The physiological state of suspension cultured cells affects the expression of the β-glucuronidase gene following transformation of tall fescue (Festuca arundinacea) protoplasts

Both the stage of the growth cycle and the age of the cell culture used to isolate protoplasts had a pronounced effect on both transient and stable expression of the GUS gene. A level of GUS gene transient expression of 9000 pmol 4MU/μg protein/h and a frequency of GUS gene stable expression of 5.72% were obtained with protoplasts isolated from suspension cultures 10–20 weeks after initiation and 3–4 days after subculturing when an optimized transformation protocol and a rice actin 1 promoter-uidA gene construct were used. The effect of the cell growth cycle on GUS gene transient expression was closely correlated with the growth rate and the rate of protein synthesis in cell cultures whereas prolonged subculturing of the cells resulted in a gradual decline in both transient and stable expression. The length of time cells were digested in cell wall digestion enzyme and the osmolarity of the transformation medium were found to critically affect both the level of transient and stable GUS gene expression. The composition and osmolarity of the protoplast culture medium was less critical for transient GUS gene expression although the osmolarity of the medium was shown to have a significant effect on stable expression of the GUS gene.     

日本曲霉产生的黑麦酮酸F对玉米的化感作用

日本曲霉(Aspergillus japonicus)是土壤和谷物种子表面的一种常见真菌.研究结果表明,日本曲霉所产生的大量黑麦酮酸F(SAF)对玉米(Zea mays)有很强的化感作用,低浓度显著促进玉米幼苗生长,高浓度则抑制.在0.0375mmol·L^-1SAF下,玉米幼苗根长增长31.7%,根数量增加13.2%,根活力提高4.73倍,并促进玉米对P、K、Ca、Mg、S等5种营养元素的吸收.高浓度SAF(0.3mmol·L^-1)下玉米根活力受抑制(72.1%),根对N、P、K、Ca、Mg、Fe等营养元素的吸收也受抑制.0.6mmol·L^-1的SAF下根完全失去活力.电镜观察表明,有SAF的情况下玉米叶绿体片层结构模糊、混乱,双层膜不完整.     

大豆fad基因反义表达载体构建及转化烟草研究

使用PCR方法从大豆基因组DNA中扩增出大豆油酸脱饱和酶基因fad2-1,连接到pMD18-T载体中,转化大肠杆菌JM109菌株.测序后,用DNAstar软件进行同源性比对.然后将正确的序列反向克隆到表达载体pBt,并转化农杆菌菌株LBA4404,经双酶切鉴定和PCR扩增检测,获得具有该基因反向序列的农杆菌工程菌,转化...     

Identification and characterisation of an RPD3 homologue from maize ( Zea mays L.) that is able to complement an rpd3 null mutant of Saccharomycescerevisiae

In mammals, yeast and Drosophila, the histone deacetylase RPD3 proteins can alter the expression of genes involved in fundamental biological processes by affecting the degree of acetylation of histones and changing chromatin structure. Here we report the isolation of a cDNA sequence encoding an RPD3 homologue from maize, which is able to complement the phenotype of an rpd3 null mutant of the yeast Saccharomyces cerevisiae. The expression of the corresponding gene(s) was assessed in different maize tissues. The number of homologous loci was estimated by Southern hybridisation to be in the range of two to three, and the chromosomal location of one of these loci was determined. Phylogenetic analysis and tests for relative divergence rates, using related RPD3 sequences from different species, were performed, and suggest that different polymorphic forms of RPD3-like proteins that evolve at distinct rates are present in the species considered. Received: 5 December 1997 / Accepted: 16 January 1998     

Mapping of 5-methylcytosine residues in Nicotiana tabacum 5S rRNA genes by genomic sequencing

Genomic sequencing was used to localise 5-methylcytosine residues in individual DNA strands of 5S rRNA genes in tobacco. The density of methylation along the sequence was high in both strands, exceeding the average methylation density of the tobacco genome. Besides methylation of CG and CNG sequences, considerable amounts of mC were found in non-symmetrical sites. Among 69 sequenced clones obtained from leaf DNA we did not detect any non-methylated clone, and Southern blot hybridisation analysis also failed to suggest the presence of methylation-free 5S rDNA units in the tobacco genome. Differences were observed among methylation patterns of individual sequenced clones. This heterogeneity reflects either heterogeneity among individual members of 5S rRNA gene cluster or differences among individual cells. Methylation of CNG and non-symmetrical sites can be efficiently reduced by treatment with dihydroxypropyladenine, an inhibitor of S-adenosylhomocysteine hydrolase. Received: 28 January 1998 / Accepted: 29 April 1998     

Differential sensitivity of CG and CCG DNA sequences to ethionine-induced hypomethylation of the Nicotiana tabacum genome

Plant DNA is distinguished from the DNA of all other organisms by its high content of 5-methylcytosine (5mC). 5mC levels may amount to 30% of total cytosines, distributed between the sequences CG and CXG. The results presented here show that the methylation status of CXG sequences could be influenced by culturing tobacco tissues on subtoxic concentrations of ethionine. The hypomethylating effect of ethionine, evaluated as the capability of MspI or HpaII to cleave the DNA, proved to be rather specific for CCG and differed from that or 5-azacytidine which did not discriminate between CG and CXG sequences.     

Immunological evidence for the presence of plant homologues of the actin- related protein Arp3 in tobacco and maize: subcellular localization to actin-enriched pit fields and emerging root hairs

Summary. The actin-nucleating and -organizing Arp2/3 protein complex is well known to be conserved throughout the eukaryotic kingdom. For higher plants, however, only limited evidence is available for the presence of the Arp2/3 complex so far. Using heterologous antibodies against the Dictyostelium discoideum and Schizosaccharomyces pombe proteins and a bovine peptide, we found immunological evidence for the presence of Arp3 homologues in plants. First, proteins with a molecular mass of about 47–50 kDa were clearly recognized in extracts of both a dicotyledonous plant (tobacco) and a monocotyledonous plant (maize) in immunoblots with the anti-Arp3 antibodies. Second, immunolocalization with these Arp3 antibodies was performed on different plant cells, selected for their diverse actin organizations and functions. On isolated plasma membrane ghosts derived from tobacco leaf protoplasts, a putative Arp3 was localized along cortical actin filaments. In the inner cortex of maize roots, Arp3 was localized to actin-rich plasmodesmata and pit fields and to multivesicular bodies in the cytoplasm. During root hair formation, distinct site-specific localization was found at the protruding apical plasma membrane portions of these tip-growing cells.Correspondence and reprints: Department of Biology, Universitaire Instelling Antwerpen, Universiteitsplein 1, 2610 Wilrijk, Belgium.Received January 3, 2003; accepted February 7, 2003; published online August 26, 2003     

Stimulatory effects of potassium iodide on auxin action in the coleoptile segments of maize (Zea mays)

Potassium iodide (KI) was found to stimulate IAA-induced elongation of coleoptile segments in maize (Zea mays L.). The promoting effects of KI on coleoptile elongation, which were optimal at 1 mM in the presence of IAA, did not occur as a result of better conservation of IAA in the incubation medium. In addition, KI did not affect fusicoccin- or epibrassinolide-induced elongation. Additionally, sodium iodide (NaI) induced similar stimulatory effects on IAA-induced elongation, however, potassium chloride (KCl) showed no effect, suggesting that iodide is the active component. KI also enhanced IAA-induced ethylene biosynthesis in maize coleoptile segments. Taken together, these results suggest the involvement of KI-sensitive step(s) in auxin action before effectors of the signal transduction pathway split to elongation growth and ethylene biosynthesis. In-yong Hwang and Soo Chul Chang contributed equally to this work.     

DNA changes during sequential leaf senescence of tobacco (Nicotiana tabacum)

Age related DNA changes in tobacco (Nicotiana tabacum) leaf nuclei were investigated by Feulgen cytophotometry, thermal denaturation, renaturation, and DNA-DNA hybridization studies during sequential leaf senescence. Cytophotometric Feulgen-DNA comparison measurements between young and senescing nuclei displayed 18% reduction in Feulgen-DNA values, with a corresponding decrease in nuclear area in senescing nuclei. Hydrolysis kinetics indicated that the loss was not due to compactness of the DNA as the curves for older nuclei were consistently lower than curves generated from younger nuclei. DNA loss in senescing nuclei was associated with a decrease in euchromatin or shift from euchromatin to facultative heterochromatin. Purified DNA from young and senescing leaf nuclei did not display different thermal profiles nor did hydroxylapatite chromatography reassociation curves. DNA-DNA hybridization in free solution from young and senescing leaf DNA performed by a Gilford thermo-programmer system indicated that DNA of senescing tobacco nuclei reassociated more slowly than DNA from young nuclei and the mixture of young and senescing leaf DNA displayed intermediate reassociation values. The study indicates that the DNA changes during senescence involve a complex phenomenon which includes the possibility of small single strand nicks undetectable by thermal denaturation, and a loss of small double strand fragments which were detectable only by precise DNA-DNA free solution reassociation and not by hydroxylapatite chromatography reassociation.     

Endophytic colonisation of tobacco,corn, wheat and soybeans by the fungal entomopathogen Beauveria bassiana (Ascomycota,Hypocreales)

We demonstrate the effectiveness of three inoculation methods (foliar spray, seed immersion and root immersion) in establishing fungal the entomopathogen Beauveria bassiana as an endophyte in tobacco, corn, wheat and soybean. Colonisation of leaves by B. bassiana was assessed 7, 14, 21 and 28 days post-inoculation. There were significant differences (p < 0.001) in endophytic colonisation among the different inoculation techniques.     

Bioreactor operation for transgenic Nicotiana tabacum cell cultures and continuous production of recombinant human granulocyte-macrophage colony-stimulating factor by perfusion culture

The production of hGM-CSF was investigated in both a flask and a 5-l bioreactor, using transgenic Nicotiana tabacum suspension cells. While the maximum cell density and secreted hGM-CSF in the flask were 15.4 g l⁻¹ and 6.5 μg l⁻¹, respectively, those in the bioreactor were 15.6 g l⁻¹ and 7.6 μg l⁻¹. No detectable growth inhibition, shorter production of hGM-CSF and reduced cell viability in the batch bioreactor were observed under the specific conditions used compared with the flask culture. To improve the productivity, a perfusion culture was carried out in the bioreactor, with three different perfusion rates (0.5, 1.0 and 2.0 day⁻¹). In all cases, the hGM-CSF in the medium was significantly increased during the overall culture period (16 days), with maximum values 3.0-, 9.4- and 6.0-fold higher than those obtained in the batch cultures, respectively, even though the intracellular hGM-CSF content was not significantly varied by the perfusion rate. In terms of the total amount of hGM-CSF secreted, 205.5, 1073.2 and 1246.3 μg accumulated in the perfusate within 16 days at the perfusion rates of 0.5, 1.0 and 2.0 day⁻¹, respectively. It was concluded that the beneficial effect of perfusion on the production of hGM-CSF originated from the reduced proteolytic degradation due to the lower protease activity caused by the perfusion. Additionally, the cell growth and physiology in the perfusion culture were somewhat negatively affected by the increased perfusion rate, although the dry cell density steadily increased, and as a result, 19.4, 22.4 and 22.9 g l⁻¹ of maximum cells were obtained with perfusion rates of 0.5, 1.0 and 2.0 day⁻¹, respectively. This work highlighted the importance of proteolytic degradation in plant cell cultures for the production of secretory proteins and the feasibility of perfusion strategies for the continuous production of foreign proteins by the prevention of protein loss due to proteolytic enzymes.     

玉米KNOX基因家族鉴定及组织和逆境表达分析

为揭示玉米转录因子KNOX家族基因功能,采用生物信息学手段在玉米基因组水平鉴定KNOX家族成员,并对家族基因逆境和组织表达谱进行分析.结果 显示:(1)玉米基因组有22个ZmKNOX基因,根据其在染色体上的位置依次命名为ZmKNOX1-ZmKNOX22;编码蛋白质亚细胞定位预测发现,除ZmKNOX5、ZmKNOX11、...     

Naturally occurring neuronal NO-synthase inactivators found in Nicotiana tabacum (Solanaceae) and other plants

NO-synthase (NOS) is a heme-containing enzyme that catalyzes the oxidation of L-arginine to nitric oxide, an important cellular signaling molecule. Recently, it was found that aqueous extracts of tobacco cigarettes cause the inactivation of the neuronal isoform of NOS (nNOS) and that this may explain some of the toxicological effects of smoking. Although the exact identity of the chemical inactivator(s) is not known, we wondered if extracts prepared from other plants, including those closely related to tobacco, Nicotiana tabacum (Solanaceae), would similarly inactivate nNOS. We examined 33 plants, representing diverse members of the plant kingdom ranging from whisk fern, Psilotum nudum (Psilotaceae) to tobacco and discovered 18 plants that contain a chemical inactivator(s) of nNOS. Of these plants, 16 are members of the core asterids flowering plant group. Of these asterids, 6 are members of the Solanaceae family, of which tobacco is a member. Based on the phylogenetic relationship of the plants, it is possible that the same chemical or related chemical inactivator(s) exist. This, in turn, may help elucidate the structure of the chemical(s), as well as provide a source of a potentially novel inactivator of nNOS. The alkaloid nicotine can be excluded as putative nNOS inhibitor.     

玉米AGO基因的克隆与表达分析

以玉米自交系‘昌7-2’三叶期前后2个时间点(种子萌发后5d和8d)幼苗不同组织部位的总RNA为研究对象,采用实时荧光定量PCR技术,对玉米中6个Argonaute(AGO)蛋白家族基因(AGO1、AGO2、AGO4、AGO5、AGO7和AGO10)在幼苗不同发育时期及不同组织部位的表达谱进行了研究。结果表明:(1)AGO1、AGO2、AGO4和AGO7在种子萌发后5d和8d幼苗不同组织中均有表达,种子萌发后5d幼苗中的平均表达量均高于萌发8d的幼苗,且在地上部分新生组织或细胞分裂比较旺盛的组织中表达较多,表明AGO1、AGO2、AGO4和AGO7可能在玉米幼苗发育早期的分生组织分裂生长中发挥调控作用。(2)AGO5和AGO10只在叶片和茎尖中表达,其他组织中不表达;其中AGO5主要集中在新生叶和种子萌发后8d的茎尖中,AGO10在玉米叶发育过程中可能存在着迁移的现象。     

烟草花青素苷转运相关基因NtAN9的分离与表达分析

为研究花青素苷的转运,利用电子克隆和RT-PCR方法,从普通烟草(Nicotiana tabacum)花中分离了1个编码谷胱甘肽转移酶(GST)基因,命名为NtAN9(GenBank登录号KX356542)。NtAN9包含一个690bp的开放阅读框,编码229个氨基酸残基,属于phi型GST。NtAN9基因组结构由3个外显子和2个内含子组成。多序列比对分析表明,NtAN9与矮牵牛(Petunia hybrida)花青素苷转运相关的GST基因PhAN9具有88%的一致性。系统进化分析显示,NtAN9与花青素苷转运相关的GST基因聚为一支,是PhAN9的直系同源基因。定量PCR分析表明,NtAN9基因在含有花青素苷的四个花发育时期中均有表达,其中在开花前的第Ⅲ期(2cm花芽4cm)表达丰度达到最高,而在不含花青素苷的根、茎和叶中不表达。由此推测,分离得到的NtAN9可能具有类似PhAN9的功能,与烟草花青素苷的转运与积累相关。NtAN9基因的分离与表达分析,为进一步研究烟草花青素苷的转运奠定了基础。     

外源基因导入对玉米叶片物理性状的影响

【目的】探讨外源基因导入对玉米叶片物理性状的影响,为转基因玉米的安全性评价提供基础资料,也为转基因玉米的科学、有效利用提供依据。【方法】在扬州大学实验农牧场种植大北农转基因(转Cry Ab和epsps基因)和大北农(对照)、IE09S034转基因(转Cry IE基因)和IE09S034(对照)、808-双抗-12-5转基因(转Cry Ab/cry2Aj和Gloevo-epsps基因)和808瑞丰-1(对照)3对玉米品种,室内测定了不同时期(苗期、穂期和花粒期)各品种叶片的蜡质含量、叶绿素含量、茸毛密度、维管束埋深及Si、K、Ca、S、P和Cl含量。【结果】转基因玉米的叶片中蜡质含量、叶绿素含量和维管束埋深较对应的常规亲本品种大,而叶片茸毛密度则较对应的常规亲本品种小。其中,穗期的大北农、IE09S034和808-双抗-12-5转基因品种叶片蜡质含量分别较对照高17.95%、48.30%和39.31%;IE09S034和808-双抗-12-5转基因品种穗期叶片的维管束埋深分别较对照高13.70%和9.21%,花粒期分别高10.81%和14.47%;IE09S034和808-双抗-12-5转基因品种穗期叶片的叶绿素含量分别较对照高18.11%和13.13%,花粒期分别高16.62%和14.61%;大北农、IE09S034和808-双抗-12-5转基因品种花粒期叶片茸毛密度分别较对照低17.70%、17.43%和17.78%。3个品种的转基因玉米叶表面元素含量均大于相应的对照。其中,与常规亲本相比,穗期大北农转基因品种叶片中Ca和S含量分别高64.71%和61.18%,IE09S034转基因品种叶片中Si、Ca、S、P和Cl含量分别高110.26%、16.67%、44.44%、46.32%和20.00%,808-双抗-12-5转基因品种叶片中Si、Ca、S和P含量分别高34.78%、50.52%、115.47%和20.41%。【结论】外源基因的导入会诱导玉米叶片相关物理性状的改变。     

Genome size and numerical polymorphism for the B chromosome in races of maize (Zea mays ssp. mays, Poaceae)

Twenty-one native populations (1120 individuals) of maize from Northern Argentina were studied. These populations, which belong to 13 native races, were cultivated at different altitudes (80-3620 m). Nineteen of the populations analyzed showed B chromosome (Bs) numerical polymorphism. The frequency of individuals with Bs varied from 0 to 94%. The number of Bs per plant varied from 0 to 8 Bs, with the predominant doses being 0, 1, 2, and 3. Those populations with varying number of Bs showed a positive and statistically significant correlation of mean number of Bs with altitude. The DNA content, in plants without Bs (A-DNA)(2n = 20), of 17 populations of the 21 studied was determined. A 36% variation (5.0-6.8 pg) in A-DNA content was found. A significant negative correlation between A-DNA content and altitude of cultivation and between A-DNA content and mean number of Bs was found. This indicates that there is a close interrelationship between the DNA content of A chromosomes and doses of Bs. These results suggest that there is a maximum limit to the mass of nuclear DNA so that Bs are tolerated as long as this maximum limit is not exceeded.     

Organization of telomeric and subtelomeric chromatin in the higher plant Nicotiana tabacum

We have examined the structure and chromatin organization of telomeres in Nicotiana tabacum. In tobacco the blocks of simple telomeric repeats (TTTAGGG)_n are many times larger than in other plants, e.g., Arabidopsis thatiana or tomato. They are resolved as multiple fragments 60–160 kb in size (in most cases 90–130 kb) on pulsed-field gel electrophoresis (PFGE) of restriction endonuclease-digested DNA. The major subtelomeric repeat of the HRS60 family forms large homogeneous blocks of a basic 180 by motif having comparable lengths. Micrococcal nuclease (MNase) cleaves tobacco telomeric chromatin into subunits with a short repeat length of 157±5 bp; the subtelomeric heterochromatin characterized by tandemly repeated sequences of the HRS60 family is cut by MNase with a 180 by periodicity. The monomeric and dimeric particles of telomeric and subtelomeric chromatin differ in sensitivity to MNase treatment: the telomeric particles are readily digested, producing ladders with a periodicity of 7 bp, while the subtelomeric particles appear to be rather resistant to intranucleosomal cleavage. The results presented show apparent similarities in the organization of telomeric chromatin in higher plants and mammals.     

Effect of rhizosphere and plant-related factors on the cadmium uptake by maize (Zea mays L.)

The characteristics of 12- and 24-day-old maize plants (Zea mays L. cv MB862) related to Cd absorption were investigated with respect to the influence of Cd concentration in the plant organs, to plant age, absorption time and competition with micronutrients. Despite high Cd concentrations in the nutrient solution, hydroponically cultivated maize did not seem to be affected by Cd toxicity, except for the highest Cd level (100 μmol L⁻¹). There was on average five times more Cd in roots than in shoots and the Cd root to shoot ratio increased with increasing Cd concentration in the nutrient solution. No significant differences were observed between influx measured for 2 h in the middle of the day light period and for a full day period. Plants with different internal Cd concentrations showed similar root absorption characteristics of this metal. The root Cd influxes were three times higher in solutions with low micronutrient contents than in the solutions with higher micronutrient contents, and almost three times higher in 12-day-old roots than in 24-day-old roots. The root Cd influx was linearly related to its concentration in the solution, showing no saturable component. Our results suggest a non-specific and unregulated transport of Cd into the maize root symplast. They also indicate a regulation of the Cd translocation from root to shoot, as well as dependence of parameters of Cd root absorption on plant and rhizosphere conditions which should be taken into account for Cd uptake modelling.