20个水稻引进资源的花药开裂及苗期耐热性评价

对20个从USDA引进的水稻资源进行了苗期耐热性鉴定和与开花期耐热性紧密相关的花药开裂性状测定。鉴定结果表明,20个资源苗期高温处理后恢复7 d的相对幼苗成活率为83.23%~100%,恢复10 d的相对幼苗成活率为68.18%~100%。其中N22、AD-1140、IR 9403-3-1-1和IR 4427-51-6-3具有较强的苗期耐热性。引进资源的花药基部开裂长度为234.9~559.0μm,花药总长度为1570.0~2351.7μm,其中IR 2006-p-P12-12-3-2和N22的花药基部开裂长度分别最短和最长。20个资源的花药基部开裂长度和高温胁迫后恢复7 d及10 d的相对幼苗成活率之间均存在极显著正相关,花药长度和高温胁迫后恢复7 d及10 d的相对幼苗成活率没有相关性。系谱分析表明,部分引进资源间具有较近的亲缘关系。花药基部开裂长度作为水稻耐热性的评价指标还需要进行进一步的深入研究。     

EMS对三个玉米自交系的诱变效应分析

EMS诱变玉米花粉是玉米化学诱变的主要技术。该研究以生产上3个常用的玉米自交系K305、21-ES、R08为材料,对其花粉用不同浓度的EMS诱变处理,探讨其EMS诱变的最佳浓度范围,明确其诱变效应。结果表明：3个自交系经过不同浓度的EMS诱变后,其结实率随着浓度的增大表现出减小的趋势,从其半致死剂量来看,EMS诱变花粉的适宜浓度范围自交系K305和R08均为0.67~1.0 mL?L-1,21-ES在1.67 mL?L-1附近。 M1代不同性状其变异幅度和变异系数与对照相比主要表现出增大的趋势,其不同性状的生物学效应在材料间表现不一致,表明性状在不同材料间对EMS的敏感性不一样,生育期表现为21-ES>K305>R08;主要株型性状表现为R08>21-ES>K305;主要雄穗性状K305和21-ES比R08敏感;主要果穗性状表现为21-ES>K305>R08。 M2代整体表现为变异谱扩大,其株高、穗位高和叶面积以及主要果穗性状的变异表现复杂,主要雄穗性状中除K305的M2株系雄穗分枝数呈双向变异外,其余M2株系整体偏向于雄穗变短,雄穗分枝数减小。该研究结果为后续研究和应用打下了基础。     

Selective potentiation of 5-methyl-2-(1-methylcyclohexyl)-4-oxazoleacetic acid (AD-4610) on glucose-induced insulin secretion

In the perfused pancreas from normal SD rats, AD-4610 (0.01-0.1 mM) potentiated biphasic insulin secretion induced by 7.5 mM of glucose. The concentration-response curve of insulin secretion to glucose was shifted leftwards with AD-4610 (0.1 mM) without altering either the threshold concentration of glucose to induce insulin secretion or the maximal insulin response to glucose, indicating increased sensitivity of the pancreatic B-cells to glucose. On the other hand, AD-4610 was 10-fold less effective in altering insulin secretion induced by arginine and glyceraldehyde. The effect of AD-4610 on insulin secretion and glucose metabolism was compared with that of tolbutamide in vivo. AD-4610 (100 mg/kg) potentiated insulin secretion induced by an intravenous glucose load, and also accelerated glucose metabolism without altering basal insulin secretion in normal rats. On the other hand, tolbutamide (20 mg/kg) increased basal insulin secretion, but slightly decreased glucose-induced insulin secretion. In yellow KK mice with hyperglycemia, AD-4610 (10-100 mg/kg) had a dose-dependent hypoglycemic action, but tolbutamide did not. Thus, AD-4610 stimulated insulin secretion in a glucose-dependent fashion and enhanced glucose metabolism in vivo. These results suggest that AD-4610 selectively potentiates glucose-induced insulin secretion by increasing the sensitivity of pancreatic B-cells to glucose and may be useful for treating human NIDDM through a different mechanism than that of tolbutamide.     

芦苇耐盐变异体与野生型植株某些特性的比较

地通过细胞工程方法得到的耐盐芦苇（Ｐｈｒａｇｍｉｔｅｓ ｃｏｍｍｕｎｉｓ Ｔｒｉｎ．）变异系Ｒ５００２－１２与野生型植株进行了分子生物学和生理生化特性比较分析。筛选到５个ＲＡＰＤ引物对变异体和野生型植株的ＤＮＡＦ的随机扩增表现出不同的多态性,表明该变异体在分子水平上发生了变化。生理生化分析的结果表明,平行生长状态下的两种株系,其可溶性蛋白和同工酶的表达水平和种类不一样,变异体在ＮａＣｌ胁迫下,表达     

AD-1, a novel ginsenoside derivative,shows anti-lung cancer activity via activation of p38 MAPK pathway and generation of reactive oxygen species

Background

Ginseng is a traditional Chinese herb that has been used for thousands of years. In the present study, effects and mechanisms of AD-1 were evaluated for its development as a novel anti-lung cancer drug.

Methods

The cytotoxic activity was evaluated by MTT assay. Flow cytometry was employed to detect cell cycle, apoptosis and ROS. Western blot and immunohistochemistry were used to analyze signaling pathways. Lung cancer xenograft models were established by subcutaneous implantation of A549 or H292 cells into nude mice.

Results

AD-1 concentration-dependently reduces lung cancer cell viability without affecting normal human lung epithelial cell viability. In A549 and H292 lung cancer cells, AD-1 induces G₀/G₁ cell cycle arrest, apoptosis and ROS production. The apoptosis can be attenuated by a ROS scavenger — N-acetylcysteine (NAC). In addition, AD-1 up-regulates the expression of p38 and ERK phosphorylation. Addition of a p38 inhibitor SB203580, suppresses the AD-1-induced decrease in cell viability. Furthermore, genetic silencing of p38 attenuates the expression of p38 and decreases the AD-1-induced apoptosis. Treatment with NAC reduces AD-1-induced p38 phosphorylation, which indicates that ROS generation is involved in the AD-1-induced p38 activation. In mice, oral administration of AD-1 (10–40 mg/kg) dose-dependently inhibited the growth of xenograft tumors without affecting body weight and decreases the expression of VEGF, MMP-9 and CD34 in tumor tissue. TUNEL staining confirms that the tumors from AD-1 treated mice exhibit a markedly higher apoptotic index.

Conclusions and general significance

These data support development of AD-1 as a potential agent for lung cancer therapy.     

Mutations at the Asc locus of tomato confer resistance to the fungal pathogen Alternaria alternata f. sp. lycopersici

The fungal pathogen Alternaria alternata f. sp. lycopersici produces host-selective AAL-toxins that cause Alternaria stem canker in tomato. Susceptibility to the disease is based on the relative sensitivity of the host to the AAL-toxins and is controlled by the Asc locus on chromosome 3L. Chemical mutagenesis was employed to study the genetic basis of sensitivity to AAL-toxins and susceptibility to fungal infection. Following the treatment of seeds of a susceptible line with ethyl methanesulphonate (EMS), resistant M₂ mutants were obtained. Most plants with induced resistances showed toxin-sensitivity responses that were comparable to those of resistant control lines carrying the Asc locus. In addition, genetic analysis of the mutagenised plants indicated that the mutations occurred at the Asc locus. Furthermore, novel mutants were identified that were insensitive to the AAL-toxins at the seedling stage but toxin-sensitive and susceptible to fungal infection at mature stages. No AAL-toxin-insensitive insertion mutants were identified following a transposon mutagenesis procedure. Molecular mechanisms involved in host defence against A a. lycopersici are discussed.     

银杏苗期生理生化指标的变异和选择研究

对银杏家系间的苗期生理生化和生长指标进行测定,并对生理生化指标与生长指标的相关关系进行研究。结果初步表明家系间生理生化指标、生长指标差异显著;叶片硝酸还原酶(NR)活性和单株生物量、苗高达到了极显著正相关水平;多酚氧化酶活性与单株生物量、苗高的相关系数达到极显著负相关水平。可以初步确定硝酸还原酶、多酚氧化酶为银杏苗木早期选择指标。     

The Cape Verde Islands allele of cryptochrome 2 enhances cotyledon unfolding in the absence of blue light in Arabidopsis

We analyzed the natural genetic variation between Landsburg erecta (Ler) and Cape Verde Islands (Cvi) accessions by studying 105 recombinant inbred lines to search for players in the regulation of sensitivity to light signals perceived by phytochromes in etiolated seedlings of Arabidopsis. In seedlings grown under hourly pulses of far-red (FR) light, we identified three quantitative trait loci (QTLs; VLF3, VLF4, and VLF5) for hypocotyl growth inhibition and three different QTLs (VLF6, VLF7, and VLF1) for cotyledon unfolding. This indicates that different physiological outputs have selective regulation of sensitivity during de-etiolation. Ler alleles, compared with Cvi alleles, of VLF3, VLF4, VLF5, VLF7, and VLF1 enhanced, whereas the Ler allele of VLF6 reduced, the response to pulses of FR. We confirmed and narrowed down the position of some QTLs by using near-isogenic lines. VLF6 mapped close to the CRY2 (cryptochrome 2) gene. Transgenic Ler seedlings expressing the Cvi allele of CRY2 showed enhanced cotyledon unfolding under hourly pulses of FR compared with the wild type or transgenics expressing the CRY2-Ler allele. This response required phytochrome A. The cry1 cry2 double mutant lacking both cryptochromes showed reduced cotyledon unfolding under FR pulses. Because the CRY2-Cvi is a gain-of-function allele compared with CRY2-Ler, cryptochrome activity correlates positively with cotyledon unfolding under FR pulses. We conclude that the blue light photoreceptor cryptochrome 2 can modulate seedling photomorphogenesis in the absence of blue light. In addition to the nuclear loci, we identified cytoplasmic effects on seedling de-etiolation.     

Zea3: a pleiotropic mutation affecting cotyledon development, cytokinin resistance and carbon-nitrogen metabolism

When photomorphogenesis takes place during early plant development, the cotyledons undergo a metabolic transition from heterotrophic sink metabolism to autotrophic source metabolism. A mutant screen was devised for seedlings affected in the regulation of nitrate assimilation during this early sink-source transition in Nicotiana plumbaginifolia. A mutant (EMS 203.6) was isolated for its inability to grow on low nitrate concentration. In contrast to wild-type (WT) plants, the mutant cotyledons remained tightly attached to each other throughout seedling development. It was found that a low carbon/nitrogen ratio (C/N ratio) in the medium was required for mutant growth. The higher the ratio was, the more the growth was inhibited. Mutant EMS 203.6 accumulated all amino acids in permissive conditions (low C/N ratio), and all amino acids and sugars also in selective (high C/N ratio) conditions. In addition, sucrose in the medium repressed light-regulated genes involved in nitrate assimilation and in photosynthesis in the mutant but not in the WT plants. The mutation was mapped to the Zea3 complementation group which confers resistance to zeatin. This zeatin resistance was associated with a hypertrophy of mutant cotyledons in response to cytokinin. Both cytokinin resistance and sensitivity to a high C/N ratio were not observed in etiolated mutant seedlings and were restricted to the jointed-cotyledon developmental stage. Previous physiological studies showed evidence for a role of cytokinins in the expression of nitrate reductase. Here, the first genetic evidence for a link between carbohydrate/nitrogen metabolism and cytokinin action during early development is provided.     

SCM-198 Alleviates Endometriosis by Suppressing Estrogen-ERα mediated Differentiation and Function of CD4+CD25+ Regulatory T Cells

Background: Endometriosis (EMS), a typical endocrine immune disorder, associates with dramatically increased estrogen production and disorganized immune response in ectopic focus. Peritoneal regulatory T cells (Tregs) expansion in women with EMS and their pathogenic role attributable to endometriotic immunotolerance has been reported. Whether local high estrogen promotes EMS by discipling Tregs needs to be further explored. Up to date, there is no effective medicine for the treatment of EMS. SCM-198 is a synthetic leonurine with multiple physiological activities. Whether SCM-198 could regulate Tregs via estrogen and facilitate the radical cure of EMS has not yet been reported.Methods: Proportion of Tregs in peritoneal fluid of patients with EMS was firstly analyzed via flow cytometry. Peritoneal estrogen concentration and the mRNA levels of estrogen receptor α (ERα) and estrogen receptor β (ERβ) of Tregs were detected by ELISA and RT-PCR, respectively. Grouped in vitro induction assays were performed to explore the effects of SCM-198 and estrogen signaling on Tregs. Cell invasion and viability assays were utilized to detect the crosstalk between Tregs and ectopic endometrial stromal cells (eESCs), with or without SCM-198 treatment. Furthermore, EMS mice models were established to verify the therapeutic effects of SCM-198.Results: Increased Tregs were found in peritoneal fluid of EMS patients, accompanied with estrogen-ERα overactivation. Estrogen-ERα triggered the expansion of Tregs and their cytokine production (IL-10 and TGF-β1), which could be reversed by SCM-198 treatment. Moreover, SCM-198 abated the invasion and viability of eESCs enhanced by Tregs. In vivo experiments confirmed that SCM-198 obviously retarded the growth of ectopic lesions and downregulated the functions of Tregs via estrogen-ERα inactivation.Conclusions: These data suggest that SCM-198 attenuates Tregs expansion via the inhibition of estrogen-ERα signaling in EMS and offer a promising therapy for such a refractory disease.     

裸燕麦EMS突变体库筛选与分析

燕麦是重要的粮饲兼用作物,构建燕麦EMS突变体库对燕麦功能基因组学研究和遗传改良有重要意义。本试验利用化学诱变剂甲基磺酸乙酯(EMS,ethyl methane sulfonate)处理燕麦品种花早2号,获得了4083株M1材料;对其中2000个单株种植了M2株行,进行全生育期调查,鉴定其表型变化;对2份黄化苗突变材料种植了M3家系,进行相关突变性状的稳定性验证。结果表明,燕麦经EMS处理后代变异巨大,在M2发现表型突变材料196份,变异率为9.8%,变异类型非常丰富,包括幼苗习性、叶片性状、分蘖、株高、穗部形态及成熟期等突变株系。M3证实突变的黄化苗特性可以稳定遗传。本研究建立了燕麦EMS诱变体系,获得的燕麦变异类型丰富,为燕麦功能基因组学研究和燕麦遗传改良奠定了材料基础。     

In vitro mutation and selection of doubled-haploid Brassica napus lines with improved resistance to Sclerotinia sclerotiorum

This paper describes a new protocol to develop doubled-haploid (DH) Brassica napus lines with improved resistance to Sclerotinia sclerotiorum. In this protocol, haploid seedlings derived from microspore cultures of B. napus were used to produce haploid calli for in vitro mutation-selection. For routine screening, mutation was induced by EMS (ethylmethane sulfonate) or occurred spontaneously, and screening for resistant mutants occurred on media with added oxalic acid (OA) as a selection agent. In tests with selected lines, the optimal concentration of EMS for mutation was determined to be 0.15%, and the optimal concentration of OA for in vitro screening was 3 mmol/l (half lethal dose was 3.1 mmol/l) for the first cycle of screening. There was an accumulated effect of OA toxicity on calli over two cycles of screening, but the growth and capacity of the surviving calli for regenerating seedlings were not affected by OA. Of the 54 DH lines produced from the in vitro mutation-selection, two DH lines of resistant mutants, named M083 and M004, were selected following seedling and glasshouse tests. The resistance of M083 and M004 to S. sclerotiorum following tests with both mycelial inoculum and OA was greater than that of their donor lines and the resistant control Zhongyou 821. In both glasshouse and field disease nurseries, disease indices on M083 and M004 were less than 50% of those of the control. The time required for M083 and M004 to mature was 14 days and 10 days shorter, respectively, than that of their donor lines. Furthermore, M083 had more pods per inflorescence, a greater 1,000 seed weight and higher yield than its donor line. Random amplified polymorphic DNA characterisation showed that M083 had DNA band patterns that differed from its donor line.     

Isolation and cross-sensitivity of X-ray-sensitive mutants of V79-4 hamster cells

The V79-4 Chinese hamster line was mutagenized and surviving clones screened for X-ray sensitivity using a replica microwell technique. One slightly sensitive clone and 3 clearly sensitive clones were isolated from approximately 5000 screened, and designated irs 1 to irs 4. The 3 more sensitive clones showed different responses to the genotoxic agents mitomycin C (MMC), ethyl methanesulphonate (EMS) and ultraviolet light (UV). irs 1 showed considerable sensitivity to all the agents tested, in the order MMC much greater than EMS greater than UV. irs 2 and irs 3 had similar sensitivities to EMS and to UV (EMS greater than UV) but irs 3 was more sensitive than irs 2 to MMC. None of these mutants is identical in phenotype to previously published mutants.     

Spatial and temporal regulation of the metabolism of reactive oxygen and nitrogen species during the early development of pepper (Capsicum annuum) seedlings

Background and Aims The development of seedlings involves many morphological, physiological and biochemical processes, which are controlled by many factors. Some reactive oxygen and nitrogen species (ROS and RNS, respectively) are implicated as signal molecules in physiological and phytopathological processes. Pepper (Capsicum annuum) is a very important crop and the goal of this work was to provide a framework of the behaviour of the key elements in the metabolism of ROS and RNS in the main organs of pepper during its development.Methods The main seedling organs (roots, hypocotyls and green cotyledons) of pepper seedlings were analysed 7, 10 and 14 d after germination. Activity and gene expression of the main enzymatic antioxidants (catalase, ascorbate–glutathione cycle enzymes), NADP-generating dehydrogenases and S-nitrosoglutathione reductase were determined. Cellular distribution of nitric oxide (^·NO), superoxide radical (O₂^·–) and peroxynitrite (ONOO^–) was investigated using confocal laser scanning microscopy.Key Results The metabolism of ROS and RNS during pepper seedling development was highly regulated and showed significant plasticity, which was co-ordinated among the main seedling organs, resulting in correct development. Catalase showed higher activity in the aerial parts of the seedling (hypocotyls and green cotyledons) whereas roots of 7-d-old seedlings contained higher activity of the enzymatic components of the ascorbate glutathione cycle, NADP-isocitrate dehydrogenase and NADP-malic enzyme.Conclusions There is differential regulation of the metabolism of ROS, nitric oxide and NADP dehydrogenases in the different plant organs during seedling development in pepper in the absence of stress. The metabolism of ROS and RNS seems to contribute significantly to plant development since their components are involved directly or indirectly in many metabolic pathways. Thus, specific molecules such as H₂O₂ and NO have implications for signalling, and their temporal and spatial regulation contributes to the success of seedling establishment.     

笃斯越桔耐弱碱突变体的离体筛选与鉴定

以笃斯越桔组培苗为试验材料,建立了EMS诱变体系,采用EMS诱变与NaHCO3共同胁迫处理的方法,筛选耐弱碱候选突变体,并进行了相关生理指标的鉴定,以明确其突变体株系的耐弱碱能力。结果显示:(1)0.4%EMS浸泡笃斯越桔茎段4h为较适宜的诱变剂量,存活率达46.7%;在含0.1%EMS的培养基中处理3d,存活率达50.0%;EMS浸泡处理后,NaHCO3适宜剂量为8mmol·L-1,经3次交替培养后,筛选出2株候选耐弱碱突变体。(2)相关生理指标鉴定表明,2株候选突变体的SOD和POD活性及游离脯氨酸含量显著高于对照,MDA含量显著低于对照。研究表明,该研究获得的笃斯越桔候选突变体具有一定的耐弱碱能力。     

Biological and biochemical characterisation of purine analogue resistant clones of V79 Chinese hamster cells

Purine analogue resistant clones have been selected from the closely related Chinese hamster lines V79A and V79S. Clones were of either spontaneous origin or induced by EMS or ultraviolet light. The majority of clones selected in 8-azaguanine showed stable cross resistance to 6-thioguanine. Clones derived from V79A and selected for 6-thioguanine resistance were cross resistant to 8-azaguanine: however a group of 6-thioguanine resistant mutants selected from V79S cells were 8-azaguanine sensitive. All clones except two were unable to grow in HAT medium. The two exceptions were 8-azaguanine resistant, showed partial sensitivity to 6-thioguanine, and also differed in other biochemical characteristics. HGPRT activity was measurable in extracts of all clones under standard conditions. In many clones, HGPRT activity increased as the hypoxanthine concentration was reduced. Whole cell uptake of [14C] hypoxanthine was low in all cases examined and was not modified by incubation in the presence of amethopterin. The heat sensitivity and electrophoretic mobility of HGPRT in extracts of some clones was compared to that in wild-type extracts. All clones tested except one, which was consistently HAT positive, showed enhanced heat sensitivity and reduced electrophoretic mobility. None of the mutants reverted spontaneously at detectable frequency but some could be induced to revert by EMS. The presence of measurable enzyme with altered properties in all clones suggests that these revertable drug resistant clones represent missense mutants.     

Characterization of a Discontinuous Neutralizing Epitope on Glycoprotein B of Human Cytomegalovirus

Human cytomegalovirus (HCMV) is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and newborn infants infected in utero. The viral envelope glycoprotein B (gB) is an attractive molecule for active vaccination and passive immunoprophylaxis and therapy. Using human monoclonal antibodies (MAbs), we have recently identified antigenic region 4 (AD-4) on gB as an important target for neutralizing antibodies. AD-4 is formed by a discontinuous sequence comprising amino acids 121 to 132 and 344 to 438 of gB of HCMV strain AD169. To map epitopes for human antibodies on this protein domain, we used a three-dimensional (3D) model of HCMV gB to identify surface-exposed amino acids on AD-4 and selected juxtaposed residues for alanine scans. A tyrosine (Y) at position 364 and a lysine (K) at position 379 (the YK epitope), which are immediate neighbors on the AD-4 surface, were found to be essential for binding of the human MAbs. Recognition of AD-4 by sera from HCMV-infected individuals also was largely dependent on these two residues, indicating a general importance for the antibody response against AD-4. A panel of AD-4 recombinant viruses harboring mutations at the crucial antibody binding sites was generated. The viruses showed significantly reduced susceptibility to neutralization by AD-4-specific MAbs or polyclonal AD-4-specific antibodies, indicating that the YK epitope is dominant for the AD-4-specific neutralizing antibody response during infection. To our knowledge, this is the first molecular identification of a functional discontinuous epitope on HCMV gB. Induction of antibodies specific for this epitope may be a desirable goal following vaccination with gB.     

Inhibition of α-Amylase Acting in Hexaploid Triticale Lines by Exogenous Abscisic Acid

Hexaploid triticale introgressive lines developed after recombination of A-genome with A^m-genome of diploid wheat (Triticum monococcum) were analysed in respect of grains responsiveness to exogenous ABA treatment. This was assessed by in vivo bioassay as grain germination indices, and by α-amylase assay as quantity of synthesised α-amylase measured with the technique of radial diffusion in agarose gel. The results showed an important diminishing of seedling length caused by ABA (variable in different lines) as well as genotype dependant variability of α-amylase synthesis inhibition. The differences of ABA responsiveness were seen both in whole grains and in embryoless half-grains as a direct reaction of the aleurone layer. Variation of grain sensitivity to ABA treatment compared with two sprouting resistance indices showed a significant correlation with Falling Number values in grains, but not with a dormant grains germination in spikes. This is an evidence that in triticale precocious starch decompose in unripened and ungerminated grains is dependent on genotype ABA-responsiveness of the aleurone layer. This revised version was published online in July 2006 with corrections to the Cover Date.