E_6分泌的单抗属IgG_1、F_3G_6、F_1B_9分泌的单抗均属IgM。以F_2E_6、F_3G_6单抗为基础的ELISA对ZR的检测灵敏度为0.05pmol(—18pg),线性检测范围为0.05—50pmol。除玉米素(Z)外,F_2E_6、F_3G_6单抗与IPA、6-BA、KT和腺苷几种ZR类似物的交叉反应值都小于0.18％。用ELISA测定F_2E_6单抗与ZR反应的亲和常数为2.36±0.99×10~(-8)M。本文还报道了包埋抗原的ELISA在植物内源细胞分裂素测定中的应用,并用此方法测定了黄化玉米(Zea mays L.)幼苗中ZR的含量。     

伤流液中玉米素的高效液相色谱测定

在植物体内,天然存在的细胞分裂素(Cytokinin)主要是玉米素和与玉米素化学结构相似的化合物6-(r.r-二甲基烯丙基氨基)—嘌呤。尤以玉米素活性最强,含量最多。其活性大约是细胞分裂     

气液色谱法定量测定植物细胞分裂素

自从Most发明了三甲基硅烷制备衍生物(以下简称TMS)方法以来,国外有许多利用该法测定细胞分裂素的报告。Horgan利用TMS衍生化方法,检测出枫树韧皮部中的玉米素核苷;Upper等用GLC配FID检测出5ng的CTK-TMS衍生物。目前,国外对细胞分裂素作定性分析测定经常采用该衍生化方法,但用于定量测     

亚种间杂交稻根系伤流液与地上部细胞分裂素的变化

本文以水稻亚种间杂交组合Ⅱ优2070及恢复系2070、Ⅱ优419及恢复系中419为材料．应用HPLC和ELISA法测定灌浆期间根系伤流液中根源细胞分裂素(CTKs)种类和数量以及稻叶和籽粒中细胞分裂素组分含量的变化,研究表明在亚种杂交稻及其恢复系灌浆起始阶段,玉米素(Z) 占总CTKs的比例高达62．8％-89．1％,是根系伤流液中细胞分裂素的主要成分,而二氢玉米素(di- HZ)和二氢玉米素核苷(diHZR)则在灌浆后期明显上升．这种变化动态与Ⅱ优2070和2070剑叶中ZRs(Z ZR)、diHZRs(diHZ diHZR)的波动变化是相符合的。与根系伤流液的主要细胞分裂素组成不同,IPAs在叶片和籽粒中占总CTKs量的比例最高。说明在灌浆期间,根系伤流液中CTKs的种类及其活性存在着变化,这种变化可能与细胞分裂素代谢酶活性变化及其相关酶基因表达的不同有关。讨论了强、弱势粒的CTKs组分的变化与籽粒结实的可能关系。     

皇冠草体细胞胚胎发生及其体胚发生过程中内源激素的变化

皇冠草是一种具有重要经济价值的大型观赏水草。以其幼叶为外植体,在MS培养基上,短期内(25d),直接诱导获得了体细胞胚。对不同的细胞分裂素及其不同组合的体胚诱导效应进行测试,发现：6-苄氨基嘌呤(6-BA,1mg/L)和玉米素(Zt,1mg/L)结合使用效果最好,诱导频率达100%,平均每个外植体可形成4.87个体细胞胚。当在含有Zt(1mg/L)的MS培养基中加入萘乙酸(NAA 0.5mg/L)时,外植体不能形成体细胞胚,而是形成大量的根。在含有吲哚乙酸(IAA,1mg/L)的MS培养基上,成熟的体胚5d后即可发育成完整的小植株。72%的小植株被成功地移栽到了水族箱中。采用HPLC法,对自外植体培养之日起到体胚发生和成熟的各个时期的内源激素含量进行了测定。发现各种内源激素的含量均不断增加,10d后达到对照的2倍左右。IAA含量在培养第10天时达到第一个峰值,第二个峰值出现在25d后;玉米素和玉米素核苷(Zt+ZR)含量在15d后达到最大值(为对照的8倍多),然后逐渐下降;赤霉素(GA-3)的变化趋势与细胞分裂素基本相同,但到体胚萌发时,其含量已恢复到对照水平;培养10d后的脱落酸(ABA)含量一直保持到了体胚成熟期。     

细胞分裂素的混合抗体型免疫亲和柱的制备与应用

内源细胞分裂素可以分成三组：异戊烯基腺苷组（ｉＰＡｓ）、玉米素核苷组（ＺＲｓ）、二氢玉米素核苷组（ＤＨＺＲｓ）。从这三组分别选ｉＰＡ、ＺＲ、ＤＨＺＲ为半抗原合成免疫原,获得的三种免抗血清基本上只识别相应组的细胞分裂素。将经初纯化的三种抗血清偶联到ＣＮＢｒ活化的Ｓｅｐｈａｒｏｓｅ４Ｂ上制成免疫亲和柱。在其中的一根柱床高２．１ｃｍ、体积３．５５ｍｌ、直径１．５ｃｍ的亲和柱上,在流速１．５ｍｌ／ｍｉｎ、８０％冷甲醇为洗脱剂的条件下测得该柱容量为３．６—４．０μｇ、回收率达９３．９％～９８．３％。用该柱对丝瓜茎木质部伤流中细胞分裂素进行了纯化．表明能够用此柱对植物粗提液中的细胞分裂素进行快速分离纯化。     

Radioimmunoassays for trans-zeatin and related cytokinins

Radioimmunoassays for the quantitation of trans-zeatin and related cytokinins have been developed. Antisera produced against bovine serum albumin conjugates of trans-zeatinriboside have a high affinity (K_a=2.4.10^-11 M) for zeatinriboside and for zeatin, but show a negligible cross reaction to isopentenyladenosine (0.1%) and cis-zeatinriboside (0.4%), only a slight cross reaction to dihydrozeatin (1.7%), and no cross reaction at all to other purines, such as adenosine and related, compounds, was observed. The assays are sensitive and measuring ranges extend from 0.06–30 pmol (0.02–10 ng) of zeatinriboside. This has been achieved by employing as tracers immunoreactive zeatin derivatives with high-specific activity, (tritiated zeatinriboside-dialcohol: 8.37.10¹¹ Bq mmol^-1 and zeatinribosyl-[¹²⁵I]tyramine: ca. 1.9.10¹³ Bq mmol^-1. The detection limit is 40 fmol (15pg) for the assay employing the tritiated tracer, and assay reproducibility is high (variation coefficients of triplicates less than 5%). Several hundred assays can be completed in one day, and, due to the high specificity of this assay, crude extracts may be used for analysis. The course of zeatin levels in developing fruits of Lycopersicon esculentum cv. Moneymaker is given.Abbreviations GC gas chromatography - LC liquid chromatography - MS mass spectroscopy - RIA radioimmunoassay - TLC thin layer chromatography - UB unspecific binding Part 12 in the Series: Use of Immunoassay in Plant Science     

Cytokinins in pathogenesis and disease resistance of Pyrenophora teres-barley and Dreschslera maydis-maize interactions during early stages of infection

Infection of Hordeum vulgare L. by Pyrenophora teres and of Zea mays by Dreschslera maydis were characterized by 'green island' formation, higher cytokinin levels and accumulation of metabolites in the infected areas. Higher cytokinin concentrations of the order 6-Y,Y-dimethylallylaminopurine > zeatinriboside > zeatin > dihydrozeatinriboside were detected at infection sites of susceptible hosts. By virtue of these cytokinins, infection sites may be acting as metabolic sinks helping proliferation of the pathogen. Existence of translocatory sinks at infection zones was confirmed from autoradiographic studies, where, accumulation of labeled metabolites was prominent at infection sites of susceptible hosts. Upon infection the lower cytokinin levels of resistant hosts decreased further with progress of infection. In the infected resistant hosts the concentrations of zeatin/zeatinriboside were the maximum among the four identified cytokinins. The pathogen is also capable of secreting cytokinins as evident from quantification of cytokinins in culture filtrate extracts using HPLC. Since detached leaves were used in the experiments the increase/decrease of various cytokinin levels may be attributed to pathogen influence. The increase in cytokinin levels in the susceptible host may be aiding the growth of the pathogen on one hand, while the decrease in the infected resistant host may signal the host to activate defenses against a potential pathogen at the early stage of infection.     

Occurrence of plant hormones in cells of the phototrophic purple bacterium Rhodospirillum rubrum 1R

Abstract Plant hormones from biomass of the purple non-sulfur bacterium Rhodospiririllum rubrum were isolated for the first time. These compounds show high physiological activities (300–330%) in the cytokinin bioassay. All three detected cytokinins are adenine derivatives, according to spectral analysis. One of them was identified as 6-(4-hydroxy-3-methyl-2-trans-2-bytenylamino)-9-ß-D-ribofuranosylpurine (zeatinriboside) as shown by thin-layer chromatography and reversed-phase high-performance liquid chromatography. The possible functions of bacterial cytokinins are also discussed.     

Cytokinins in pathogenesis and disease resistance of Pyrenophora teres-barley and Dreschslera maydis-maize interactions during early stages of infection

Infection of Hordeum vulgare L. by Pyrenophora teresand of Zea mays by Dreschslera maydis were characterized by green island formation, higher cytokinin levels and accumulation of metabolites in the infected areas. Higher cytokinin concentrations of the order 6-Y,Y-dimethylallylaminopurine > zeatinriboside > zeatin >dihydrozeatinriboside were detected at infection sites of susceptible hosts. By virtue of these cytokinins, infection sites may be acting as metabolic sinks helping proliferation of the pathogen. Existence of translocatory sinks at infection zones was confirmed from autoradiographic studies,where, accumulation of labeled metabolites was prominent at infection sites of susceptible hosts. Upon infection the lower cytokinin levels of resistant hosts decreased further with progress of infection. In the infected resistant hosts the concentrations of zeatin/zeatinriboside were the maximum among the four identified cytokinins. The pathogen is also capable of secreting cytokinins as evident from quantification of cytokinins in culture filtrate extracts using HPLC. Since detached leaves were used in the experiments the increase/decrease of various cytokinin levels may be attributed to pathogen influence. The increase in cytokinin levels in the susceptible host may be aiding the growth of the pathogen on one hand, while the decrease in the infected resistant host may signal the host to activate defenses against a potential pathogen at the early stage of infection.This revised version was published online in October 2005 with corrections to the Cover Date.     

Identification of Cell Division Inducing Compounds from Coconut Milk

The use of chromatographic techniques coupled with mass spectrometry, infra-red analysis and nuclear magnetic resonance revealed that the amino acid phenylalanine might be responsible for a large proportion of the cell division activity of coconut milk as determined by the soybean bioassay. The observation that zeatin can be removed very readily by partitioning against ethyl acetate at alkaline pH values, cautions against inclusion of this method as a purification step for extracting cytokinins from plant extracts. Mass spectrometry revealed the presence of an unidentified cell division substance with a molecular ion of 279 and which co-chromatographed with zeatinriboside on Sephadex LH-20.     

二氢玉米素核苷组细胞分裂素的放射免疫测定法

本文报道二氢玉米素核苷（ＤＨＺＲ）组细胞分裂素放射免疫测定法（ＲＩＡ）的研制结果。以（３Ｈ）二氢玉米素作示踪剂,测得免抗ＤＨＺＲ抗血清的效价为１：１３７０（Ｂ％＝３０％）。该抗血清主要与本组细胞分裂素发生交叉反应。回收率为９６．９％,灵敏度为１２ｆｍｏｌ ＤＨＺＲ／管,检测线性范围为０．１￣１００ｐｍｏｌＤＨＺＲ／管。批内误差ＣＶ＝４．３,批间误差ＣＶ＝２．０％。对基于同一免抗血清的ＤＨＺＲ组细胞     

Immunoenzyme Analysis of Cytokinins as an Assay for Cytokinin Oxidase Activity

A new highly sensitive method of measuring cytokinin oxidase activity was worked out on the basis of an immunoenzyme test-system for isopentenyladenosine (IPA) assay. The enzyme activity is determined by immunoenzyme assay as the difference between the IPA quantities in the reaction mixture before and after incubation. The specificity of the assay was verified by using the well-known enzyme activators and inhibitors and by monitoring the formation of other cytokinins.     

The aromatic cytokinins

After the discovery of kinetin (Miller et al. 1956, J. Am. Chem. Soc. 78: 1345–1350) there was a flurry of syntheses that led to the finding of 6-benzylaminopurine (BA), an active and easily obtainable cytokinin. Much research into cytokinin physiology was subsequently done with this substance. Further, the isolation and unequivocal identification of natural BA and the high biological activity of its meta -hydroxylated analogues stimulated the search for other natural aromatic cytokinins. Screening was accomplished by ELISA of HPLC fractions using antisera against ortho - and meta -hydroxybenzyladenosine. Subsequent isolation and decisive identification by mass spectrometry led to discovery of a broad spectrum of endogenous plant growth substances structurally similar to a highly active compound, meta -topolin (6-[3-hydroxybenzyl-amino]purine), and to its less active analogue, ortho -topolin (6-[2-hydroxybenzyl-amino]purine). The structures of such aromatic cytokinins suggest considerably different biosynthetic pathways from that of zeatin and related isoprenoid cytokinins. From a physiological viewpoint, aromatic cytokinin metabolism can be classified under four main headings analogous to isoprenoid cytokinins: interconversion, hydroxylation, conjugation, and oxidative degradation. This review attempts to put into context what is known about 9-alkyl-BAs and compares their metabolism in regard to the practical use of cytokinins in agriculture and biotechnology. The recently discovered unusual specificity of additionally C2,N9-disubstituted aromatic cytokinins toward cell cycle kinases, suggests that these cytokinin-derived growth regulators may selectively inhibit certain steps of the cell cycle. The functional overlap of the aromatic cytokinins with those of their isoprenoid counterparts and cytokinin inhibitors, in relation to growth and developmental processes in plants, has yet to be determined.     

A rapid radioimmunoassay for serum testosterone

Sekihara et al. have proposed that it is possible to combine two antisera, each of which is unsatisfactory for a clinical radioimmunoassay due to cross-reactivity problems, and obtain an assay which is of sufficiently good specificity for practical application. The present report provides a theoretical analysis of this problem in a "reduced" case of minimal complexity. We assume infinitesimal concentration of tracer, equilibrium of reactants, perfect separation of bound and free, and that each of the two antisera contain only a single class of antibody sites which can bind to the desired ligand or to a cross-reacting species. Numerical methods are used to generate "ideal" dose response curves. The specificity is evaluated by three criteria: 1) as the ratio of ligand concentrations resulting in 10 or 50% reduction of binding of labeled ligand to antibody, i.e. %B/BO = 90 or 50%; 2) the %B/BO or %B/T at an arbitrary dose level; or 3) the apparent amount of ligand present, for an arbitrary dose of crossreacting ligand. Results indicate that mixing of two nonspecific antisera (each cross-reacting with a different ligand) results in a radioimmunoassay system with a specificity intermediate between that obtained with either of the antisera used alone. Whether this will provide a "satisfactory" assay depends on the purposes for which it is intended, the expected concentrations of cross-reacting ligands, etc. Computer simulation studies may be utilized to select the optimal ratio of the two antisera being used for the assay.     

Identification of zeatin and zeatin riboside in cones of the hop plant and their possible role in cone growth

Cytokinin contents of fertilized cones, unfertilized cones andseeds of the hop plant (Humulus lupulus L.) were found to be0.08 ppm (Benzyladenine equivalent), 0.03 ppm and 0.5 ppm respectively.Major cytokinin in the former two was determined to be zeatinriboside based on gas chromatography-mass spectrometry afterseveral purification steps. Two more cytokinins were detectedin fertilized cones, one of which was determined to be zeatin.The possible role of endogenous cytokinin in cone growth isdiscussed in terms of quantitative and qualitative data obtained.Furthermore, effective purification techniques introduced inthis work are also discussed. (Received December 2, 1977; )