甜叶菊同源四倍体离体诱导及鉴定

用不同浓度秋水仙素溶液处理甜叶菊不定芽,诱导同源四倍体,并进行解剖学、染色体鉴定和流式细胞仪鉴定倍性。结果表明:(1)用0.20%的秋水仙素溶液浸泡甜叶菊不定芽12h,同源四倍体诱导率最高,可达32.14%。(2)同源四倍体植株与二倍体(对照)相比,其气孔、叶片等均表现巨大性,且叶片变厚、叶色浓绿、叶片皱缩。(3)对照植株染色体2n=2x=22,四倍体植株染色体2n=4x=44;流式细胞仪倍性鉴定结果显示,对照DNA相对含量为100,四倍体DNA相对含量为200。(4)该研究共鉴定出48株甜叶菊同源四倍体植株,为进行倍性植株的诱导奠定了技术基础,为进一步开展甜叶菊同源四倍体新品种的选育提供了实验材料。     

铁皮石斛四倍体离体诱导和鉴定及生理特性研究

以铁皮石斛原球茎为材料,经不同质量浓度的秋水仙素(C₂₂H₂₅O₆)和0.02 g·mL^-1二甲基亚砜(DMSO)混合水溶液处理后进行组织培养,通过对变异株进行形态学、细胞学及流式细胞仪鉴定,以期获得稳定的四倍体植株并分析其生理特性。结果表明:用2.0 g·L^-1秋水仙素和0.02 g·mL^-1 DMSO混合水溶液处理铁皮石斛原球茎36 h后,植株诱导率达20%;诱导四倍体植株在形态上明显矮化、茎秆粗壮、叶片变小增厚、气孔直径增大;细胞遗传学观察发现,四倍体植株染色体2n=4x=76,二倍体植株染色体2n=2x=38;流式细胞仪分析显示,DNA相对含量四倍体为400,二倍体仅为200;四倍体植株叶片中叶绿素含量、可溶性蛋白、可溶性糖含量均高于二倍体,分别为5.03、3.59、2.98 mg·g^-1;四倍体叶片中主要抗氧化酶POD和SOD活性均显著高于二倍体,分别为9.08、180.4 U·mg^-1,且四倍体植株明显降低了MDA含量累积。研究认为,2.0 g·L^-1秋水仙素和0.02 g·mL^-1 DMSO混合水溶液处理原球茎36 h可提高诱导成功率、降低嵌合体比例,此组合为诱导四倍体较佳诱导条件。     

光皮桦组织培养离体再生研究

采用正交试验等设计,系统开展了光皮桦组织培养高效再生体系研究。结果表明:光皮桦茎段外植体最佳诱导培养基及激素组合为MS+0.50mg.L-1 6-BA+0.10mg.L-1 TDZ+30mg.L-1蔗糖+5.50g.L-1琼脂,丛生芽最佳生根培养基为1/2MS+1mg.L-1 IBA+20g.L-1蔗糖+5.50g.L-1琼脂;丛生芽在最佳生根培养基上培养15d后获健壮生根苗,移栽成活率达90%。该实验结果为光皮桦的优良品种快繁以及遗传转化体系建立奠定了良好的基础。     

粤紫萁离体再生技术研究

粤紫萁(Osmunda mildei)为新近形成的自然杂交种,其分布区狭窄、种群个体数量少、有性繁殖能力极低.该文以孢子为外植体,以MS、3/4MS、1/2MS和1/4MS为基本培养基,探讨不同生长调节剂对粤紫萁孢子萌发、原叶体增殖和孢子体形成的影响.结果表明:粤紫萁孢子中极少的可育孢子在植物生长调节剂的作用下,能够萌...     

In vitro assembly of gap junctions

Gap junction structures were assembled in vitro from octyl-β- -glucopyranoside-solubilized components of lens fiber cell membranes. Individual pore structures (connexons), short double-membrane structures, and other amorphous material were evident in the solubilized mixture. Following the removal of the detergent by dialysis, these connexons associated to form single- and double-layered, two-dimensional hexagonal arrays (unit cell size a = B = 8.5 nm). The formation of larger arrays was dependent on the lipid-to-protein ratio and the presence of Mg²⁺ ions. Crystallographic analysis of electron micrographs revealed that lens junctional connexons consisted of six subunits surrounding a stain-filled channel. Upon further detergent treatment, in vitro assembled gap junctions were insoluble and formed three-dimensional stacks while other components were solubilized. SDS-PAGE and mass data from scanning transmission electron microscopy strongly suggest that a 38-kDa polypeptide, which is a processed form of the lens specific gap junction protein MP70, is a major component of the arrays. The in vitro assembly of gap junctions opens new avenues for the structural analysis of gap junctions and for the study of the intermolecular interactions of connexons during junctional assembly.     

pNEgr-mIL-12真核表达载体的体外和体内生物学活性检测

肿瘤的基因-放射治疗是近年来发展起来的新技术.分别于体外和体内检测含辐射敏感启动子和mIL-12基因的真核表达载体（pNEgr-mIL-12）的生物学活性.体外经酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)法检测转染pNEgr-mIL-12重组质粒的COS-7和B16细胞可经辐射诱导mIL-12 p70表达,于1.5～2.0 Gy照射后表达增高最明显,COS-7细胞于照后4 h 达峰值,B16细胞的表达水平随照射后时间延长而逐渐增高;pNEgr-mIL-12重组质粒联合电离辐射治疗小鼠移植肿瘤,单次或多次注射pNEgr-mIL-12重组质粒,联合局部照射能够抑制小鼠移植肿瘤生长,与单纯照射组比较肿瘤生长速度减慢,瘤重降低,尤以多次给予质粒治疗组效果明显.为进一步探讨最佳治疗方案及临床肿瘤病人的基因放射治疗提供了初步依据.     

hhlim对心肌肥大的影响及其作用机制探讨

hhlim是从胎儿心脏中新近分离和克隆得到的与心脏发生相关的基因,其表达产物作为转录因子参与多种基因的转录调控和细胞的发育与分化过程.用细胞转染方法将外源性hhlim基因导入原代心肌细胞,发现该基因强制性表达可使心肌细胞体积明显增大.RT-PCR和蛋白质印迹结果表明,hhlim促心肌细胞肥大与诱导α-肌动蛋白(α-actin)过表达及重新启动胚胎期表达基因脑钠肽（BNP）表达有关.用可表达hhlim反义RNA的真核表达载体转染心肌细胞后,致心肌细胞肥大因子ET-1对BNP和α-actin表达的诱导受到显著抑制.这些结果表明,ET-1促进BNP和α-actin表达及引发心肌肥大的效应可能由hhlim所介导,提示hhlim表达与心肌细胞肥大的启动有关.单独或共转染转录因子hhlim、Nkx2.5、GATA-4表达质粒和BNP转录调控区指导的报告基因结果显示,hhlim强制性表达不仅能直接激活BNP基因表达,而且与NKx2.5具有协同作用.结果表明,hhlim可以通过直接或与Nkx2.5协同作用激活BNP基因的表达.     

In vitro production of solasodine from Solanum trilobatum

Sucrose concentration in the culture medium affected chlorophyll content, trichome development and amount of solasodine in regenerated plantlets of Solanum trilobatum. High chlorophyll content and glandular trichomes were observed in the plants grown on Murashige and Skoog basal medium supplemented with 131.85 mM sucrose. The solasodine was quantified using reverse phase high performance liquid chromatography. The plantlets cultivated on this medium yielded 35.97 mg g⁻¹ (d.m.) solasodine whereas the field plants used as control yielded only 2.32 mg g⁻¹ (d.m.) of solasodine.     

In vitro biohydrogenation of four dietary fats

This study was designed to determine in vitro rates of biohydrogenation of dietary unsaturated fatty acids by a mixed population of rumen microbes. The four dietary fats [Alifet High-Energy^® (AHE), Alifet-Repro^® (AR), Megalac^® (MG), and Energy Booster^® (EB)] differ in method of preparation, fatty acid composition, or both of these factors. Dietary fats (20 mg) were incubated with 4 mL strained rumen fluid diluted with 16 mL of medium, 0.8 mL of reducing solution buffer, and 200 mg of a synthetic diet (370 g cellulose, 370 g starch, and 160 g casein per kg DM) at 37 °C. Total contents were collected after 0, 6, 12, 24, or 36 h and change in fatty acid content determined. Disappearance of oleic acid was minimal (0.05–0.20) in AR and MG but moderate (about 0.60) in AHE and EB after 36 h of incubation. Rate of biohydrogenation of linoleic and linolenic acids from AR were similar (0.025 ± 0.009 h⁻¹) and 0.65 of these fatty acids remained intact after 36 h. Rate of biohydrogenation of linoleic acid was four times greater than for oleic acid (0.040 ± 0.013 h⁻¹ versus 0.009 ± 0.002 h⁻¹) in MG. Thus, 0.65 of the linoleic acid but only 0.20 of the oleic acid had disappeared from MG after 36 h. Trans-11 and trans-12 were the predominant trans-isomers in AHE and AR cultures whereas trans-9 and trans-10 were the predominant trans-isomers in EB and MG cultures. None of the dietary fats contained conjugated linoleic acid (CLA) but CLA was present in the incubation inoculum. The amount of CLA decreased with time but this was not affected by source of dietary fat. Most (0.90–0.95) of the long-chain fatty acids eicosapentaenoic (EPA) and docosahexaenoic (DHA) in AR remained after 36 h of incubation. Results demonstrate that biohydrogenation varied among fatty acids and among source of dietary fat and indicate that AR can be used to increase post-ruminal supply of linolenic, EPA and DHA.     

In vitro organogenesis of Citrus volkameriana and Citrus aurantium

In vitro organogenesis of Citrus volkameriana and C. aurantium was studied considering three explant types: epicotyl segment, internodal segment, and hypocotyl segment with attached cotyledon fragment. The explants were cultured in medium according to Grosser and Gmitter (EME) supplemented with 0, 0.5, 1.0, 1.5, and 2.0 mg dm^{− 3} 6-benzyl-aminopurine (BAP), incubated firstly in darkness for 4 weeks, and then transferred to 16-h photoperiod for 2 weeks. Comparing epicotyl and internodal segments, a higher percentage of responsive explants and a higher number of shoots per explant were obtained with epicotyl segments, regardless of the BAP concentration. For C. volkameriana the highest percentage of responsive epicotyl segments (42 %) was obtained in EME with 1.0 mg dm⁻³ BAP, while for C. aurantium (59 %) in EME with 0.5 mg dm⁻³ BAP. The organogenesis efficiency was the best with the use of the hypocotyl segment with attached cotyledon fragment (77 % for C. volkameriana and to 75 % for C. aurantium). With this explant the morphogenesis occurred only in the hypocotyl region. The in vitro organogenesis was characterized by histological analyses showing that the morphogenic process started in the cambium region near the explant cut end.     

广藿香不同外植体离体培养的研究

以广藿香叶片、带节茎、不带节茎及根尖为材料进行离体培养,对影响离体再生的因素进行了研究。结果表明:BA有利于广藿香外植体出芽,浓度以0.1～0.5mg/L效果较好;不同外植体的出芽能力有较大差异,其中以叶片和带节茎出芽能力较强,出芽率均达100%;外植体在培养基上的接种方式对出芽也有一定影响,带节茎以形态学下端垂直插入,可以缩短出芽时间及增加单个外植体出芽的数量。无根苗生根以MT+IBA0.2mg/L培养基为好,苗的生长较为健壮。     

以转录因子AP-1为靶的decoy核酸体内外抗肿瘤研究

合成了双链寡聚核苷酸——decoy核酸,其与靶转录因子AP-1有高亲和性,可进入细胞作为decoy顺式元件,通过抑制特异的转录因子和调控区域的结合,调控基因转录而改变基因的表达.在体内外抗肿瘤试验中, decoy核酸有显著抑制肿瘤细胞增殖的作用,可以成为潜在性的肿瘤基因治疗药物.     

In vitro effects of propolis on Giardia duodenalis trophozoites

In order to improve the current chemotherapy of Giardia infection, potential antigiardial agents have been screened, including natural products. Propolis, a resinous hive product collected by bees, has attracted attention as a useful and popular substance with several therapeutic activities. The present study was carried out aiming to evaluate the in vitro effects of an ethanolic extract of propolis on the growth and adherence of Giardia duodenalis trophozoites. Propolis inhibited the growth of trophozoites and the level of inhibition varied according to the extract concentration and incubation times. The highest reduction of parasite growth was observed in cultures exposed to 125, 250 and 500 microg/ml of propolis, in all incubation periods (24, 48, 72 and 96 h). Growth reduction by 50% was observed in 125 microg/ml propolis-treated cultures, while the concentrations of 250 and 500 microg/ml were able to inhibit growth by more than 60%. Propolis also inhibited parasite adherence and all assayed propolis concentrations promoted the detachment of trophozoites. Light microscope observations revealed changes of the pear-shaped aspect of the cell and reduction of flagellar beating frequency in the great part of the trophozoites. Our results hold the perspective for the utilization of propolis as an antigiardial agent.     

In vitro chromosome doubling of nineZantedeschia cultivars

Tetraploid plants have been produced from nineZantedeschia cultivars usingin vitro colchicine treatment. Rapidly-multiplying shoot cultures were treated on a medium containing 0.05% colchicine for 1, 2 or 4 days to induce chromosome doubling. Following the treatment, most shoots were killed but the surviving shoots were multiplied for several subcultures. These shoots were then rootedin vitro and transferred to a greenhouse. Plants were screened 2 months later by measuring stomatal length, and 110 out of 565 plants were selected as putative tetraploids with a stomatal length significantly greater than in diploid control plants. Chromosome counts were carried out on root tips from 44 plants and confirmed that 38 were tetraploids, 2 were chimeras (predominantly tetraploid with a few octoploid cells), and 4 were diploids. Stomatal length has been rechecked in mature tetraploid plants of the cultivar Black Magic, demonstrating that stomatal length is a good indicator of ploidy level inZantedeschia. This study has shown that multiplying colchicine-treated shootsin vitro for several subcultures prior to transfer to soil produced very few chimeras. The stomatal length measurements are non-destructive and allow the rapid screening of a population for tetraploids.     

Chelating agent inhibition of Trypanosoma cruzi epimastigotes In vitro

A number of chelating agents and some of their derivatives are as effective as, or superior to, benznidazole, the compound currently in clinical use, in the suppression of the reproduction of epimastigotes of Trypanosoma cruzi, the protozoa that causes Chagas' disease. All compounds were examined at a culture concentration of 5 μg/mL. The most effective compounds included N, N, N′, N′-tetrakis(2-pyridylmethyl)ethylenediamine, sodium diethylamine-N-carbodithioate, piperidine-N-carbodithioate and several of its analogs, a number of other carbodithioates with two nonpolar groups on the nitrogen, and tetraethylthiuram disulfide, a prodrug of sodium diethylamine-N-carbodithioate and widely used in the treatment of alcoholism. The introduction of additional ionic or nonionic polar groups on the chelating molecule generally results in a loss of tyrpanocidal activity. Common commercially available chelating agents which exhibited no activity included -penicillamine, meso-2,3-dimercaptosuccinic acid, and triethylenetetramine tetrahydrochloride. Dose-response data on the culture indicated that some of these compounds exhibited inhibition of Trypanosoma cruzi epimastigotes at concentrations as low as 0.625 μg/mL. It is proposed that the mechanism of action of these compounds is based on their ability to interface with the essential metal metabolism at intracellular sites of the epimastigote involving iron, copper, or zinc. The results also indicate that a certain degree of hydrophobicity may be necessary for the groups attached to the literal metal-bonding structure if the compounds are to successfully inhibit the epimastigotes of Trypanosoma cruzi. The development of antiprotozoal drugs which are chelating agents specifically designed to selectively disrupt the essential metal metabolism of Trypanosoma cruzi should furnish a new generation of drugs which can be used in the treatment of Chagas' disease.     

In vitro cloning of Azadirachta indica from root explants

In vitro cultures of Azadirachta indica A. Juss. were raised by first culturing the root segments on modified Murashige and Skoog (MS) medium supplemented with 8.88 μM 6-benzylaminopurine (BAP), 9.84 μM N⁶-(2-isopentenyl) adenine (2iP), 5.71 μM indole-3-acetic acid (IAA), 81.43 μM adenine hemisulphate and 2.27 μM putrescine for 2 d followed by their transfer to the same medium except containing one-tenth of the initially used concentrations of BAP, 2iP and IAA. The regenerated shoots sustained proliferation in the basal medium supplemented with 1.11 μM BAP, 1.43 μM IAA and 135.72 μM adenine hemisulphate. The isolated shoots were rooted to produce plantlets in the presence of 2.46 μM indole-3-butyric acid (IBA). The plantlets showed uniform luxuriant growth under field conditions. True-to-type nature of the field-grown root-regenerated plants was ascertained by random amplified polymorphic DNA (RAPD) analysis.     

斑鸠霉素氧化酶CftA体外生物活性研究

[背景]斑鸠霉素及其氧化衍生物属于多环特特拉姆酸大环内酰胺类化合物,具有良好的生物活性,挖掘更多新颖斑鸠霉素类似物具有重要意义.细胞色素P450氧化酶CftA被认为能催化斑鸠霉素的氧化反应,但未有相关体外实验数据的报道.[目的]通过体外生化实验鉴定氧化酶CftA的功能,并探索其催化斑鸠霉素氧化反应的机制.[方法]利用基...     

In vitro responses of grape rootstocks to NaCl

An investigation was undertaken to adjudge the in vitro salt tolerance and biochemical changes due to NaCl on four grape rootstocks (Dogridge, SO4, H-144 and 3309C). The in vitro two-node micro-cuttings from the established cultures were sub-cultured on rooting medium comprising Murashige and Skoog (MS) basal medium supplemented with 0.5 μM indolebutyric acid (IBA), 200 mg dm⁻³ activated charcoal and different NaCl concentrations ranging from 0 to 125 mM. The surviving and proliferated cultures were further sub-cultured four times on respective media. Dogridge and H-144 tolerated 125 and 100 mM NaCl, respectively, while SO4 and 3309C survived only up to 75 mM NaCl. Contents of proteins, proline, K⁺ and Na⁺ in tissue increased in all genotypes due to NaCl supplementation, while contents of chlorophyll and total soluble sugars declined. Higher K⁺/Na⁺ ratio was registered in Dogridge and H-144 than in SO4 and 3309C. The relative NaCl tolerance for different grape rootstocks under study could be ranked as Dogridge > H-144 > SO4 and 3309C.     

In vitro chiral nematic ordering of chitin crystallites

Microfibrillar fragments of purified crab and shrimp chitin were prepared by hydrolysis in 3 HCl at its boiling point (104°C). After removal of the acid by centrifugal washing and dialysis, an ultrasound treatment converts the residual product to a colloidal suspension stabilized by NH₃⁺ charges. When dewatered to a critical concentration, spontaneous formation of a two-phase equilibrium system occurs. The upper phase (lower concentration) is isotropic and the lower phase is anisotropic. The latter displays chiral nematic order and dries to a solid film which mimics the helicoid organization characteristic of the chitin microfibrils in the cuticle of arthropods.