低能离子束介导转基因技术

通过与常规的植物转基因方法进行比较,阐述了低能离子束介导转基因的原理和优点,以及该技术自开创以来在应用领域中取得的成果。     

低能离子束修饰蛹虫草菌株高产虫草素

虫草素具有抗肿瘤、免疫调节、抗炎等多种功效。为了更好地开发蛹虫草资源,选择合适剂量的低能离子束注入蛹虫草,优化虫草素的提取工艺条件,采用紫外分光光度法检测注入前后菌株中虫草素的含量。结果表明:最佳注入剂量为2.60×1015ions/cm2,虫草素最佳的微波-超声波提取工艺为:乙醇浓度70%,提取功率200W,提取时间110s,料液比1:240。选育出虫草素含量较高的15株菌株,最高含量达(11.924±0.063)mg/g,比原始菌株增长了近30%。     

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     

低能离子注入短命植物多伞阿魏(Ferula ferulaeoides)抗性生理特征

以能量30 keV、不同剂量N 离子注入多伞阿魏(Ferula ferulaeoides)种子用以研究多伞阿魏抗性生理指标的变化,以期为保护短命植物多伞阿魏提供理论依据.试验结果表明:随离子注入剂量增大,多伞阿魏种子发芽率和发芽指数逐渐下降;超氧化物歧化酶(SOD)活性增加、过氧化物酶(POD)、和过氧化氢酶(CAT)活性逐渐升高.剂量过大时,SOD、POD和CAT活性下降;游离脯氨酸含量逐渐降低,但在6×1016N /cm2剂量时含量升高;可溶性蛋白质含量先升高再降低,6×1016 N / cm2剂量时为最高值.通过对发芽率、发芽指数的结果进行方差分析,得出低剂量N 离子注入可破除多伞阿魏种子休眠,促进种子萌发;适当剂量N 离子注入可激活保护酶、脯氨酸和可溶性蛋白的表达.     

Improvement of astaxanthin production by a newly isolated Phaffia rhodozyma mutant with low-energy ion beam implantation

Aims: Isolation, characterization and identification of Phaffia sp. ZJB 00010, and improvement of astaxanthin production with low‐energy ion beam implantation. Methods and Results: A strain of ZJB 00010, capable of producing astaxanthin, was isolated and identified as Phaffia rhodozyma, based on its physiological and biochemical characteristics as well as its internal transcribed spacer (ITS) rDNA gene sequence analysis. With low‐energy ion beam implantation, this wild‐type strain was bred for improving the yield of astaxanthin. After ion beam implantation, the best mutant, E5042, was obtained. The production of astaxanthin in E5042 was 2512 μg g^?1 (dry cell weight, DCW), while the wild‐type strain was about 1114 μg g^?1 (DCW), an increase of 125·5%. Moreover, the fermentation conditions of mutant E5042 for producing astaxanthin were optimized. The astaxanthin production under the optimized conditions was upscaled and studied in a 50‐l fermentor. Conclusions: A genetically stable mutant strain with high yield of astaxanthin was obtained using low‐energy ion beam implantation. This mutant may be a suitable candidate for the industrial‐scale production of astaxanthin. Significance and Impact of the Study: Astaxanthin production in Phaffia rhodozyma could be fficiently improved by low‐energy ion beam implantation, which is a new technology in the mutant breeding of micro‐organisms. The mutant obtained in this work could potentially be utilized in industrial production of astaxanthin.     

SOS induction by vacuum,desiccation and low-energy ion beam mock-irradiation in bacteria

In this work, we compared the SOS response induced by vacuum-drying, desiccation (wind-spray-drying) and low-energy ion beam mock-irradiation with that of mitomycin C, UV induction. The induction factor induced by vacuum-drying and low-energy ion beam mock-irradiation was relatively higher than that of desiccation in Sa194 and JC19008 strain, respectively. These findings revealed that the SOS response produced by low-energy ion beam mock-irradiation was mainly induced by the step of vacuum-drying, unlikely by the step of wind-spray-drying. The mutation frequencies of rifampicin resistance gene in AB1157 andlacI gene in W3110 increased significantly by vacuum treatment and low-energy ion beam mock-irradiation, but had no remarkable change by desiccation treatment. Meanwhile, the mutation frequency of rifampicin resistance gene in 1C400 strain was not significantly influenced by these treatments. These results implied that the SOS response played an important role in the mutations induced by vacuum treatment and low-energy ion beam mock-irradiation.     

Radiobiological effects and dose-LET spectra

Summary To advance our knowledge about radiobiological cell effects typical different cell reactions must be discriminated and studied individually. Today the 2-component theory which distinguishes between repairable-reactions and nonrepairable-reactions, is the first step on this road. Such specialized investi gations give a more detailed and clearer picture of the cell reactions than the usual RBE comparisons carried out with different radiation types. This is espe cially important for clinical radiotherapy where it is essential to increase the selectivity between reactions on normal and tumor cells.-values for different types of radiation are communicated.     

Biological effects of low-energy C ion implantation on sesame (Sesamum indicum L.)

Field cultivation experiments on white sesame (Sesamum indicum L.)seeds implanted with low-energy C ion showed that different dosages of C ion implantation produce different biological effects.Sesame plants in 6 different dosage groups with C ion density respectively at 1×1011,1×1012,1×1015,5×1015,1×1016,5×1016 ion/cm2 were superior to the control group in plant height,leaf number,stalk diameter and leaf size.Further,sesame plants in these groups flower and seed earlier than those in the control group,and single plant yield also increased.Of all the groups,the 5×1015 ion/cm2 dosage group yielded the best effect,whereas the 1×1017/cm2 dosage group showed an evident inhibitory effect of ion implantation on the germination and growth of the sesame seeds.     

Mutational spectrum of the lacI gene in Escherichia coli K12 induced by low-energy ion beam

The mutational spectrum of the genomic lacI gene induced by low-energy nitrogen ion irradiation in wild type Escherichia coli strain W3110 were compared with the spontaneous and the vacuum controls. The mutant frequency of irradiated group was dose-dependent and reached 26.3 × 10⁻⁶ at dose of 31.2 × 10¹⁴ ions/cm², which was about 18-fold over the background (1.5 × 10⁻⁶) and 10-fold over the vacuum controls (2.6 × 10⁻⁶). This result indicated that the low-energy ion irradiation was one of many effective mutagens, though the vacuum condition of low-energy ions contributed some low-level gene mutations. It was found that the difference between the spontaneous and the vacuum control was the increases of base-pair substitutions in the vacuum control group. The spectra of irradiated group were quite similar to that of oxygen free-radical induced in the same strain, suggesting free-radicals and other adducts generated by low-energy ions might play an important role in the mutagenesis in vivo. When the spontaneous and the vacuum control group were compared, base-pair substitutions, deletions and additions of the irradiated group were significantly increased, and the +TGGC or −TGGC at hot spot was decreased from 82 to 48%. But the remarkable increase in absolute MF of the +TGGC or −TGGC at hot spot in the irradiated group suggested that low-energy ions did induce the mutations of this type. The spectra of our irradiated group had relative low-level base-pair substitutions, high-level ±TGGC and high proportion additions than those of γ-radiation induced, implying there were some different effects or processes between them.     

A novel approach for improving the productivity of ubiquinone-10 producing strain by low-energy ion beam irradiation

Agrobacterium tumefaciens ATCC4452 cells were irradiated by nitrogen ion beam, a new mutagen, with energy of 10 keV and fluence ranging from 2.6×10¹⁴ ions/cm² to 6.5×10¹⁵ ions/cm². A similar “saddle shape” survival curve due to ion beam irradiation appeared again in this study. Some mutants with high yield of ubiquinone-10 were induced by ion implantation. High mutation rate and wide mutation spectrum were also observed in the experiment. These results suggested that the mutagenic effect of such low-energy ion influx into bacterium cells could result from multiple processes involving direct collision of particles with cytoplasm, nucleolus, and cascade atomic and molecular reactions due to plentiful primary and secondary particles.     

Biological effects of low-energy C ion implantation on sesame ( Sesamum indicum L.)

Field cultivation experiments on white sesame (Sesamum indicum L.) seeds implanted with low-energy C ion showed that different dosages of C ion implantation produce different biological effects. Sesame plants in 6 different dosage groups with C ion density respectively at 1 × 10¹¹, 1 × 10¹², 1 × 10¹⁵, 5 × 10¹⁵, 1 × 10¹⁶, 5 × 10¹⁶ ion/cm² were superior to the control group in plant height, leaf number, stalk diameter and leaf size. Further, sesame plants in these groups flower and seed earlier than those in the control group, and single plant yield also increased. Of all the groups, the 5 × 10¹⁵ ion/cm² dosage group yielded the best effect, whereas the 1 × 10¹⁷/cm² dosage group showed an evident inhibitory effect of ion implantation on the germination and growth of the sesame seeds. __________ Translated from Journal of Beijing Normal University (Natural Science), 2006, 42 (1): 95–97 [译自: 北京师范大学学报 (自然科学版)]     

Radiobiological effects of total body irradiation on the spinal cord

Total body Irradiation (TBI) is often used for conditioning, prior to bone marrow transplantation. Doses of 8–14 Gy in 1–8 fractions over 1–4 days are administered using low dose rate external beam radiotherapy (EBRT). When necessary, consolidation EBRT using conventional doses, fractionation and dose rate is given. The irradiated volume usually contains critical organs such as spinal cord. The purpose of this study was to assess the biologic effect of TBI on the spinal cord in terms of EQD₂ (equivalent dose given in fractions of 2 Gy). EQD₂ values were calculated using the linear-quadratic generalized incomplete repair (IR) model that incorporates IR between fractions and low dose rate irradiation corrections and accounts for mono and bi-exponential repair. Three fractionation schemes were studied as function of dose rate: 8 Gy in 1 and 2 fractions and 12 Gy in 8 fractions. For the 12 Gy in 8 fractions scheme, the influence of dose rate on EQD₂ was limited because the effect of IR between fractions dominates. For the 8 Gy in 1 fraction scheme, significant sparing of the spinal cord may be achieved for low dose rate (5–20 cGy/min). The extent of effects depends on the parameters used. The IR model provides a useful mathematical framework for examination of the effects of fractionated treatments of varying dose rate. Reliable experimental data are needed for accurate assessment of radiation damage to the spinal cord following fractionated low dose rate TBI.     

Radiobiological effects and medical applications of non-ionizing radiation

Radiation is used in medicine to diagnose and treat diseases but it can also cause harm to the body by burning or mutation. This depends on whether the radiation is ionizing or nonionizing. Despite its vast applications in surgery, dermatology and cosmetics, little is taught and thus known about non-ionizing radiation.This review article discusses the fundamentals of non-ionizing electromagnetic radiations. The main aim is to extensively explain the different types of non-ionizing radiation. This will equip students and medical personnel with knowledge on different medical applications and expose them to a variety of specializations in medicine that utilize non-ionizing radiation. The article discusses the physics, hazard, means of protection and medical application of each type of radiation: ultraviolet radiation, light (both visible light and LASER), infrared radiation, microwaves and extremely low frequency radiation separately. It presents these terms in a simple manner that avoids rigors mathematics and physics, which makes them comprehensible for medical students.The development of new diagnostic and therapeutic approaches could also lead to increased hazards to the body unless they are treated with precaution. If not adequately monitored, a significant health risk may be posed to potentially exposed employees. Hence proper dosage should be used for non-ionizing radiation. This is only possible through understanding of the risks/benefits of these radiations by studying the physics and radiobiological effects of each individual radiation.     

Possibility of using a low-energy proton beam for particle-induced X-ray emission microanalysis.

The present analysis concentrates on the utilization of low-energy proton accelerators of several hundred kilovolts for particle-induced X-ray emission microanalysis. Experiments on stainless-steel and carbon matrices for thick target conditions have been carried out at several proton energies. The ionization cross sections for some elements are calculated using the ECPSSR theory. The effective cross section and minimum detection limits are calculated for the two matrices. With the availability of ultra LE-Ge detectors low-Z elemental detection is discussed.     

Radiobiological aspects of intraoperative radiotherapy (IORT) with isotropic low-energy X rays for early-stage breast cancer

The purpose of this study was to model the distribution of biological effect around a miniature isotropic X-ray source incorporating spherical applicators for single-dose or hypo-fractionated partial-breast intraoperative radiotherapy. A modification of the linear-quadratic formalism was used to calculate the relative biological effectiveness (RBE) of 50 kV X rays as a function of dose and irradiation time for late-reacting normal tissue and tumor cells. The response was modeled as a function of distance in the tissue based on the distribution of equivalent dose and published dose-response data for pneumonitis and subcutaneous fibrosis after single-dose conventional irradiation. Furthermore, the spatial distribution of tumor cell inactivation was assessed. The RBE for late reactions approached unity at the applicator surface but increased as the absorbed dose decreased with increasing distance from the applicator surface. The ED50 for pneumonitis was estimated to be reached at a depth of 6-11 mm in the tissue and that for subcutaneous fibrosis at 3-6 mm, depending on the applicator diameter and whether the effect of recovery was included. Thus lung tissue would be spared because of the thickness of the thorax wall. The RBE for tumor cells was higher than for late-reacting tissue. The applicator diameter is an important parameter in determining the range of tumor cell control in the irradiated tumor bed.     

Molecular templates for bio-specific recognition by low-energy electron beam lithography

Protein patterning has become an important topic as advances are made in biologically integrated devices and protein chip technology. Versatile and effective patterning requires substrates that can be quantified, with active presentation of proteins and control over protein density and orientation. Herein we describe a model system and the use of low-energy electron beam lithography to pattern molecular templates for immobilization of antibodies through ligand recognition. The templates were patterned over a background of poly(ethylene glycol) (PEG) modified silicon oxide (SiO _x ). These substrates were exposed to a low-voltage (2 keV) electron beam to remove PEG selectively from exposed regions. These regions were then functionalized with a dinitrophenyl (DNP) ligand and tested for specific binding of fluorescently labeled anti-DNP antibodies. The PEG modified regions in conjunction with ligand-presenting regions in the patterned arrays substantially reduces non-specific adsorption of proteins, yielding a specific/nonspecific ratio of approx 10. The surface coverage of the biologically active DNP groups on SiO _x and the amount of immobilized antibody on DNP were measured with a fluorescence-based, enzyme-linked immunosorbent assay. The specificity of the interaction between DNP ligand and fluorescently labeled anti-DNP antibodies was evaluated with fluorescence microscopy. This approach to patterning of molecular templates and assays for quantification are generally applicable to immobilization of any ligand-receptor pair on a wide range of substrates.     

Development of beam-plasma instability during the injection a low-energy electron beam into the ionospheric plasma

Results are presented from an active experiment on the injection of charged particle beams into the ionospheric plasma. The experiment was carried out in 1992 onboard the Intercosmos-25 satellite and the Magion-3 daughter satellite (APEX). A specific feature of this experiment was that both the ion and electron beams were injected upward, in the same direction along the magnetic field. The most interesting results are the excitation of HF and VLF-LF waves and the generation of fast charged particle flows, which were recorded on both satellites.     

Nitrite in the root zone and its effects on ion uptake and growth of wheat seedlings

The immediate and posteffects of various concentrations of NaNO₂ on ion uptake of wheat ( Triticum aestivum L. cv. GK Öthalom) seedlings were studied at different pH values. Without pretreatment, the higher the concentration of NaNO₂ the greater was the decrease in uptake of K⁺ into the roots, both at pH 4 and pH 6. At pH 6 but not at pH 4 the reverse was true when the seedlings were pretreated with NaNO₂. Due to the high Na⁺ content of the roots, an effect of Na⁺ in this process cannot be excluded. Nitrite was taken up by the roots more rapidly than nitrate. Nitrite at 0.1 m M in the medium induced the development of an uptake system for both NO⁻₂ and NO⁻₃ in wheat roots. At higher concentrations pretreatment with NO⁻₂ decreased NO⁻₃ uptake by the roots, but NO₃ did not inhibit the uptake of NO₂. The toxic effect of NO⁻₂ was strongly pH dependent. Lower pH of the external solution led to an increased inhibition by NO⁻₂ of both ion uptake and growth of seedlings. The inhibitory effect of NO⁻₂ differed considerably for roots and shoots. The roots and especially the root hairs were particularly sensitive to NO⁻₂ treatment.     

Effect of low-energy electron irradiation on DNA damage by Fe3+ ion

We investigated the combined effects of low-energy electron irradiation and Fe(3+) ion on DNA damage. We used lyophilized pBR322 plasmid DNA films with various concentrations (0 ~ 7 mM) of Fe(3+) ions and irradiation with monochromatic, low-energy 3 or 5 eV electrons for these studies. DNA-Fe(3+) films were recovered and analyzed by agarose gel electrophoresis to identify and compare the effects of Fe(3+) ions and/or low-energy electrons alone or in combination on DNA damage. In nonirradiated DNA-Fe(3+) films, there was little DNA damage observed (less than 10% of the total DNA loaded on the gel appeared damaged) for Fe(3+) ion up to 7 mM concentration. In irradiated DNA films without Fe(3+) ions, there was also very little DNA damage observed (less than 3% of the total DNA loaded on the gel appeared damaged). However, when DNA-Fe(3+) films, were irradiated with low-energy electrons, DNA damage was significantly increased compared to the sum of the damage caused both by either Fe(3+) ion or low-energy electrons irradiation alone. We proposed that both DEA and/or electron transfer processes might play a role in the enhanced DNA damage when DNA-Fe(3+) films were irradiated by low-energy electrons.