Bioeffects of Low Energy Ion Beam Implantation:DNA Damage,Mutation and Gene Transter

Low-energy ion beam implantation （10 - 200 keV） has been proved to have a wide range of biological effects and is broadly used in the breeding of crops and micro-organisms.To understand its mechanisms better and facilitate its applications, the developments in the bioeffects of low energy ion beam implantation in the past twenty years are summarized in this paper.

Preliminary Studies on Base Substitutions and Repair of DNA Mismatch Damage Stimulated by Low Energy N^+ Ion Beam Implantation in Escherichia coli

Ever since the low energy N+ ion beam has been accepted that the mutation effects of ionizing radiation are attributed mainly to direct or indirect damage to DNA. Evidences based on naked DNA irradiation in support of a mutation spectrum appears to be consistent, but direct proof of such results in vivo are limited. Using mutS, dam and/or dcm defective Eschericha coli imitator strains, an preliminary experimental system on induction of in vivo mutation spectra of low energy N+ ion beam has been established in this study. It was observed that the mutation rates of rifampicin resistance induced by N+ implantation were quite high, ranging from 9.2 x 10~8 to 4.9× 10~5 at the dosage of 5.2×1014 ions/cm2. Strains all had more than 90-fold higher mutation rate than its spontaneous mutation rate determined by this method. It reveals that base substitutions involve in induction of mutation of low energy nitrogen ion beam implantation. The mutation rates of mutator strains were nearly 500-fold (GM2929), 400-fold (GM5864) and 6-fold larger than that of AB1157. The GM2929 and GM5864 both lose the ability of repair DNA mismatch damage by virtue of both dam and dcm pathways defective (GM2929) or failing to assemble the repair complex (GM5864) respectively. It may explain the both strains had a similar higher mutation rate than GM124 did. It indicated that DNA cytosine methylase might play an important role in mismatch repair of DNA damage induced by N+ implantation. The further related research were also discussed.

Creation of High-Yield Polyhydroxyalkanoates Engineered Strains by Low Energy Ion Implantation

Polyhydroxyalkanoates （PHAs）, as a candidate for biodegradable plastic materials, can be synthesized by numerous microorganisms. However, as its production cost is high in comparison with those of chemically synthesized plastics, a lot of research has been focused on the efficient production of PHAs using different methods. In the present study, the mutation effects of PHAs production in strain pCB4 were investigated with implantation of low energy ions. It was found that under the implantation conditions of 7.8×10^14 N^＋/cm^2 at 10 keV, a high-yield PHAs strain with high genetic stability was generated from many mutants. After optimizing its fermentation conditions, the biomass, PHAs concentration and PHAs content of pCBH4 reached 2.26 g/L, 1.81 g/L, and 80.08% respectively, whereas its wild type controls were about 1.24 g/L, 0.61 g/L, and 49.20%. Moreover, the main constituent of PHAs was identified as poly-3-hydroxybutyrates （PHB） in the mutant stain and the yield of this compound was increased up to 41.33% in contrast to that of 27.78% in the wild type strain.

Damaging Effect of Low Energy N^+ Implantation on Aspergillus niger Spores

The mutant effects of a keV range nitrogen ion （N＋） beam on enzyme-producing probiotics were studied, particularly with regard to the induction in the genome. The electron spin resonance （ESR） results showed that the signal of ESR spectrum existed in both implanted and non-implanted spores, and the yields of free radicals increased in a dose-dependent manner. The ionic etching and dilapidation of cell wall could be observed distinctly through the scanning electron microscope （SEM）. The mutagenic effect on genome indicated that N＋ implantation could make base mutation. This study provided an insight into the roles low-energy ions might play in inducing mutagenesis of micro-organisms.

Evidence for Base Substitutions and Repair of DNA Mismatch Damage Induced by Low Energy N^+ Ion Beam Implantation in E. coli

Ever since the low energy N + ion beam has been accepted, the mutations of ionizing radiation are attributable mainly to avoidance of DNA damages repair. Evidences based on in vivo proof results are limited. Using the E.coli wild type and mutator strains, the mutant frequencies suggest that base substitutions in rpoB gene are induced by the N + implantation. A highly conserved region is selected to get the direct evidence for base substitutions by sequence of the high fidelity PCR amplification products in mutants. Most of the mutants (90.9%, 40/44) have at least one base substitution in the amplification region. The evidences for CG to TA (55%, 22/40), AT to GC (20%, 8/40) and TA to CG (5%, 2/40) transitions are identified. The transversions are AT to TA (15%, 6/40) and GC to CG (5%, 2/40). It is suggested that DNA cytosine methylase might play an important role in mismatch repair of DNA damage induced by N + implantation by analysis of the mutant frequencies of mutator strains.

Modeling of Inner Surface Modification of a Cylindrical Tube by Plasma-Based Low-Energy Ion Implantation

The inner surface modification process by plasma-based low-energy ion implantation（PBLEII）with an electron cyclotron resonance（ECR）microwave plasma source located at the central axis of a cylindrical tube is modeled to optimize the low-energy ion implantation parameters for industrial applications.In this paper,a magnetized plasma diffusion fluid model has been established to describe the plasma nonuniformity caused by plasma diffusion under an axial magnetic field during the pulse-off time of low pulsed negative bias.Using this plasma density distribution as the initial condition,a sheath collisional fluid model is built up to describe the sheath evolution and ion implantation during the pulse-on time.The plasma nonuniformity at the end of the pulse-off time is more apparent along the radial direction compared with that in the axial direction due to the geometry of the linear plasma source in the center and the difference between perpendicular and parallel plasma diffusion coefficients with respect to the magnetic field.The normalized nitrogen plasma densities on the inner and outer surfaces of the tube are observed to be about 0.39 and 0.24,respectively,of which the value is 1 at the central plasma source.After a 5μs pulse-on time,in the area less than 2 cm from the end of the tube,the nitrogen ion implantation energy decreases from 1.5 keV to 1.3 keV and the ion implantation angle increases from several degrees to more than 40°;both variations reduce the nitrogen ion implantation depth.However,the nitrogen ion implantation dose peaks of about 2×10^（10）-7×10^（10）ions/cm^2 in this area are 2-4 times higher than that of 1.18×10^（10）ions/cm^2 and 1.63×10^（10）ions/cm^2 on the inner and outer surfaces of the tube.The sufficient ion implantation dose ensures an acceptable modification effect near the end of the tube under the low energy and large angle conditions for nitrogen ion implantation,because the modification effect is mainly determined by the ion implantation dose,just as the mass transfer process in PBLEII is dominated by low-energy ion implantation and thermal diffusion.Therefore,a comparatively uniform surface modification by the low-energy nitrogen ion implantation is achieved along the cylindrical tube on both the inner and outer surfaces.

A Preliminary Study on DNA Mutation Induction of Maize Pollen Implanted by Low Energy N^+ Beam

The maize pollens were implanted with seven different doses of 30 keV N+ beam respectively, The genomic DNA polymorphism from treated pollens were analyzed with 104 primers by using RAPD respectively. The results showed that N^+ beam-induced mutation of maize pollens can result in the change of their DNA bases. The mutation is not properly random and its frequency increases with a rise in 30 keV N+ beam doses. It is conformed with A-G transformation, which is one of the most important factors in DNA bases induced by N+ beam.

High-Temperature Annealing Induced He Bubble Evolution in Low Energy He Ion Implanted 6H-SiC

Bubble evolution in low energy and high dose He-implanted 6H-SiC upon thermal annealing is studied. The （0001）-oriented 6H-SiC wafers are implanted with 15keV helium ions at a dose of 1×10^17 cm^-2 at room temperature. The samples with post-implantation are annealed at temperatures of 1073, 1173, 1273, and 1473K for 30rain. He bubbles in the wafers are examined via cross-sectional transmission electron microscopy （XTEM） analysis. The results present that nanoscale bubbles are almost homogeneously distributed in the damaged layer of the as-implanted sample, and no significant change is observed in the He-implanted sample after 1073 K annealing. Upon 1193 K annealing, almost full recrystallization of He-implantation-induced amorphization in 6H-SiC is observed. In addition, the diameters of He bubbles increase obviously. With continually increasing temperatures to 1273K and 1473 K, the diameters of He bubbles increase and the number density of lattice defects decreases. The growth of He bubbles after high temperature annealingabides by the Ostwald ripening mechanism. The mean diameter of He bubbles located at depths of 120-135 nm as a function of annealing temperature is fitted in terms of a thermal activated process which yields an activation energy of 1.914＋0.236eV.

Penetration depth and concentration distribution for implanted heavy ions with low energies in plant seeds by SEM and EDS

The penetration depth and concentration distribution of implanted ions have been studied for low energy heavy ions implanted in the dry seeds of plant, such as peanut, mung beau, sunflower, wheat and radish seeds, etc. by SEM+EDS. The results .how that the maximum penetration depth is about 12μm for V+ with an energy 200 keV implanted in cotyledon of the peanut, 18pm, spin, 20μm for V2+ with 90 keV implanted in sunflower, wheat, radish seeds, respectively. The penetration depth of implanted Cu2+ with 80 keV is about 90μm in the remainder funicle derivative of the mung beau seeds. The experimental result of the maximum penetration depth of implanted V+ in the peanut seeds was compared with the calculated value of the TRIM95.

Multi-effects and Mechanism of Broad Beau M_1 Root-tip Cells Implanted by Low Energy N^+ Beam

Broad beans were divided into six groups and implanted with N+ beam of 30 KeV, 8 × 1016/cm2 per time for various radiating times respectively. Besides the statistics of its vigor of germination, the M1 root-tip cells of these broad beans were systematically analyzed on their changes in mitotic percentage, morphology and behavior of chromosomes, along with the structure o f cytoskeletons, including microtubule and intermediate filament. Based on all results of these studies, our opinions have been expressed in the report on the mechanism of low-energy N+ beams effecting on higher dicotyledons such as broad beau.

A Mutant of Bacillus Subtilis with High-Producing Surfactin by Ion Beam Implantation

In order to generate a mutant of Bacillus subtilis with enhanced surface activity through low energy nitrogen ion beam implantation, the effects of energy and dose of ions implanted were studied. The morphological changes in the bacteria were observed by scanning electron microscope （SEM）. The optimum condition of ions implantation, 20 keV of energy and 2.6 × 10^15N^＋/cm^2 in dose, was determined. A mutant, B.s-E-8 was obtained, whose surface activity of 50-fold and 100-fold diluted cell-free Landy medium was as 5.6-fold and 17.4-fold as the wild strain. The microbial growth and biosurfactant production of both the mutant and the wild strain were compared. After purified by ultrafiltration and SOURCE 15PHE, the biosurfactant was determined to be a complex of surfactin family through analysis of electrospray ionization mass spectrum （ESI/MS） and there was an interesting finding that after the ion beam implantation the intensities of the components were different from the wild type strain.

Etching and Damage Action on Microbes' Cells by Low Energy N^+ Beam

The action of etching and damage by 20 keV N+ beam on the cells of Deinococcus radiodurans and Escherichia coli was investigated by scanning electron microscope (SEM) and the electron spin resonance (ESR) spectrum of free radicals. The results showed that N+ implantation exerted the direct action of etching and damage of momentum transferring and the indirect action of the free radicals of energy deposition on their cells, many microholes were found on the surface of cells' wall and /or membrane by SEM, the damaged DNA was determined using DNA unwinding technique, and the signal of free radicals was measured by ESR. The degree of damage to cells by ion beam gradually increased with the increase implantation dose. With the post-treatment of 2 mmol/l caffeine and 0.5 mmol / l Na2-EDTA, the survival rate of D.radiodurans and E.coli further decreased in the order of caffeine > Na2-EDTA > control, and this suggested that low energy ion beam could be implanted into nucleus, doing a damage to DNA and resulting in the mutation of organisms.

Improvement of L(+)-Lactic Acid Production of Rhizopus Oryzae by Low-Energy Ions and Analysis of Its Mechanism

The wild type strain Rhizopus oryzae PW352 was mutated by means of nitrogen ion implantation （15 keV, 7.8×10^14 ～ 2.08 ×10^15 ions/cm^2） to find an industrial strain with a higher L（＋）-lactic acid yield, and two mutants RE3303 and RF9052 were isolated. In order to discuss the mechanism primarily, Lactate Dehydrogenase of Rhizopus oryzae was studied. While the two mutants produced L（＋）-lactic acid by 75% more than the wild strain did, their specific activity of Lactate Dehydrogenase was found to be higher than that in the wild strain. The optimum temperature of Lactate Dehydrogenase in Rhizopus oryzae RF9052 was higher. Compared to the wild strain, the Michaelis constant （Km） value of Lactate Dehydrogenase in the mutants was Changed. All these changes show that L（＋）-lactic acid production has a correlation with the specific activity of Lactate Dehydrogenase. The low-energy ions, implanted into the strain, may improve the specific activity of Lactate Dehydrogenase by influencing its gene structure and protein structure.

Improvement of Vitamin K_2 Production by Escherichia sp.with Nitrogen Ion Beam Implantation Induction

Low-energy ion implantation as a novel mutagen has been increasingly applied in the microbial mutagenesis for its higher mutation frequency and wider mutation spectra. In this work, N^＋ ion beam implantation was used to enhance Escherichia sp. in vitamin K2 yield. Optimization of process parameters under submerged fermentation was carried out to improve the vitamin K2 yield of mutant FM5-632. The results indicate that an excellent mutant FM5-632 with a yield of 123.2±1.6 μg/L, that is four times that of the original strain, was achieved by eight successive implantations under the conditions of 15 keV and 60 ×2.6 ×10^13 ions/cm^2. A further optimization increased the yield of the mutant by 39.7%, i.e. 172.1±1.2 μg/L which occurred in the mutant cultivated in the optimal fermentation culture medium composed of （per liter）： 15.31 g glycerol, 10 g peptone, 2.89 g yeast extract, 5 g K2HPO4, 1 g NaCl, 0.5 g MgSO4·7H2O and 0.04 g cedar wood oil, incubated at 33 ℃, pH 7.0 and 180 rpm for 120 h.

Anomalous Phenomena in E<18 keV Hydrogen Ion Beam Implantation Experiments on Pd and Ti

Implantation experiments of low energy（1 keV【E【18 keV）hydrogen ion beamson hydrogen loaded metals are performed with high beam density(Jmax1.2 mA/cm2)and lowbeam density(Jmin0.02 mA/cm2).Palladium and titanium foils（plates）are bombarded withproton and deuteron beams in order to compare the atomic and nuclear interactions between dif-ferent ion beams.X-ray and charged particles are measured,and neutron and gamma doses arealso monitored during the implantation experiments.An anomalous peak in X-spectra,whoseenergy is about four times the beam energy,is observed during the high beam density experi-ment.The peak moves from about 40 keV to 62 keV and FWHM reduces rapidly,while thebeam energy and intensity increase.Another wide peak with over twice the beam energy is mea-sured in experiment with low beam density.It is located between 16 keV and 30 keV,and itspeak energy increases with the increase of implantation dose（implantation time）.Some anoma-lous intensities of neutrons correlated with a charged particle peak（2.93,3.85 MeV）are alsoobserved in the deuteron-palladium experiment.The highest neutron intensity reaches about8×104 n/s,while the beam energy and intensity are about 15 keV and 1.0 mA,respectively.

低能N^(+)注入诱变耐诺氟沙星的金黄色葡萄球菌及其机制分析

利用低能N^(+)注入诱变筛选耐诺氟沙星的金黄色葡萄球菌,通过基因组测序、耐药表型和生物被膜分析,探索低能N^(+)注入驱动金黄色葡萄球菌产生诺氟沙星耐药性的机制。结果表明,利用低能N^(+)注入诱变获得81株耐诺氟沙星的金黄色葡萄球菌,其中44株耐药菌16S rRNA或耐药基因出现片段缺失、插入、点突变和拷贝数变化;且基因变化高的耐药菌的药物外排蛋白、生物被膜的生物量和密度显著增加,对诺氟沙星的耐药性提高了8~16倍,并对5~8种抗菌药物产生了多重耐药。说明低能N^(+)注入可能通过介导金黄色葡萄球菌16S rRNA或相关耐药基因变异,调控细菌药物外排蛋白表达量增加和生物被膜形成能力增强,进而介导菌株产生耐药性。

低能N^(+)注入诱导大肠杆菌的16S rRNA遗传进化

为了探究低能N^(+)注入对大肠杆菌16S rRNA遗传进化与耐药表征的作用,本研究利用低能N^(+)注入诱变筛选耐药大肠杆菌,通过基因组de novo测序获得其16S rRNA基因序列,通过K-B法检测诱变菌株的耐药特征。结果共诱变获得了25株耐药菌株,其中5株诱变菌16S rRNA基因分别出现片段缺失,点突变(A257C),GC%含量增高,二级结构变异,并获得多药耐药特性。结果提示:低能N^(+)注入可以驱动大肠杆菌16S rRNA基因的随机突变和进化,进而调节耐药基因从头合成或变异,使大肠杆菌耐药性改变。

一株耐盐石油降解菌的鉴定及低能N^+注入诱变

通过分离、初筛和复筛从盐渍化石油污染土壤中分离出一株高效耐盐的石油降解菌株YJ-8,经形态学、生理生化和16S rRNA序列分析,该菌株被鉴定为粘质沙雷氏菌Serratia marcescens,其石油烃的降解率为64%。耐盐性实验结果表明,该菌可在NaCl浓度为50 g/L的培养基中生长。为进一步增强该菌株耐受高浓度NaCl和降解石油烃的能力,采用低能N^+注入技术对其进行诱变,最终获得1株遗传性能稳定的突变株。该突变株在原油发酵液中可将表面张力降至27.5 mN/m,石油烃的降解效率为74%,可耐受80 g/L浓度的NaCl。作为一株耐盐的石油烃高效降解菌,该菌在石油污染的盐渍化土壤修复中具有广阔的应用前景。

低能N^+注入对紫花苜蓿生理生化的影响

研究了低能N+注入紫花苜蓿后,对其发芽率、发芽势、苗高、直株重、叶绿素含量、粗蛋白含量以及幼苗过氧化物酶同工酶酶谱的影响.实验结果表明,发芽率、发芽势和过氧化物酶同工酶活性随剂量变化呈现明显的"马鞍型"变化曲线,而且过氧化物酶同工酶酶谱发生变异.

低能离子注入木聚糖酶产生菌黑曲霉(Aspergillus Niger)育种中参数的优化

本文对离子束技术的注入工艺和在不同能量下的不同注入剂量下黑曲霉的存活率和突变率进行了研究,目的是为了找到恰当的注入工艺和良好的注入参数,使诱变达到较好的效果。结果发现:当注入离子为氮离子时,在10keV的能量下注入剂量为5.2×1014ions/cm2和1.56×1015ions/cm2时,诱变效果较好。在这注入条件下,通过培养基优化,使木聚糖酶酶活达到600IU/ml。