Color recovery after photoconversion of H2B::mEosFP allows detection of increased nuclear DNA content in developing plant cells

Many higher plants are polysomatic whereby different cells possess variable amounts of nuclear DNA. The conditional triggering of endocycles results in higher nuclear DNA content (C value) that in some cases has been correlated to increased cell size. While numerous multicolored fluorescent protein (FP) probes have revealed the general behavior of the nucleus and intranuclear components, direct visualization and estimation of changes in nuclear-DNA content in live cells during their development has not been possible. Recently, monomeric Eos fluorescent protein (mEosFP) has emerged as a useful photoconvertible protein whose color changes irreversibly from a green to a red fluorescent form upon exposure to violet-blue light. The stability and irreversibility of red fluorescent mEosFP suggests that detection of green color recovery would be possible as fresh mEosFP is produced after photoconversion. Thus a ratiometric evaluation of the red and green forms of mEosFP following photoconversion could be used to estimate production of a core histone such as H2B during its concomitant synthesis with DNA in the synthesis phase of the cell cycle. Here we present proof of concept observations on transgenic tobacco (Nicotiana tabacum) Bright Yellow 2 cells and Arabidopsis (Arabidopsis thaliana) plants stably expressing H2B::mEosFP. In Arabidopsis seedlings an increase in green fluorescence is observed specifically in cells known to undergo endoreduplication. The detection of changes in nuclear DNA content by correlating color recovery of H2B::mEosFP after photoconversion is a novel approach involving a single FP. The method has potential for facilitating detailed investigations on conditions that favor increased cell size and the development of polysomaty in plants.     

Changes in chloroplast DNA during development in tobacco, Medicago truncatula, pea, and maize

We examined the DNA from chloroplasts obtained from young and fully expanded leaves of tobacco (Nicotiana tabacum L.), Medicago truncatula, pea (Pisum sativum L.), and maize (Zea mays L.). The changes in plastid DNA content and structure were monitored by four independent methods: 4′,6-diamidino-2-phenylindole (DAPI) staining with intact chloroplasts, in situ DAPI staining of cytological sections, ethidium bromide staining at the single-molecule level after exhaustive deproteinization of lysed chloroplasts, and pulsed-field gel electrophoresis. During leaf development, we found a decline of chloroplast DNA (cpDNA) in all four plants. For tobacco, for which plants can readily be regenerated from somatic cells, cpDNA persisted longer than in the other three plants. We also found a striking progression from complex multigenomic DNA molecules to simple subgenomic molecules during plastid development. Although the decrease in molecular size and complexity paralleled the decrease in DNA content per plastid, 6% of the chloroplasts in a fully expanded tobacco leaf still contained DNA in complex branched structure, whereas no such complex structures were found in mature leaves for the hard-to-regenerate maize.     

The mustard trypsin inhibitor 2 affects the fertility of Spodoptera littoralis larvae fed on transgenic plants

The effects of mustard trypsin inhibitor MTI-2 expressed at different levels in transgenic tobacco lines have been evaluated by feeding the lepidopteran Spodoptera littoralis throughout its larval life. Specific conditions were selected to study the long-term effects of feeding larvae on transgenic plants expressing the inhibitor at various levels. The data obtained led to the establishment of three relevant parameters to be considered during the experimentation: (i) the PI content of the plant lines to be used; (ii) the developmental stage of larvae sensitive to that PI content; (iii) the ratio of MTI-2/proteases sufficient to inhibit gut proteases. The experimental data obtained from feeding S. littoralis larvae using these conditions led to two main results. First, when L2 S. littoralis larvae were fed on high MTI-2 expressing tobacco plants, no effects on larval development were detected but there was a significantly reduced fertility. When the same larvae were fed on low expressing MTI-2 tobacco plants, only a less marked lowering of fertility was observed. Second, after the first generation, no differences in protease activity were observed in insects derived from larvae fed on high or low MTI-2 expressing tobacco lines, suggesting that genetic traits observed in previous studies were not inherited.     

T-DNA induced anomalies of flowers and male sterility in transgenic tobacco plants: morphometric and cytological analysis

Morphometric and cytological analysis have been carried out in transgenic tobacco (Nicotiana tabacum L.) plants possessing male sterility and abnormalities of flowers. Mutation affected flower structure and conducted to long pistil and male sterility. It was established that development of microspores was blockaded on different stages of gametogenesis but the first injuries were detected for tetrad stage.     

Variation in nuclear DNA content of isonicotinic acid hydrazide-resistant cell lines and mutant plants of Nicotiana tabacum

Summary Isonicotinic acid hydrazide (INH)-resistant lines of Nicotiana tabacum have been maintained in callus culture for six years and mutant plants have been regenerated from a number of these lines. This study examines variations in DNA content in nuclei of several of these callus cultures, regenerated plants, and secondary callus from the regenerated plants. The lines selected for study include three easily regenerated lines (I 21, I 24, and I 9) and two lines of poor regenerating capacity (I 1 and I 18). Two of the regenerating lines eventually led to fertile plants and the third produced only sterile plants. In general, the range of total nuclear variability was not as high as anticipated from other studies of long-term tobacco callus cultures. The majority of nuclei in all the distributions were between 3 and 20 pg, and the most frequently encountered distributions concentrated in the 7–18 pg region corresponding to 2–5C by our estimate of the C value for tobacco. Distributions were not identical for plants regenerated from the same culture simultaneously, and the nuclear DNA content of secondary callus cultures from one of the plants examined did not reflect the quantitative DNA pattern of the plant from which it was derived. The greatest degree of variability and highest DNA content for individual nuclei were observed in the primary callus of the poorly- and non-regenerating lines. The variability in DNA content was not associated with the INH-resistant trait.     

Functional cybrid plants possessing a Nicotiana genome and an Atropa plastome

Summary Mesophyll protoplasts of plastome chlorophyll-deficient, streptomycin-resistant Nicotiana tabacum were fused with those of wild type Atropa belladonna using the polyethylene-glycol/high pH/high Ca⁺⁺/dimethylsulfoxide method. Protoplasts were cultured in nutrient media suitable for regeneration of tobacco but not Atropa cells. In two experiments, a total of 41 cell lines have been selected as green colonies. Cytogenetic (chromosomal number and morphology) and biochemical (isozyme analyses of esterase, amylase and peroxidase) studies were used to evaluate the nuclear genetic constitution of regenerated plants. To study plastid genetic constitution, restriction endonuclease analysis of chloroplast DNA was performed. Three groups of regenerants have been identified: (a) nuclear hybrids (4 cell lines); (b) Atropa plants, most probably arising from rare surviving parental protoplasts (4 lines) and (c) Nicotiana/Atropa cybrids possessing a tobacco genome and an Atropa plastome (33 lines). Most of cybrids obtained were diploid, morphologically normal plants phenotypically similar to tobacco. Some plants flowered and yielded viable seeds. Part of cybrid regenerants were variegated, variegation being transmitted to sexual progeny. Electron microscopic analysis of the mesophyll cells of variegated leaves revealed the presence of heteroplastidic cells. Analysis of thylakoid membrane polypeptides shows that in the cybrids the content of at least one of the major polypeptides, presumably a chlorophyll a/b binding protein is drastically reduced.     

Antisense expression of a gene encoding a calcium-binding protein in transgenic tobacco leads to altered morphology and enhanced chlorophyll

Entamoeba histolytica contains a novel calcium-binding protein like calmodulin, which was discovered earlier, and we have reported the presence of its homologue(s) and a dependent protein kinase in plants. To understand the functions of these in plants, a cDNA encoding a calcium-binding protein isolated from Entamoeba histolytica (EhCaBP) was cloned into vector pBI121 in antisense orientation and transgenic tobacco plants were raised. These plants showed variation in several phenotypic characters, of which two distinct features, more greenness and leaf thickness, were inherited in subsequent generations. The increase in the level of total chlorophyll in different plants ranged from 60% to 70%. There was no major change in chloroplast structure and in the protein level of D1, D2, LHCP and RuBP carboxylase. These morphological changes were not seen in antisense calmodulin transgenic tobacco plants, nor was the calmodulin level altered in EhCaBP antisense plants. The results of this paper have been granted US Patent No. 6,791,009.     

ipt基因定位表达对转基因烟草育性的影响

杂种优势是生物界的一种普遍现象。作物杂种优势的利用是培育高产、抗逆、优质新品种的重要手段之一。然而 ,常规育种技术获得配套三系的难度很大 ,由此限制了杂种优势利用的发展。近年来应用转基因技术创造雄性不育植株已有不少报道[1- 5] 。有研究表明 ,自然突变的雄性不育株     

Effects on Fertility in Transgenic Tobacco by Localized Expression of ipt Gene

The isopenteryl transferase (ipt) gene from Agrobacterium tumefaciens (Smith et Townsend) Conn was driven under the tobacco TA29 promoter and introduced into tobacco ( Nicotiana tabacum L.) plants by A. tumefaciens mediated transformation. PCR and Southern blot analysis confirmed that the ipt gene has integrated into the genomes of tobacco plants. The expression pattern of this chimeric TA29-ipt gene in the transgenic plants was studied, and the endogenous cytokinin level in different organs was assayed by ELISA method. The results showed that the cytokinin content in the androecium of transgenic plants increased 3-4 times as compared with the control, and some changes of the fertility of the TA29-ipt transgenic plants have been observed.     

Floral induction of vegetative plants supplied a purified fraction of deoxyribonucleic Acid from stems of flowering plants

It has been found that floral induced stems of flowering tobacco (Nicotiana tabacum cv. Wis. 38) plants contain large amounts of rapidly renaturing DNA, whereas noninduced stems of vegetative plants contain only small amounts. In addition, it has been shown that the striking qualitative difference in DNA between stems of flowering and vegetative plants mimics the over-all quantitative difference in DNA content (on a fresh weight basis). Therefore, the extra DNA in stems of flowering plants seems, at least in part, to represent preferential synthesis of rapidly renaturing DNA.     

Complete nucleotide sequence and the genome organization of tobacco leaf curl geminivirus from Japan

The genomic DNA of tobacco leaf curl geminivirus (TLCV) from tomato plants with leaf curl disease in Japan has been sequenced. The single circular DNA molecule comprises 2,761 nucleotides. TLCV DNA contains six open reading frames (ORFs) capable of encoding proteins with a molecular weight greater than 10 K. In total nucleotide sequence comparisons with other geminiviruses, TLCV was most closely related to tomato leaf curl virus from Taiwan (TwToLCV) (76% identity), tomato leaf curl virus from Bangalore (ToLCV-Ba) (74%) and agerantum yellow vein virus (AYVV) (74%), all possessing a monopartite genome. The significant but relatively low sequence similarity in the genomic DNA between TLCV and other geminiviruses suggests it is a distinct geminivirus in genus Begomovirus.     

Expression of a functional human adenosine deaminase in transgenic tobacco plants

An inherited disorder, adenosine deaminase deficiency is a form of severe combined immunodeficiency, which is ultimately caused by an absence of adenosine deaminase (ADA), a key enzyme of the purine salvage pathway. The absence of ADA-activity in sufferers eventually results in a dysfunctional immune system due to the build-up of toxic metabolites. To date, this has been treated with mixed success, using PEG-ADA, made from purified bovine ADA coupled to polyethylene glycol. It is likely, however, that an enzyme replacement therapy protocol based on recombinant human ADA would be a more effective treatment for this disease. Therefore, as a preliminary step to produce biologically active human ADA in transgenic tobacco plants a human ADA cDNA has been inserted into a plant expression vector under the control of the CaMV 35S promoter and both human and TMV 5′ UTR control regions. Plant vector expression constructs have been used to transform tobacco plants via Agrobacterium-mediated transformation. Genomic DNA, RNA and protein blot analyses have demonstrated the integration of the cDNA construct into the plant nuclear genome and the expression of recombinant ADA mRNA and protein in transgenic tobacco leaves. Western blot analysis has also revealed that human and recombinant ADA have a similar size of approximately 41 kDa. ADA-specific activities of between 0.001 and 0.003 units per mg total soluble protein were measured in crude extracts isolated from transformed tobacco plant leaves.     

极性生长的细胞中胞嘧啶甲基化的动态变化及其对GABA信号的响应

DNA胞嘧啶(C)的甲基化(5m C)在植物发育过程中具有重要的调节作用,多种环境因子如逆境胁迫、植物内/外源性因子等均会触发DNA甲基化的变化。为探讨γ-氨基丁酸(GABA)对植物发育的可能调节机制,本研究以极性生长的烟草花粉管和拟南芥根为材料,分析5m C的含量及其对GABA信号的响应。结果表明,1.0 mmol/L GABA能显著促进烟草花粉管和拟南芥根的极性生长;同时,GABA处理使烟草花粉管和拟南芥根的基因组中5m C含量显著降低、5-羟基胞嘧啶(5hm C)含量显著增加。5hm C是5m C去甲基化途径中的一个重要中间产物,本研究证实了GABA可以作为一种重要的外源信号调节DNA甲基化的动态变化。     

Flower Formation in Excised Tobacco Stem Segments: III. Deoxyribonucleic Acid Content in Stem Tissue of Vegetative and Flowering Tobacco Plants

A method has been developed that extracts DNA from stem tissue of flowering tobacco plants, Nicotiana tabacum cv. Wis. 38. The DNA content of stem tissue from a flowering tobacco plant is correlated with its capacity to flower in vitro. Stem segments known to form 100% floral buds contain 10 times more DNA per gram fresh weight than segments that form 5% floral buds and 95% vegetative buds, and in the uppermost 28 centimeters of flowering tobacco plant stems the DNA content decreases roughly in parallel with the floral gradient.     

Morphological changes and hypomethylation of DNA in transgenic tobacco expressing antisense RNA of the S-adenosyl-l-homocysteine hydrolase gene

S-adenosyl-l-homocysteine hydrolase (SAHH) is a key enzyme in the regulation of intracellular methylation reactions. To investigate the role of SAHH in methylation reactions and morphogenesis in planta, we have made transgenic plants expressing antisense RNA of tobacco SAHH. The transgenic plants displayed distinct morphological changes including a floral homeotic change. We hypothesized that the changes were caused by increased levels of cytokinin. In those transgenic plants, we observed that a repetitive DNA sequence appeared less methylated than controls. We speculated that altered gene expressions by the hypomethylation of DNA might be involved in the changes.     

蓄势待发的印度生物技术产业

构建的重组表达质粒pPIC9K-HSA-IFNα-2b经限制性内切酶SalI酶切线性化后电转化巴斯德毕赤酵母菌（P.Pastoris）SMD1168,将阳性转化子用甲醇诱导表达并进行PCR鉴定和Mut表型鉴定,再用Western-Blot法及Wish细胞-VSV病毒系统来鉴定表达蛋白的免疫原性及生物活性,同时,在摇瓶培养条件下,初步研究了甲醇诱导浓度及菌体诱导的起始OD值两因素对目的蛋白表达量的影响,本研究成功表达出具有高生物学活性的白蛋白融合干扰素蛋白（HSA-IFNα-2b）,对摇瓶发酵表达条件的初步摸索也为进一步在大规模发酵中提高目的蛋白在P.Pastoris的表达水平提供一个参考依据。     

Insertion of vector sequences in the genome of transgenic plants

Insertion of vector sequences of different lengths in the genome of transgenic tobacco and carrot plants occurring at a frequency of 35.2% was shown. No significant differences in insertion frequencies between the two species were observed. Integrated fragments of the vector DNA were stably inherited in the following generation resulted from self-pollination of original transformants.