Detection of changes in DNA methylation induced by low-energy ion implantation in Arabidopsis thaliana

This study evaluated changes in DNA methylation in Arabidopsis thaliana plants grown from seeds implanted with low-energy N(+) and Ar(+) ions. Methylation-sensitive amplified polymorphism (MSAP) testing revealed altered DNA methylation patterns after ion implantation at doses of 1 × 10(14) to 1 × 10(16) ions/cm(2). Comparison of the MSAP electrophoretic profiles revealed nine types of polymorphisms in ion-implanted seedlings relative to control seedlings, among which four represented methylation events, three represented demethylation events, and the methylation status of two was uncertain. The diversity of plant DNA methylation was increased by low-energy ion implantation. At the same time, total genomic DNA methylation levels at CCGG sites were unchanged by ion implantation. Moreover, a comparison of polymorphisms seen in N(+) ion-implanted, Ar(+) ion-implanted, and control DNA demonstrated that the species of incident ion influenced the resulting DNA methylation pattern. Sequencing of eight isolated fragments that showed different changing patterns in implanted plants allowed their mapping onto variable regions on one or more of the five Arabidopsis chromosomes; these segments included protein-coding genes, transposon and repeat DNA sequence. A further sodium bisulfite sequencing of three fragments also displayed alterations in methylation among either different types or doses of incident ions. Possible causes for the changes in methylation are discussed.     

Study of the permeability of tomato pericarp etched by low-energy ion beams based on α-particles Irradiation

Low-energy ion beam bio-technology has been applied in the biological field and has gained remarkable success in crop and microbe breeding. However, to understand how low-energy ion beams interact with biological materials remains a challenge for researchers who work for the development of ion-beam bio-technology. In this work, tomato pericarp was used as the target sample to study the effect of ion beams on the permeability of biological objects. A series of experiments were conducted via irradiating tomato pericarp samples with low-energy (10 keV ～ 25 keV) ion beams followed by measuring the pericarp’s permeability using transmissive α particles. The transmissive spectra of α particles and the measurement of the tip number in CR39 gave a quantitative evaluation of the sputtering effect caused by low-energy ions. Meanwhile, natural red dye was used to examine the permeability of irradiated tomato pericarp samples. It was found that the sputtering effect is not only proportional to the ion energy and dose, but dependent on the ion type as well. The damage caused by Ar ions due to sputtering was much more severe than that caused by N ions sputtering with the same dose. Therefore, this study not only demonstrates the permeability difference of biological membranes before and after ion irradiation, but also provides the information on how to optimize the experimental conditions for application of the low-energy ion beam in biology.     

Interaction of wheat high-mobility-group proteins with four-way-junction DNA and characterization of the structure and expression of HMGA gene

Plant high-mobility-group (HMG) chromosomal proteins are the most abundant and ubiquitous nonhistone proteins found in the nuclei of higher eukaryotes. There are only two families of HMG proteins, namely, HMGA and HMGB in plants. The cDNA encoding wheat HMGa protein was isolated and characterized. Wheat HMGA cDNA encodes a protein of 189 amino acid residues. At its N terminus, there is a histone H1-like structure, which is a common feature of plant HMGA proteins, followed by four AT-hook motifs. Polymerase chain reaction results show that the gene contains a single intron of 134 bp. All four AT-hook motifs are encoded by the second exon. Northern blot results show that the expression of HMGA gene is much higher in organs undergoing active cell proliferation. Gel retardation analysis show that wheat HMGa, b, c and histone H1 bind to four-way-junction DNA with high binding affinity, but affinity is dramatically reduced with increasing Mg(2+) and Na(+) ion concentration. Competition binding studies show that proteins share overlapping binding sites on four-way-junction DNA. HMGd does not bind to four-way-junction DNA.     

离子束介导大豆DNA导入小麦后其蛋白质和酯酶同工酶分析

对由离子束介导大豆DNA导入豫麦52获得的第四代两个转化株系9004和9002中的可育株籽粒和矮秆不育株杂交籽粒以及受体豫麦52籽粒,进行酯酶和可溶性蛋白的聚丙烯酰胺凝胶电泳分析。结果表明,可育株籽粒可溶性蛋白电泳在谱带上与受体存在差异,矮秆不育株材料的酯酶和可溶性蛋白电泳图谱谱带差异更明显。由此推测,外源大豆DNA导入受体后某些片段有可能插入受体基因组,从而导致基因突变或受体基因表达发生改变。     

The Arabidopsis HKT1 gene homolog mediates inward Na(+) currents in xenopus laevis oocytes and Na(+) uptake in Saccharomyces cerevisiae

The Na(+)-K(+) co-transporter HKT1, first isolated from wheat, mediates high-affinity K(+) uptake. The function of HKT1 in plants, however, remains to be elucidated, and the isolation of HKT1 homologs from Arabidopsis would further studies of the roles of HKT1 genes in plants. We report here the isolation of a cDNA homologous to HKT1 from Arabidopsis (AtHKT1) and the characterization of its mode of ion transport in heterologous systems. The deduced amino acid sequence of AtHKT1 is 41% identical to that of HKT1, and the hydropathy profiles are very similar. AtHKT1 is expressed in roots and, to a lesser extent, in other tissues. Interestingly, we found that the ion transport properties of AtHKT1 are significantly different from the wheat counterpart. As detected by electrophysiological measurements, AtHKT1 functioned as a selective Na(+) uptake transporter in Xenopus laevis oocytes, and the presence of external K(+) did not affect the AtHKT1-mediated ion conductance (unlike that of HKT1). When expressed in Saccharomyces cerevisiae, AtHKT1 inhibited growth of the yeast in a medium containing high levels of Na(+), which correlates to the large inward Na(+) currents found in the oocytes. Furthermore, in contrast to HKT1, AtHKT1 did not complement the growth of yeast cells deficient in K(+) uptake when cultured in K(+)-limiting medium. However, expression of AtHKT1 did rescue Escherichia coli mutants carrying deletions in K(+) transporters. The rescue was associated with a less than 2-fold stimulation of K(+) uptake into K(+)-depleted cells. These data demonstrate that AtHKT1 differs in its transport properties from the wheat HKT1, and that AtHKT1 can mediate Na(+) and, to a small degree, K(+) transport in heterologous expression systems.     

A novel approach to microbial breeding—low-energy ion implantation

Low-energy ions exist widely in the natural world. People had neglected the interaction between low-energy ions and material; it was even more out of the question to study the relation of low-energy ions and the complicated organism until the biological effects of low-energy ion implantation were discovered in 1989. Nowadays, the value of low-energy ion beam implantation, as a new breeding way, has drawn extensive attention of biologists and breeding experts. In this review, the understanding and utilization of microbial breeding by low-energy ion beam irradiation is summarized, including the characteristics of an ion beam bioengineering facility, present status of the technology of low-energy ions for microbial breeding, and new insights into microbial biotechnology.     

无菌条件下小麦氨基酸态氮及铵态氮营养效应研究

对铵态氮(硫酸铵)、氨基酸态氮(甘氨酸,谷氨酸及赖氨酸)和缺氮无菌砂培条件下小麦单株干物重、全氮量及根、叶谷草转氨酶和谷丙转氨酶活性作了研究．结果表明,铵态氮和氨基酸态氮均可被小麦吸收,且吸收量相当．培养30d后,甘氨酸和谷氨酸处理的小麦干物重显著高于缺氮及铵态氮处理,而铵态氮、赖氨酸及缺氮处理的干物重相近．低浓度铵态氮(0．7mmol·L^1)培养15d的小麦仅根的GPT活性显著高于缺氮处理,而高浓度(35．7mmol·L^1)处理6h对这两种转氨酶活性影响不大.不同种类、不同浓度的氨基酸态氮培养15d或处理6h后,小麦植株根、叶的GOT或GPT活性变化趋势有较大差异,这反映出小麦外源氨基酸主要同化部位及同化量,与氨基酸种类及浓度有较大关系．     

A hypothesis on the role of transposons

Genomic transposable elements, or transposons, are sequences of DNA that can move to different positions in the genome; in the process, they can cause chromosomal rearrengements and changes in gene expression. Despite their prevalence in the genomes of many species, their function is largely unknown: for this reason, they have been labelled “junk” DNA. “Epigenetic Tracking” is a model of development that, combined with a standard evolutionary algorithm, become an evo-devo method able to generate arbitrary shapes of any kind and complexity (in terms of number of cells, number of colours, etc.). The model of development has been also shown to be able to produce the artificial version of key biological phenomena such as the phenomenon of ageing, and the process of carcinogenesis. In this paper the evo-devo core of the method is explored and the result is a novel hypothesis on the biological role of transposons, according to which transposition in somatic cells during development drives cellular differentiation and transposition in germ cells is an indispensable tool to boost evolution. Thus, transposable elements, far from being “junk”, have one of the most important roles in multicellular biology.     

RAPD分析氮离子注入甜菊种子后的幼苗基因组DNA变异

应用ＲＡＰＤ 技术检测经低能氮离子注入甜菊纯系种子引起的幼苗基因组ＤＮＡ 变异。筛选出ＯＰＪ系列中的１５ 种引物对实验及对照基因组ＤＮＡ 进行了ＰＣＲ 扩增,共获扩增片段１０３ 条,分子量在０．３ － ３ｋｂ 之间,其中５ 种引物ＯＰＪ－ １ ,７ ,９,１１ ,１２ 扩增出差异片段１２ 条。结果表明,低能氮离子注入甜菊种子可引起体内基因组ＤＮＡ 发生突变;ＲＡＰＤ 技术是检测基因组ＤＮＡ 发生诱变的一种简便、有效方法。本文同时探讨了离子强度和Ｔａｇ ＤＮＡ 聚合酶用量对甜菊ＲＡＰＤ 分析结果的影响,以及氮离子注入诱变效应的可能机制。     

Formation of plasmid DNA strand breaks induced by low-energy ion beam: indication of nuclear stopping effects

Plasmid pGEM 3zf(+) was irradiated by nitrogen ion beam with energies between 20 and 100 keV and the fluence kept as 1×10¹² ions/cm². The irradiated plasmid was assayed by neutral electrophoresis and quantified by densitometry. The yields of DNA with single-strand and double-strand breaks first increased then decreased with increasing ion energy. There was a maximal yield value in the range of 20–100 keV. The relationship between DNA double-strand breaks (DSB) cross-section and linear energy transfer (LET) also showed a peak-shaped distribution. To understand the physical process during DNA strand breaks, a Monte Carlo calculation code known as TRIM (Transport of Ions in Matter) was used to simulate energy losses due to nuclear stopping and to electronic stopping. It can be assumed that nuclear stopping plays a more important role in DNA strand breaks than electronic stopping in this energy range. The physical mechanisms of DNA strand breaks induced by a low-energy ion beam are also discussed. Received: 30 July 1997 / Accepted in revised form: 18 January 1998     

Kismeth: Analyzer of plant methylation states through bisulfite sequencing

Background  

There is great interest in probing the temporal and spatial patterns of cytosine methylation states in genomes of a variety of organisms. It is hoped that this will shed light on the biological roles of DNA methylation in the epigenetic control of gene expression. Bisulfite sequencing refers to the treatment of isolated DNA with sodium bisulfite to convert unmethylated cytosine to uracil, with PCR converting the uracil to thymidine followed by sequencing of the resultant DNA to detect DNA methylation. For the study of DNA methylation, plants provide an excellent model system, since they can tolerate major changes in their DNA methylation patterns and have long been studied for the effects of DNA methylation on transposons and epimutations. However, in contrast to the situation in animals, there aren't many tools that analyze bisulfite data in plants, which can exhibit methylation of cytosines in a variety of sequence contexts (CG, CHG, and CHH).     

Cross-genome map based dissection of a nitrogen use efficiency ortho-metaQTL in bread wheat unravels concerted cereal genome evolution

Monitoring nitrogen use efficiency (NUE) in plants is becoming essential to maintain yield while reducing fertilizer usage. Optimized NUE application in major crops is essential for long-term sustainability of agriculture production. Here, we report the precise identification of 11 major chromosomal regions controlling NUE in wheat that co-localise with key developmental genes such as Ppd (photoperiod sensitivity), Vrn (vernalization requirement), Rht (reduced height) and can be considered as robust markers from a molecular breeding perspective. Physical mapping, sequencing, annotation and candidate gene validation of an NUE metaQTL on wheat chromosome 3B allowed us to propose that a glutamate synthase (GoGAT) gene that is conserved structurally and functionally at orthologous positions in rice, sorghum and maize genomes may contribute to NUE in wheat and other cereals. We propose an evolutionary model for the NUE locus in cereals from a common ancestral region, involving species specific shuffling events such as gene deletion, inversion, transposition and the invasion of repetitive elements.     

植物CACTA转座子的研究进展

转座子是染色体上可移动的DNA序列,根据转座机制可将其分为：通过RNA中间体进行转座的逆转录座子（Retrotransposon）和通过DNA中间体进行转座的转座子（Transposon）。En／Spm家族转座子是后者中的一类,它的末端反向重复序列（Terminal inverted repeats,TIRs）具有保守的5个碱基CACTA,所以通常又称为CACTA转座子。除此之外,其靶位点一般为3bp的同向重复（Target site duplication,TSD）;亚末端区域分布着若干正向或反向的重复序列（Subterminal repeat,STR）。迄今为止,CACTA转座子仅发现于植物基因组。过去一直认为由于其相对保守的转座机制而拷贝较少,但最近研究发现,该因子多拷贝存在于某些禾本科植物基因组中。由于该家族在基因组中分布的广泛性,具有用作分子指纹的应用前景。本文就其结构、转座机制和应用前景等做一综述。