Rapid and efficient transformation of diploid Medicago truncatula and Medicago sativa ssp. falcata lines improved in somatic embryogenesis

We describe a simple and efficient protocol for regeneration-transformation of two diploid Medicago lines: the annual M. truncatula R108-1(c3) and the perennial M. sativa ssp. falcata (L.) Arcangeli PI.564263 selected previously as highly embryogenic genotypes. Here, embryo regeneration of R108-1 to complete plants was further improved by three successive in vitro regeneration cycles resulting in the line R108-1(c3). Agrobacterium tumefaciens-mediated transformation of leaf explants was carried out with promoter-gus constructs of two early nodulins (MsEnod12A and MsEnod12B) and one late nodulin (Srglb3). The transgenic plants thus produced on all explants within 3–4 months remained diploid and were fertile. This protocol appears to be the most efficient and fastest reported so far for leguminous plants. Received: 18 March 1997 / Revision received: 25 June 1997 / Accepted: 15 July 1997     

蒺藜苜蓿株高控制基因COSTIN的克隆及功能研究

株高是影响植物株型建成的重要农艺性状之一,直接决定作物的倒伏性和生物产量,但目前关于苜蓿等豆科牧草株高性状形成的分子调控机制尚不清楚。通过定向筛选豆科模式植物蒺藜苜蓿Tnt1逆转座子插入突变体库,分离鉴定了一个蒺藜苜蓿矮化突变体compact stalk internodes(costin),该突变体的矮化表型是由于茎节伸长受到抑制所致。通过基因表型连锁分析成功克隆了COSTIN基因,该基因编码一个钙离子交换蛋白,与拟南芥的CALCIUM EXCHANGER 7(CAX7) 基因高度同源。qRT-PCR检测发现COSTIN基因在茎、叶和果荚等组织中有较高的表达。进一步研究发现在costin突变体中赤霉素合成途径关键基因MtCPS、MtKAO1、MtGA20ox4、MtGA20ox7和MtGA3ox1表达下调;外施赤霉素GA3可以恢复costin突变体的矮化表型。上述研究表明COSTIN基因通过影响植物激素赤霉素的生物合成来调控蒺藜苜蓿的茎节伸长。     

天麻种子与真菌共生萌发的蛋白组学研究

天麻Gastrodia elata是典型的腐生型兰科药用植物,其种子萌发需要小菇属Mycena真菌的侵染和共生,目前天麻种子共生萌发分子机制是该领域的热点问题。我们首次对天麻种子共生萌发过程进行了系统的蛋白质组学研究。采用iTRAQ标记的液质联用技术,成功鉴定了天麻成熟种子和萌发后原球茎的蛋白质组,共鉴定蛋白1 769个（global FDR 1%）。两组进行了差异蛋白质组学研究,获得差异蛋白269个。差异蛋白GO注释结果表明,在天麻种子共生萌发过程中,差异蛋白参与的功能和生物过程多样,以催化和结合为主,还参与感知环境刺激、分子信号等功能。KEGG代谢通路分析表明,差异蛋白还主要参与了转导、能量代谢、次生代谢和环境适应等过程。我们发现,一些参与内吞作用的蛋白在共生萌发过程中存在差异表达,表明内吞可能参与到二者互作过程中。对差异蛋白质组的深入解析和研究有利于揭示天麻种子共生萌发的分子机制,具有较强的理论和现实意义。     

Temporal expression of ornithine decarboxylase in developing embryos of Musca domestica

The objective of this study was to partially characterize and follow the temporal expression of the enzyme ornithine decarboxylase (ODC) throughout embryonic and early larval development of Musca domestica. Enzymatically active ODC was shown to be present at detectable levels in the embryos only during the latter stages of embryogenesis. This temporally expressed enzyme displayed maximum activity at the time of hatching, and the activity rapidly declined in the newly hatched larvae. The half-life of ODC activity in extracts at the time of hatching and 30 min after hatching was 57 min and 12 min, respectively. The subunit molecular weight of the embryonic ODC was determined to be 46,000, and the apparent native molecular weight was determined to be 276,000. The concentrations of the polyamines putrescine, spermidine, and spermine also were determined throughout embryogenesis. Spermidine was found to be present in the embryos at about 10-fold higher concentrations than spermine and at about 100-fold higher concentrations than putrescine. These polyamines did not undergo major changes in concentration throughout development of the embryos.     

Integration of the barley genetic and seed proteome maps for chromosome 1H, 2H, 3H, 5H and 7H

Two-dimensional gel electrophoresis was used to screen spring barley cultivars for differences in seed protein profiles. In parallel, 72 microsatellite (simple sequence repeat (SSR)) markers and 11 malting quality parameters were analysed for each cultivar. Over 60 protein spots displayed cultivar variation, including peroxidases, serpins and proteins with unknown functions. Cultivars were clustered based on the spot variation matrix. Cultivars with superior malting quality grouped together, indicating malting quality to be more closely correlated with seed proteomes than with SSR profiles. Mass spectrometry showed that some spot variations were caused by amino acid differences encoded by single nucleotide polymorphisms (SNPs). Coding SNPs were validated by mass spectrometry, expressed sequence tag and 2D gel data. Coding SNPs can alter function of affected proteins and may thus represent a link between cultivar traits, proteome and genome. Proteome analysis of doubled haploid lines derived from a cross between a malting (Scarlett) and a feed cultivar (Meltan) enabled genetic localisation of protein phenotypes represented by 48 spot variations, involving e.g. peroxidases, serpins, α-amylase/trypsin inhibitors, peroxiredoxin and a small heat shock protein, in relation to markers on the chromosome map. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

木兰科四种植物种子油的提取及脂肪酸成分分析

采用超声波辅助提取法和微波辅助提取法同时提取白玉兰、凹叶厚朴、深山含笑和醉香含笑四种木兰科植物的种子油,种子油甲酯化后,运用气相色谱—质谱联用技术测定其脂肪酸成分。结果表明:四种植物种子油的提取率不同,白玉兰平均为27.35%、凹叶厚朴23.34%、深山含笑31.66%,醉香含笑9.27%。不同提取方法所得到的种子油脂肪酸成分和相对含量不同,但四种种子油的主要脂肪酸成分相同,包括油酸、亚油酸、硬脂酸和棕榈酸。     

植物蛋白质组学研究进展Ⅱ.蛋白质组技术在植物生物学研究中的应用

蛋白质组技术已广泛应用于植物遗传、发育和生理生态等诸多生物学领域, 主要研究植物的遗传多样性、植物发育(如种子成熟与发芽过程)、组织器官的分化过程、不同亚细胞结构的新蛋白组分的发现及其功能鉴定、植物对非生物逆境(包括高温、低温、高盐和干旱等)和生物逆境(病虫害)的适应机制和植物与微生物(根瘤共生体)相互作用机制。同时对植物蛋白质组学的发展前景进行了讨论。     

Proton N.M.R. study of the conformational dynamics of porcine pancreatic colipase. Titration of aromatic residues.

The low-field portion of the 360 MHz proton N.M.R. spectrum of native porcine pancreatic colipase has been studied as a function of pH over the pH range 2-12. Resonances associated with the 26 protons of the aromatic rings of the two histidines, two phenylalanines and three tyrosines have been identified and tentatively assigned to specific residues. Titrations of pH yielded apparent pKa's of 7.9, 6.9, 10.4, 10.3 and 11.3 for His I (His 30), His II (His 86), Tyr I (Tyr 56 or 57), Tyr II (Tyr 56 or 57) and Tyr III (Tyr 53) respectively (tentative assignments). The high pKa value of His 30 is attributed to the vicinity of Asp 31. The mobility of the aromatic ring of Tyr 53 is hindered and an upper bound of 500 s-1 on the rate of rotation can be estimated. The aromatic rings of the 2 other tyrosine residues and of the 2 phenylalanine residues can rotate freely on the N.M.R. time scale. The study of perturbations in titration profiles and chemical shift values reveals a specific interaction of His 86 with Tyr I and, to a lesser extent, Tyr II. The existence of this interaction indicates that the protein folding brings in close spatial vicinity two distant regions of the covalent structure to form a "hydrophobic-aromatic" site which might be involved in the binding of bile salt micelles to pancreatic colipase.     

Evaluation of allergenic potential for rice seed protein components utilizing a rice proteome database and an allergen database in combination with IgE-binding of recombinant proteins

Among 131 rice endosperm proteins previously identified by MS-based proteomics, most of the proteins showed low or almost no sequence similarity to known allergens in databases, whereas nine proteins did it significantly. The sequence of two proteins showed high overall identity with Hsp70-like hazel tree pollen allergen (Cor a 10) and barley α-amylase (Hor v 16), respectively, whereas the others showed low identity (28–58%) with lemon germin-like protein (Cit l 1), corn zein (Zea m 50 K), wheat chitinase-like xylanase inhibitor (Tri a XI), and kinase-like pollen allergen of Russian thistle (Sal k 1). Immuno-dot blot analysis showed that recombinant proteins for these rice seed homologs were positive in the IgE-binding, but not necessarily similarity dependent, from some allergic patients. These results suggest that utilization of proteome and sequence databases in combination with IgE-binding analysis was effective to screen and evaluate allergenic potential of rice seed protein components.     

Involvement of Enterobacter cloacae in the mortality of the fish, Mugil cephalus

Aims:  To identify the causative agent of the mortality in the fish, Mugil cephalus , in Muttukadu lagoon.
Methods and Results:  An enteric bacterium from the kidneys of moribund fish M. cephalus , was isolated and identified as Enterobacter cloacae (MK). Mugil cephalus was experimentally infected by this isolate and was re-isolated from the kidneys of the moribund fish. Enterobacter cloacae isolates from the lagoon water (MW1, MW2 and reference strain ATCC 13047) and the reference strain were not able to induce similar pathogenesis. The putative factor imparting pathogenicity to the MK isolate was identified as a cationic molecule, which migrated towards the cathode on agarose gel electrophoresis.
Conclusions:  The Ent. cloacae (MK) isolate harbouring a cationic factor was the causative agent for the mortality of M. cephalus , found in Muttukadu lagoon.
Significance and Impact of the Study:  This study reveals that human enteric bacteria MK which is considered as nonpathogenic to fish, may become pathogenic to fish when it harbours this cationic factor. This cationic factor is found to be pathogenic to the fish M. cephalus leading to mortality. It was also found to be pathogenic to mice. Therefore, the shuttling of Ent. cloacae , harbouring cationic factor, between human and fish may be of human health importance.     

肝癌亚细胞结构的蛋白质组分比较分析

运用亚细胞蛋白质组学的研究策略,分离纯化亚细胞结构,可以提高低丰度蛋白质在双向电泳中检出的数量。通过对比分析肝癌细胞与正常肝细胞线粒体、细胞核蛋白质组的差异表达情况,为肝癌发病机理的研究提供更多、更有价值的信息。以体外培养的人体肝癌细胞QGY-7703与正常肝细胞LO2为研究模型,通过超离心的方法分离细胞的线粒体和细胞核。双向电泳分离线粒体和细胞核的蛋白质,图像分析筛选差异表达蛋白斑点,MALDI-TOF-MS鉴定蛋白质。从线粒体、细胞核的蛋白质电泳图谱中筛选出54个候选差异表达的蛋白质斑点,质谱鉴定出22种差异表达蛋白质,其中17种在肝癌细胞中表达上调,5种在肝癌细胞中表达下调。筛选出的差异表达蛋白质涉及到细胞的能量代谢、蛋白质合成、细胞骨架与核骨架的改变、mRNA的加工成熟及凋亡调控等许多方面,表明癌变细胞的组织结构和代谢状态都发生了很大的变化。     

6-DMAP和胚胎干细胞代数对异种重构卵体外发育的影响(简报)

核移植技术已经广泛应用于动物克隆,但是克隆动物的成活率仍然很低。许多克隆胚胎死于妊娠期,少部分能发育到期,正常出生,但多数在出生后由于心肺和消化道的问题,很快就夭折,有些克隆动物有异常表型,如出生时体重和胎盘过大等。研究发现,在同种克隆动物实验中用胚胎干细胞(Embryonic stem cell,ES细胞)作为核供体,发育到期的克隆动物比例明显高于体细胞,并且用杂交一代的小鼠ES细胞为核供体,绝大多数克隆仔     

我国鼎湖山小皮伞属的分类研究

本文报道了广东鼎湖山小皮伞属(Marasmius Fr．)的34个种,其中新种4个,国内新记录20个。新种为拟花味小皮伞(M·pseudo-euosmus Zheng et Bi sp nov.),近刚毛小皮伞(M. subsetiger Bi et Zheng sp. Nov.),脐状小皮伞(M．Umbilieatus Bi et Zheng sp．nov．)及近血红小皮伞(M. subaimaru Bi sp．nov.)。