甜瓜S-腺苷甲硫氨酸脱羧酶基因的克隆及白粉病诱导表达分析

为探讨S-腺苷甲硫氨酸脱羧酶(S-adenosylmethionine decarboxylase,SAMDC)基因在甜瓜抵御白粉病菌中的作用,根据已知EST序列和甜瓜基因组数据库,在甜瓜抗白粉病品种‘Yuntian930’中克隆获得该基因的全长编码序列,命名为CmSAMDC(GenBank登录号为KF151861)。生物信息学分析表明,CmSAMDC的主开放阅读框(mORF)长1 095 bp,编码364个氨基酸,预测分子量为40 kDa。聚类分析表明,CmSAMDC预测蛋白与黄瓜和四季橘中该蛋白的同源关系最近,并与其他双子叶植物聚为一类。原核表达分析表明,CmSAMDC以融合蛋白形式表达,相对分子量约为40 kDa,与预测一致。实时定量表达分析表明,CmSAMDC基因受白粉病诱导表达,在接种后48 h表达量达到峰值,为接种前的7倍,并且在甜瓜的根、茎、叶、卷须中均有表达。结果提示该基因可能参与了甜瓜的抗白粉病反应。     

Characterization of an NBS-LRR resistance gene homologue from soybean

Conserved motifs such as the nucleotide-binding site (NBS) were found in many characterized plant disease resistance genes. Based on the NBS domain, resistance gene analogs have been isolated in our previous study and were used as probes to screen a soybean (Glycine max) cDNA library. A full-length cDNA, KR4, was isolated by screening the library and rapid amplification of cDNA ends method. Sequence analysis revealed that the cDNA was 3818 bp in length and the open reading frame coded for a polypeptide of 1211 amino acids with an NBS and five leucine-rich repeats domains, which were identified by Pfam protein analysis. Sequence alignment showed that KR4 was similar to 12 protein of tomato. Southern analysis indicated that the KR4 gene had low copies in soybean genome and it was mapped on the molecular linkage group E. Its expression was also investigated and it was found that KR4 was induced by exogenous salicylic acid and responded upon infection of soybean mosaic virus strain N3.     

Indole-3-acetic acid, ethylene, and abscisic acid metabolism in developing muskmelon (Cucumis melo L.) fruit

Hormonal metabolism associated with fruit development in muskmelon was investigated by measuring IAA, ABA, and ACC levels in several tissues at various stages of development. In addition, levels of conjugated IAA and ABA were determined in the same tissues. Ethylene production, which is believed to signal the ripening and senescence of mature fruit, was also measured. Ethylene production was highest in the outer tissue near the rind and gradually declined during maturation, except for a dramatic increase in all fruit tissues at the climacteric. In contrast to ethylene production, ACC levels increased during maturation and remained equal throughout the fruit until the climacteric, when levels in the outer tissues increased nearly 5-fold over levels in the inner tissues. The consistent presence of ACC indicates that ACC oxidase rather than the availability of ACC regulates ethylene production in developing fruits. ABA and ABA esters generally declined during maturation, however an increase in ABA esters associated with the outer mesocarp tissue was observed in fully mature, climacteric fruit. IAA and IAA conjugates were only found in the outer tissue near the rind, and their levels remained low until the fruit was fully mature and entering the climacteric. At that time, increased levels of conjugates were detected. The late burst of hormonal metabolism in the outer mesocarp tissue appeared to signal its degeneration and the deterioration that typically occurs in ripening fruit. The tissue-specific conjugation of IAA and ABA, in addition to the production of climacteric ethylene, may represent part of the signaling mechanism initiating ripening and eventual deterioration of tissues in muskmelon fruits.Abbreviations ABA abscisic acid - ACC 1-aminocylopropane-1-carboxylic acid - DAP days after pollination - IAA indole-3-acetic acid     

Phenolic glycosides from Cucumis melo var. inodorus seeds

A new phenolic glycoside (E)-4-hydroxycinnamyl alcohol 4-O-(2′-O-β-d-apiofuranosyl)(1″ → 2′)-β-d-glucopyranoside (1) was isolated and identified from Cucumis melo seeds together with benzyl O-β-d-glucopyranoside (2), 3,29-O-dibenzoylmultiflor-8-en-3α,7β,29-triol (3) and 3-O-p-amino-benzoyl-29-O-benzoylmultiflor-8-en-3α,7β,29-triol (4). Their structures were elucidated by extensive NMR experiments including ¹H–¹H (COSY, TOCSY, ROESY) and ¹H–¹³C (HSQC and HMBC) spectroscopy and chemical evidence. The multiflorane triterpene esters were identified as new melon constituents.     

Characterization of an abscisic acid carrier in suspension-cultured barley cells

The uptake of [³H]-abscisic acid in barley (Hordeum vulgareL. cv. Heartland) cell cultures was found to be mediated throughboth non-saturable and saturable components. The kinetic parametersof the saturable component, determined at pH 4.5 and 21 °C,showed a K_m for natural or (+ )-ABA of 1.3±0.7µMand an apparent V_mex of 7.0 ± 2.8 nmol g^–1 cellsh^–1. The carrier showed a strong preference for the naturalenantiomer of ABA as compared to the unnatural one. Other substancestested, e.g. amino acids, organic acids, and other growth regulators,did not appear to interfere with the carrier-mediated uptakeof ABA. At low external concentrations of ABA (below 2.0 µM),the saturable component was greater than the diffusion component.Similarly, between pH 4.0 and 6.0, the saturable uptake wasresponsible for more than 50% of the total uptake. The carriermay be important in vivo for mediating uptake when endogenouslevels of ABA are low (c. 1 µM). The carrier specificity was evident in inhibition experimentsdone with ABA analogues. Our data showed that the carrier couldaccommodate small modifications in the ABA structure. Four analogueswere able to compete efficiently with ( + )-ABA for the bindingsite of the carrier. Three of these competitors were of the(+)-series. Only one ( –)-analogue, (–)-ABA, wasable to inhibit markedly the saturable uptake of ( + )-ABA.The induction of the ABA-respons-ive gene WCS120 (Houde et al.,1992) presented stricter requirements for the ABA molecule thanthe carrier, although with a similar preference for the ( +)-analogues. Besides ( + )-ABA itself, only two of the analoguestested, both ( + )-series, were able to induce the WCS120 geneafter a 24 h incubation period. The absence of correlation betweenthe activity of the analogues as ABA inhibitors in the carriersystem, and their capacity to induce the WCS120 gene tend tosuggest that the carrier is not directly involved in the signaltransduction pathway leading to the induction of this specificgene. Key words: Abscisic acid, barley, gene induction, Hordeum vulgare, uptake carrier     

Expression of an antisense 1-aminocyclopropane-1-carboxylate oxidase gene stimulates shoot regeneration in Cucumis melo

The role of ethylene in shoot regeneration was investigated using transgenic Cucumis melo plants expressing an antisense 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene. ACC oxidase catalyses the last step of ethylene biosynthesis. Leaf and cotyledon explants from the transgenic plants exhibited low ACC oxidase activity and ethylene production, whereas the regeneration capacity of the tissues was greatly enhanced (3.5- and 2.8-fold, respectively) compared to untransformed control tissues. Addition of ethylene released by 50 or 100 μm 2-chloroethylphosphonic acid dramatically reduced the shoot regeneration rate of the transgenic tissues. The results clearly demonstrate that ethylene plays an important role in C. melo morphogenesis in vitro. Received: 23 April 1997 / Revision received: 9 June 1997 / Accepted: 2 July 1997     

ACC脱氨酶基因转化白兰瓜的初步研究

用带有ACC脱氨酶基因和卡那霉素抗性基因（NptⅡ,作为报告基因）的工程根癌农杆菌转化白兰瓜子叶。通过组织培养,得到具有卡那霉素（Km)抗性的再生小苗,经NptⅡ报告基因的PCR扩增,ACC脱氨酶基因的Southern点杂交以及ACC脱氨酶活性的生理生化检测可知,ACC脱氨酶基因已成功转入白兰瓜子叶再生小苗,而且不同植株的真叶中表现出不同水平的ACC脱氨酶活性。     

Isolation and identification of the gibberellins of Cucumis sativus and Cucumis melo

Summary Thin-layer chromatography, gas-liquid chromatography, and mass spectrometry were used to identify gibberellins isolated from mature seeds of both Cucumis sativus (cucumber) and Cucumis melo (muskmelon). The gibberellins were extracted and purified by organic solvent fractionation, paper and thin-layer chromatography, and crystallization. Seeds of C. sativus were found to contain gibberellins A₁, A₃, A₄, and A₇ with A₁ the predominant species. Seeds of C. melo contained gibberellins A₁ and A₃ and a trace of A₅. Direct probe mass spectrometry of the gibberellins proved successful for identification purposes. Distinctive molecular ions and fragmentation patterns were obtained for each gibberellin.Journal Article No. 5664 from the Michigan Agricultural Experiment Station. This work was supported in part by a grant from the Herman Frasch Foundation.Portions were taken from a thesis submitted in partial fulfillment of the requirements for the Ph.D. degree, Michigan State University, 1971     

甜瓜的组织培养与植株再生

1　植物名称　甜瓜 (Ｃｕｃｕｍｉｓｍｅｌｏ)品种“伽师瓜”。2　材料类别　成熟甜瓜种子无菌苗的子叶。3　培养条件　种子萌发培养基 :(1 ) 1 / 2ＭＳ(大量元素减半 ) 2 %蔗糖。愈伤组织诱导培养基 :(2 )Ｍｉｌｌｅｒ 6 ＢＡ 5 .0ｍｇ·Ｌ- 1 (单位下同 ) ＺＴ 0 .0 1 3 %蔗糖。丛生芽诱导培养基 :(3 )Ｍｉｌｌｅｒ 6 ＢＡ 2 .0 3 %蔗糖 ;(4)Ｍｉｌｌｅｒ 6 ＢＡ 0 .5 3 %蔗糖。芽伸长培养基 :(5 )Ｍｉｌｌｅｒ 6 ＢＡ 0 .1 3 %蔗糖 ;(6 )Ｍｉｌｌｅｒ 6 ＢＡ 0 .0 1 3 %蔗糖。生根培养基 :(7) 1 /2ＭＳ(大 ) ＮＡＡ 0 .0…     

Galactosyl-sucrose metabolism and UDP-galactose pyrophosphorylase from Cucumis melo L. fruit

In muskmelon ( Cucumis melo L.), sink tissues receive stachyose, raffinose and sucrose through phloem translocation of carbohydrates that are formed as products of leaf photosynthesis. Melon fruits accumulate sucrose massively during the final stages of maturation. This sucrose is derived partially from the catabolism of raffinose saccharides. Rapid galactose metabolism is required, because liberation of free galactose is the first step in the metabolic utilization of the raffinose sugars. The current study demonstrates that the enzyme UDP-glucose-hexose-1-P uridylyltransferase (EC 2.7.7.12), the central enzyme in the classical Lelior pathway, is not the central enzyme in galactose metabolism in muskmelon fruit. Rather, a broad substrate specificity UDP-galactose pyrophosphorylase (PPase) serves the same functional role. This enzyme accepts either UDP-galactose or UDP-glucose as a substrate and is different from a UDP-glucose PPase with more strict substrate specificity for UDP-glucose that is also present in melon tissue. UDP-galactose PPase was purified 113-fold from melon tissue and was shown to be a 54 kDa (size exclusion chromatography) to 68 kDa (SDS-PAGE) protein that is enzymatically active as a monomer. We also present evidence that the enzyme likely accepts UDP-galactose and UDP-glucose at the same catalytic site. Polyclonal antibodies prepared against this protein reacted with numerous other antigens in melon extracts, apparently as a result of the presence of common antigenic epitopes.     

甜瓜STK类R基因同源序列的克隆与分析

以植物丝氨酸/苏氨酸蛋白激酶类( serine-threonine kinase,STK)抗病基因产物催化结构域I和Ⅸ的保守氨基酸序列( FGK/V/L/SVYK/RG,DY/IYSF/YGV/I/M)设计简并引物,对甜瓜(Cucumis melo L.)基因组DNA进行PCR扩增,得到大约500 bp的目的条带,通过重组质粒克隆并经PCR检测后得到12条不同的DNA序列,命名为tg1～tg12,其中tg2、tg5、tg9和tg12(Genbank登录号为JN646853 ～JN646856)可以编码完整的氨基酸序列.Blast分析结果显示:4条序列均具有ATP结合部位、底物结合部位和激酶结构域的活化环(A-loop)等,属于典型的蛋白激酶基因家族,可能是STK类R基因的同源序列片段;4条序列与蓖麻(Ricinus communisL.)的STK同源性均较高.氨基酸序列比对结果显示tg2、tg5、tg9和tg12均具有R基因的9个保守结构域,为STK类候选抗病基因类序列.分子系统树显示tg2、tg5、tg9和tg12与已知的R基因(Pto、Lr10和Lectin)在氨基酸水平上的相似性仅为33.5％ ～53.4％,且4个甜瓜同源序列的氨基酸相似性也较低,表明甜瓜RGAs标记可能具有较高的特异性.     

Expression of an abscisic acid responsive promoter in Picea abies (L.) Karst. following bombardment from an electric discharge particle accelerator

The 1.5 kilobase promoter sequence upstream of Dc8, a late embryo abundant gene of Daucus, fused to the reporter -glucuronidase gene was introduced into several tissues of Picea abies via a custom-made electric-discharge particle accelerator. Transient expression was measured histochemically as spot number 2 d after bombardment. Embryogenic suspensions gave higher levels of expression depending upon cell line than embryogenic callus or zygotic embryos. Expression was enhanced when cultures were treated with abscisic acid for 3 d before bombardment. A mean and maximum of 17 and 34 spots/disk, respectively, were observed with the best cell line, which was comparable with the level of expression driven by an enhanced 35S promoter.Abbreviations ABA Abscisic acid - BA Benzyladenine - 2,4-D 2, 4-dichlorophenoxyacetic acid     

Regeneration of plants from leaf explants of Cucumis melo cv. Pusa Sharbati

Leaves of three different sizes excised from 14, 21, 28 and 35-day-old seedlings of Cucumis melo were cultured on a MS medium supplemented with a range and combination of growth regulators. Maximum shoot differentiation from the leaf explants occurred in the combined presence of BAP and 2iP at equimolar concentration of 1 M. Regeneration potential of leaves declined with increasing size of the leaves and the age of the donor seedlings. For elongation the shoots were transferred to MS+BAP [1 M]. Such shoots were rooted with 75% frequency on MS+IAA [0.5 M]. The plants have been established in pots.Abbreviations BM Basal Medium - MS Murashige and Skoog - BAP 6-Benzyl amino-purine - 2iP 6- V, V -dimethylallylamino purine - IAA Indole-3-acetic acid