Sequence of the cellular T-DNA in the untransformed genome of Nicotiana glauca that is homologous to ORFs 13 and 14 of the Ri plasmid and analysis of its expression in genetic tumors of N. glauca x N. langsdorffii

A region homologous to the TL-DNA of Agrobacterium rhizogenes was previously detected in the genome of untransformed Nicotiana glauca and designated cellular T-DNA (cT-DNA). Subsequently, part of this region was sequenced and two genes, which corresponded to rolB and rolC and were named NgrolB and NgrolC, were found. We have now sequenced a region of the cT-DNA other than the region that includes NgrolB and C and we have found two other open reading frames (ORFs), NgORF13 and NgORF14. These ORFs correspond to ORFs 13 and 14 of the TL-DNA of A. rhizogenes and exhibit a high degree of homology to these ORFs, without having a nonsense codon. We have not found any sequence homologous to rolD (ORF15). The two genes, NgORF13 and 14, as well as the NgrolB and C genes, are expressed in genetic tumors of hybrids between N. glauca and N. langsdorffii but not in leaf tissues of the hybrid.     

Cloning and morphological properties of Nicgl;CYCD3;1 gene in genetic tumors from interspecific hybrid of N. langsdorffii and N. glauca

Plant genetic tumors represent neoplastic growths, which arise spontaneously in hybrid plants without apparent external induction. To understand the molecular nature of unregulated cell proliferation, a cyclin D cDNA clone encoding a cyclin D of 1104bp was isolated from a genetic tumor and designated Nicgl;CYCD3;1 gene. DNA gel blot analysis suggested that there are two copies of Nicgl;CYCD3;1 in the genetic tumors. Northern analysis showed that this gene had the highest expression level in genetic tumor compared to Nicotiana glauca, N. langsdorffii and hybrid plants. Plant morphology of hybrid plant was an intermediate between N. glauca and N. langsdorffii and was altered in the genetic tumors. The cell cycle distribution in N. glauca was G0/G1, 90.59; S, 0.60; G2/M, 8.81; in N. langsdorffii it was G 0/G1, 86.22; S, 6.90; G2/M, 6.88; in hybrid plants it was G 0/G1, 96.40; S, 1.79; G2/M, 1.81; and in genetic tumors G 0/G1, 74.70; S, 2.35; G2/M, 22.94. These data provide new insights into the molecular mechanisms underlying genetic tumor formation from interspecific hybrid between N. langsdorffii and N. glauca.     

Expression analysis of the NgORF13 promoter during the development of tobacco genetic tumors

We investigated the expression pattern of the promoter of Nicotiana glauca (Ng) ORF13 in the hybrids between N. glauca and N. langsdorffii harboring the NgORF13-beta-glucuronidase (GUS) chimeric gene. The promoter of NgORF13 of N. glauca had lower activities than the promoter of RiORF13 of Agrobacterium rhizogenes agropine-type root-inducing (Ri) plasmid. However, the localization of GUS activity in the NgORF13 transgenic plants was similar to that in the RiORF13 transgenic plants. The GUS activity of NgORF13-GUS was high in genetic tumors cultured in vitro or developed spontaneously on F1 plants with aging or by wounding. The GUS activity in tumors was observed in bud primordia, vascular bundles and leaves in the buds. While the activity was lower than in tumors, NgORF13-GUS was also expressed in vascular bundles and the parenchymatous tissues in plants regenerated from tumors. Furthermore, the promoter activity of NgORF13 was induced by wounding and activated by exogenous application of methyl jasmonate. During tumorization, NgORF13 was induced at an early stage and showed expression patterns similar to both NgrolB and NgrolC whose expression were investigated by Nagata et al. (1996) Plant Cell Physiol. 37: 489-498. It is thought that Ngrol genes might be involved in the formation of genetic tumors, and, moreover, NgORF13 might work in cooperation with NgrolB and NgrolC.     

Anxiolytic and antidepressant-like activity of a standardized extract from Galphimia glauca

An infusion prepared with aerial parts from Galphimia glauca has been widely used in Mexican traditional medicine as a remedy for nervous excitement. The sedative activity of a methanolic extract from this plant has been demonstrated by neuropharmacological tests. This effect was attributed to the nor-secotriterpene named galphimine B (GB). In the present work, the anxiolytic and antidepressant-like effects of G. glauca methanolic extract (standardized on GB content, 8.3mg/g) were assayed by using the elevated plus-maze, light-dark test and the forced swimming paradigm, on ICR albino mice. This extract, administered orally, three times (24, 18 and 1h before the test), and in different doses (125, 250, 500, 1,000 and 2,000 mg/kg) was able to increase significantly (p<0.05) the number of entries, as well as the time spent in the open arms of the elevated plus-maze, indicating an anxiolytic-like effect. A similar effect was observed in the light-dark paradigm test, the time spent in the light box was increased in treated mice. Nevertheless, this treatment was unable to change any parameter in the forced swimming test. Altogether, these results suggest an anxiolytic-like effect to the methanolic standardized extract of G. glauca on ICR inbred mice.     

CYP72B1基因和AUR3基因响应光、生长素和油菜素内酯的转录调控机制研究

为研究光、生长素和油菜素内酯在基因层次上的互作机制,开发了转录调控元件识别工具OCMMat,其中,在对共表达基因信息和直系同源基因信息进行整合时,利用了转录调控元件在直系同源基因启动子中的富集性.利用该方法发现,CYP7281基因和AUR3基因启动子含有3个相同的调控模序GAGACA、AAGAAAAA、ATCATG,它们分别承担了AuxRE元件、GT元件和GT辅助元件的功能.其中,ATCATG模序是目前尚未报道过的调控元件,与AAGAAAAA模序的距离相对恒定.基于调控元件识别结果,构建了CYP7281基因和AUR3基因响应光、生长素和油菜素内酯的转录调控模型,模型显示:光信号和生长素、油菜素内酯信号在CYP72B1基因和AUR3基因的转录调控元件上相互交叠,而生长素和油菜素内酯信号则在转录因子ARF水平上相交.     

Analysis of nuclear and organellar DNA of somatic hybrid calli and plants between Lycopersicon spp. and Nicotiana spp.

Protoplast fusion experiments between Lycopersicon esculentum or L. peruvianum and Nicotiana tabacum or N. plumbaginifolia were performed to investigate the possibility of producing symmetric and asymmetric somatic hybrids between these genera. These fusions, which involved 1.7 × 10⁸ protoplasts, yielded 35 viable hybrid calli. Plant regeneration was successful with two calli. One of these regenerants flowered, but developed no fruits. Analysis of the nuclear DNA by means of dot blot hybridization with species-specific repetitive DNA probes combined with flow cytometry, revealed that the nuclei of most hybrid calli contained the same absolute amount of Nicotiana DNA as the Nicotiana parent or (much) less, whereas the amount of Lycopersicon DNA per nucleus was 2–5 times that of the parental genotype. Eighteen of the 34 hybrids analyzed possessed Lycopersicon chloroplast DNA (cpDNA), whereas the other 16 had DNA from Nicotiana chloroplasts. The cpDNA type was correlated with the nuclear DNA composition; hybrids with more than 2C Nicotiana nuclear DNA possessed Nicotiana chloroplasts, whereas hybrids with 2C or less Nicotiana nuclear DNA contained Lycopersicon chloroplasts. Mitochondrial DNA (mtDNA) composition was correlated with both nuclear DNA constitution and chloroplast type. Hybrids possessed only or mainly species-specific mtDNA fragments from the parent predominating in the nucleus and often providing the chloroplasts. The data are discussed in relation to somatic incompatibility which could explain the low frequency at which hybrids between Lycopersicon and Nicotiana species are obtained and the limited morphogenetic potential of such hybrids.     

Genetic variation and divergence of the sibling pair of cyprinid fishes, Notropis cornutus and N. chrysocephalus

Horizontal starch gel electrophoresis was used to estimate the amount of genetic divergence between Notropis cornutus and N. chrysocephalus. Measures of genetic identity (1) and distance (D) were 0.881 and 0.127 ± 0.055 (s.e.), respectively. These estimates correspond more closely to the sibling species status of these taxa than other previously reported estimates. Notropis cornutus was found to be significantly more variable than N. chrysocephalus electrophoretically and morphologically. Assuming the existence of an electrophoretic clock, the time of divergence was calculated to be roughly 1.9–2.5 million years. This estimate corresponds very closely to a previously hypothesized late Pliocene divergence.     

Flowering of strict photoperiodic <Emphasis Type="Italic">Nicotiana</Emphasis> varieties in non-inductive conditions by transgenic approaches

The genus Nicotiana contains species and varieties that respond differently to photoperiod for flowering time control as day-neutral, short-day and long-day plants. In classical photoperiodism studies, these varieties have been widely used to analyse the physiological nature for floral induction by day length. Since key regulators for flowering time control by day length have been identified in Arabidopsis thaliana by molecular genetic studies, it was intriguing to analyse how closely related plants in the Nicotiana genus with opposite photoperiodic requirements respond to certain flowering time regulators. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and FRUITFULL (FUL) are two MADS box genes that are involved in the regulation of flowering time in Arabidopsis. SOC1 is a central flowering time pathway integrator, whereas the exact role of FUL for floral induction has not been established yet. The putative Nicotiana orthologs of SOC1 and FUL, NtSOC1 and NtFUL, were studied in day-neutral tobacco Nicotiana tabacum cv Hicks, in short-day tobacco N. tabacum cv Hicks Maryland Mammoth (MM) and long-day N. sylvestris plants. Both genes were similarly expressed under short- and long-day conditions in day-neutral and short-day tobaccos, but showed a different expression pattern in N. sylvestris. Overexpression of NtSOC1 and NtFUL caused flowering either in strict short-day (NtSOC1) or long-day (NtFUL) Nicotiana varieties under non-inductive photoperiods, indicating that these genes might be limiting for floral induction under non-inductive conditions in different Nicotiana varieties.     

嫁接西瓜对西瓜枯萎病菌侵染的防御机制研究

为了揭示嫁接提高西瓜抗枯萎病的机制,该研究以嫁接西瓜为材料,采用扫描电镜观察了枯萎病菌侵染下寄主的组织结构变化,荧光定量分析了相关防卫基因的表达,比较了嫁接西瓜对枯萎病菌侵染的抗感反应。结果显示:(1)枯萎病菌侵染后,与自根西瓜相比,嫁接西瓜的根部木质部导管通过快速形成膜状物、侵填体及细胞壁增厚阻塞菌丝入侵;自根西瓜防御反应较嫁接西瓜晚,严重侵染时薄壁细胞降解,导管组织脱落导致维管系统空洞,从而使植株呈现萎蔫症状,该现象在嫁接西瓜中没有发现。(2)枯萎病菌侵染后,嫁接西瓜比自根西瓜具有较高的防卫基因表达水平,其中:嫁接西瓜中,CHI、APX和PPO基因的表达随枯萎病菌侵染时间的延长而升高,而PAL呈现先升高后降低的表达趋势,但仍高于本底表达;自根西瓜中,仅PPO基因在枯萎病菌侵染后表达上调,而其他基因的表达则是先升高后降低,与嫁接西瓜中的PAL基因表达一致。研究表明,嫁接植株一方面通过快速的组织结构响应,另一方面从转录水平提高了相关防卫基因的表达,最终使植株具有抗病性;推测防御基因在嫁接植株与枯萎病菌互作中的强烈诱导响应可能是嫁接植株抗病的分子机制之一。     

中国假百合属亲缘关系及假百合遗传多样性的ISSR分析

利用7条ISSR引物对中国假百合属植物24个居群255个个体的遗传多样性进行了初步探讨。结果表明:(1)UPGMA聚类结果显示,假百合(Notholirion bulbuliferum)、钟花假百合(N.campanulatum)与大叶假百合(N.macrophyllum)分别聚为三支,在分子水平上出现了明显的分化,揭示三者是独立物种。(2)N.bulbuliferum×N.campanulatum与钟花假百合聚为一支,这可能与其母系遗传和极强的无性繁殖体系有关,但两者间基因流(N_m=0.216 0)很低,假百合与N.bulbuliferum×N.campanulatum的基因流(N_m=0.144 9)也极低,说明N.bulbuliferum×N.campanulatum正在分化。(3)假百合种下各居群间按照地理结构可明显分为4支,经Mantel检测表明存在显著的谱系地理结构(r=0.410,P=0.01)。(4)AMOVA分析显示,假百合居群间遗传变异为77.12%(P<0.01),而居群内为22.88%(P<0.01),同样表明居群间遗传分化大于居群内。研究结果揭示了横断山区假百合属植物的亲缘关系,从分子水平上为其鉴定提供了依据;并从遗传结构上为药用植物假百合的可持续利用和开发奠定了理论基础。     

Morphometric and biochemical characterization of red beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) hairy roots obtained after single and double transformations

It is known that T-DNA of Agrobacterium rhizogenes affects processes of plant development and activates the synthesis of secondary metabolites in transformed plant cells. In the present investigation, we provide evidence that different strains of A. rhizogenes significantly affect morphometric, morphological and functional characteristics of hairy roots of red beet (Beta vulgaris L.). Infection with four strains of A. rhizogenes (A4, A 2/83, A 20/83 and LMG-150) resulted in ten clones of hairy roots, which were named accordingly as A4(1), A4(2), A4(3), A 2/83(1), A 2/83(2), A 2/83(3), A 20/83(1), A 20/83(2), A 20/83(3) and LMG-150. Their growth characteristics, pigment content, levels of endogenous auxin and T-DNA copy number showed significant differences probably due to the physiological status of the host cell rather than the T-DNA copy number. Although A 2/83 showed highest hairy root induction capacity, the best hairy root clone was obtained with strain LMG-150 that produced highest biomass and pigments. In this root clone, the enzyme peroxidase was found involved in altering the endogenous auxin pool. When root clone LMG-150 was re-transformed to insert additional individual rol genes, two double transformed clones were obtained, one for rolABC and the other for rolC gene where the former produced higher biomass and betalaine than the latter. Despite the established fact that rol genes of T-DNA influence endogenous phytohormones, no direct correlation among the single transformants and the double transformants was found. This is the first report, in our knowledge, where a hairy root clone has been used to obtain double transformants.     

珍稀植物华木莲的现代濒危机制:探析与展望

华木莲(Sinomanglietia glauca)在探讨木兰科系统演化与被子植物起源等方面具有重要科学价值,但其分布区域狭窄、种群规模较小,被列为我国濒危植物和极小种群植物。该文在系统综述华木莲生物生态学特性和更新维持机制的基础上,对华木莲的现代濒危原因进行了总结:(1)华木莲属古老残遗濒危植物,环境变迁、瓶颈效应、高频自交和遗传漂变,导致其遗传多样性下降、适应性差;(2)喜光喜肥的生物生态学特性和群落种间竞争驱赶作用,"林—窗"游击式更新是华木莲种群延续的重要方式,然而当前人类干扰导致有效林窗供给不足、种子传播效率下降、"游击"机会减少,更新难以完成;(3)毛竹扩张与林下箬竹过度增长严重影响华木莲开花结实,并妨碍其幼苗更新。今后应加强华木莲谱系地理与群落系统发育、群落环境对华木莲生长发育的影响及其应答、林窗更新机制与种子长距传播、群落结构调整与保护技术、遗传复壮与迁地保护等方面研究,为华木莲以及相似生物生态学特征的濒危植物保护提供理论指导。     

Possible involvement of auxin-induced ethylene in an apoptotic cell death during temperature-sensitive lethality expressed by hybrid between Nicotiana glutinosa and N. repanda

Interspecific hybrids of Nicotiana glutinosa L. x N. repanda Willd. express temperature-sensitive lethality induced by apoptotic cell death. Hybrid seedlings cultured at 28 degrees C began to exhibit lethal symptoms during early growth stages, and then they showed a high level of endogenous auxin compared with those of parental seedlings. Meanwhile, the level of auxin in hybrid seedlings cultured at 32 degrees C, which is a condition suppressing the lethality of this cross combination, was equal to or lower than those of parental seedlings. Administration of 2,3,5-triiodobenzoic acid (TIBA) as an auxin transport inhibitor into the hybrid seedlings suppressed lethal symptoms and had a life-extending effect. Additionally, TIBA has an effect to suppress DNA fragmentation, which is one of characteristics of apoptosis and has been detected in the hybrid seedlings expressing the lethality. Administration of aminooxyacetic acid (AOA) as an ethylene synthesis inhibitor, which could inhibit ethylene production, also showed the same effects as TIBA for the lethality. From these results, we suggested that auxin and ethylene were involved in an apoptotic cell death during the lethality, and the abnormal increase of endogenous auxin may lead to the ethylene production in hybrid seedlings during early growth stages.     

Somatic hybridization of Nicotiana tabacum and Petunia hybrida

Summary Leaf mesophyll protoplasts of a nitrate reductase deficient streptomycin resistant mutant of Nicotiana tabacum were fused with cell suspension protoplasts of wild type Petunia hybrida. Somatic hybrid cell colonies were selected for streptomycin resistance and nitrate reductase proficiency. Six independent cell lines, capable of growth in selection medium, were analysed by electrophoresis of callus peroxidases and leucine aminopeptidases and also by hybridization with rDNA and a chloroplast encoded gene as molecular probes. The results show that all six lines represented nuclear somatic hybrids, possessing the chloroplast of N. tabacum, at an early stage of development. However, after 6–12 months in culture, genomic incompatibility was observed resulting in the loss of most of the tobacco nuclear genome in the majority of the cell lines. One of the latter cell lines regenerated plants which possessed the chloroplast of N. tabacum in a predominantly P. hybrida nuclear background.     

Restoration of shooty morphology of a nontumorous mutant of Nicotiana glauca x N. langsdorffii by cytokinin and the isopentenyltransferase gene

The shooty morphology of a nontumorous amphidiploid mutant of Nicotiana glauca Grah. x N. langsdorffii Weinm. was restored by cytokinins, whether exogenously applied or endogenously produced by transformation of the mutant with a transfer DNA (T-DNA) cytokinin-biosynthesis gene (isopentenyltransferase; ipt). Auxins alone did not confer this effect. Similar transformation was not achieved for the parental species. In the case of transformation with the ipt gene, selection of the transformed tissues was based on its hormone-independent growth in the presence of the antibiotic kanamycin. Transformed tissues exhibited a shooty morphology, indistinguishable from that of wildtype genetic tumors N. glauca x N. langsdorffii. This altered phenotype was caused by the presence and constitutive expression of the ipt gene. The insertion and expression of this gene in transformed tissues was confirmed by using the polymerase chain reaction (PCR) technique as well as conventional molecular hybridization analysis. Expression of the ipt gene led to an elevated level of cytokinin in the transformed mutant tissues. This evidence supports the notion that genetic tumors are caused, at least in part, by elevated levels of cytokinin in interspecific hybrids.