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.

Mutation Breeding of β-carotene Producing Strain B. trispora by Low Energy Ion Implantation

Ion beam bioengineering technology as a new mutation approach has been widely used in the biological breeding field. In this paper the application of low energy nitrogen ion implantation in the β-carotene producing strain, Blakeslea trispora（-） was investigated. The effects of different fermentation conditions on β-carotene production by a high yield strain were examined. Results showed that two β-carotene high yielding strains B.trispora（-） BH3-701 and BH3-728 were screened out and the averaged production of β-carotene was raised by 178.7% and 164.6% respectively after five passages in the shaking flasks. Compared with the original strain, the highest yield strain BH3-701 was potent in accumulating β-carotene, especially in the later stage, and greatly increased production efficiency.

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.

Screening of Biocontrol Strain Bacillus subtilis by N^+ Ion Beam Implantation

[Objective] This study was to investigate the effect of N＋ ion beam implantation on the survival rate and mutation rate of biocontrol strain Bacillus subtilis. [Method] The factors influencing B. subtilis ion beam implantation, including culture time, dilution concentration, solvent, drying time of mycoderm were optimized. B. subtilis cells were implanted by using ion beam at dose of 2.0×10^14～4.0×10^14 ions/cm2 and the energy of 30 kev. Then the methods of culturing colonies confronting each other on plate and Oxford cup diffusion were used to screening strains. [Result] The optimal parameters were found as follows： culture in liquid for 20-24 h, dilution with sterile water to 106 cells/ml and drying time of 60 min for sample preparation; the optimal N＋ ion beam implantation dose of 2.0×10^14～4.0×10^14 ions/cm2 at the energy of 30 kev, the survival rate of 8.43%-26.71% and the mutation rate of 3.50%-5.43%. [Conclusion] This study provided reference for ion beam implantation mutation of B. subtilis.

Biological Effects on Fruit Fly by N^+ ion Beam Implantation

Mutation induced by low energy ion beam implantation has been applied widely both in plants and microbes. However, due to the vacuum limitation, such ion implantation into animals was never studied except for silkworm. In this study, Pupae of fruit fly were irradiated with different dosage N+ ions at energy 20 KeV to study the biological effect of ion beam on animal. The results showed a saddle like curve exists between incubate rate and dosage. Damage of pupae by ion beam implantation was observed using scanning electron microscope. Some individuals with incomplete wing were obtained after implantation but no similar character was observed in their offspring. Furthermore, about 5.47% mutants with wide variation appeared in M1 generation. Therefore, ion beam implantation could be widely used for mutation breeding.

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.

Differential Expression of Retrotransposon WIS 2-1A Response to Vacuum, Low-Energy N^+ Implantation and ^(60)Coγ-ray Irradiation in Wheat

Mutagenesis and retrotransposons have a close relationship, but little attention has been paid yet to the activity of retrotransposons produced by physical mutagens. The variation of retrotransposon WIS 2-1A activity in wheat （Triticum aestivum L.） embryos at three different growth times （30 h, 45 h and 60 h） was investigated after they had been treated with N^＋ implantation in a vacuum of 5× 10^-2 Pa and irradiation by ^60Coγ-ray respectively. For each of the three growth times the expression of WIS 2-1A showed almost entirely a same trend of downregulation, upregulation, then downregulation, and upregulation again with the increase in dose of N^＋ implantation, but the expression appeared irregular with the increase in irradiation of ^60Coγ-ray. In conclusion, the acutely activating effect of WIS 2-1A stimulated by vacuum and high dose N^＋ implantation within a shorter incubation time may provide a convenient tool to advance the research on mutagenic breeding and function genes.

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.

Screening of Bioflocculant-Producing Strain by Ion Implantation and Flocculating Characteristics of Bioflocculants

A bioflocculant-producing mutator strain, NIM-192, was screened out through nitrogen ion implanting into F J-7 strain. The results showed that NIM-192 had good genetic stability and high flocculating activity, and the flocculating rate increased by 34.26% than that of the original. Sucrose, complex nitrogen source contained yeast extract, urea and pH 7.0～ 9.0 were chosen as the best carbon source, nitrogen source and initial solution pH for bioflocculant production, respectively. The bioflocculant kept high and stable flocculating activity at alkalinous reaction mixture with a pH beyond 7.0, while the flocculating activity was remarkably reduced when the reaction pH was lower than 7.0. Addition of many cations could obviously increase the flocculating rate, among which Ca^2＋ demonstrated the best effect. The bioflocculant had very strong acid-base stability and thermo-stability.The flocculating rate kept over 86% when pH of the bioflocculant was in a range of 3.0 ～ 12.0, and the change of flocculating activity was not great when heated at 100℃ for 60 rain.

GERM CELL MUTATIONS IN MICE:STANDARDS FOR PROTECTING THE HUMAN GENOME

The most extensive set of information on the factorsaffecting mutation induction by chemical mutagens inmice has been obtained by use of the dominant lethalassay and the specific locus method.Dominant lethalmutations are caused by chromosomal aberrations,and they occur shortly before or after implantation.

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.

Mutagenic Effects of BM302:GO112 Induced by Low-Energy Ion Beam Implantation

Mutant strains of GO112 and BM302 with a high 2-keto-L-gulonic acid （2KLG） transformation rate induced by ion beam implantation were separately and combinatorially compared with the original strains GO29 and BM80 to study the mutagenic effects of ion beam implantation. Both the sole GOl12 and mixed BM302：GOl12 demonstrated improved SNDH activity and 2KLG yield compared to the original strains. The mutant combinations of BM302：GOl12 showed a longer stationary phase and higher biomass than BM80：GO29. The mutant BM302 exhibited a stronger capacity to maintain a stable pH environment at mixed fermentation with Gluconobacter oxydans （G. oxydans） for 2KLG transformation and facilitated the growth of G. oxydans compared with the original strain BM80. The promotive capacity to L-sorbosone dehydrogenase （L-SNDH） from the supernate of BM302 was 1.6-fold higher than that of BM80. Genes encoded SNDH in GO29 and GOl12 were amplified and sequenced, and mutations including three transitions （CG →TA, CG →TA, GC → AT） and one transversion （AT→ TA） were confirmed from GO29 to GOl12. The corresponding amino acid was changed as Leu →Phe, Arg → Gln and Asn → Lys.

N^+离子注入阿维拉霉素产生菌诱变效应的研究

选用低能(30keV)N+离子注入阿维拉霉素产生菌SV-56,研究其诱变效应。试验结果表明:SV-56菌株存活率曲线是典型的“马鞍型”剂量-效应曲线,“马鞍型”区域内具有高的正突变率(20．9％-26．2％)。通过5×1015cm-2N+注入诱变处理、链霉素抗性筛选,最终获得一株稳定性良好、阿维拉霉素产量稳定在79．6-82．9 mg·L-1之间、较出发菌株提高41．4％-47．2％的突变株SVN-116。

N^+注入选育高毒力球孢白僵菌菌株及对3种油茶害虫的毒力测定

以低能氮离子注入(N+)作为诱变手段对球孢白僵菌进行诱变,并对3种油茶主要害虫进行了毒力测试。结果表明:不同剂量的氮离子注入后白僵菌存活率呈"马鞍曲线",其峰值即150×1013ions.cm-2为诱变最佳剂量。氮离子注入对白僵菌的菌落形态、产孢量、孢子萌发率等生长特性均有影响,通过产孢量、Pr1蛋白酶活和几丁质酶活指标筛选我们得到3株优良菌株,其中BbⅢ22菌株的Pr1蛋白酶活和几丁质酶活分别达0.230、0.137(OD值),为出发菌株的近2倍水平。BbⅢ22菌株对油茶叶蜂、油茶毒蛾有较强的致病力,在孢子浓度为107孢子.mL-1时,致死率达到了80%以上,而对油茶象甲的致死率相对较弱为60%左右。对油茶叶蜂、油茶毒蛾和油茶象甲的LD50对数浓度分别为5.2960、5.6347、6.3555。

N^＋注入中性蛋白酶高产菌株诱变选育的研究

利用低能N+注入技术对产中性蛋白酶的枯草芽孢杆菌(Bacillus subtilis)进行诱变选育,以提高该菌株的产酶能力.试验结果表明,菌株的存活率曲线是典型的马鞍型"剂量-效应曲线,在注入剂量为(30~50)×1014ions·cm-2的区域内具有高的正突变率.在注入能量为30keV、注入剂量为50×1014 ions·cm-2的条件下,经过筛选最终获得1株遗传稳定性良好的高产菌株NP131,其所产中性蛋白酶活力稳定在15230 U·mL-1左右,较出发菌株提高了71.2%.比较突变菌株NP131与出发菌株的产酶曲线,发现2条曲线的变化规律一致.说明离子注入技术是一种比较理想的微生物菌种选育方法.

低能N^+离子注入对加工番茄的辐照效应

本研究以87-5制罐番茄种子为处理材料,用N+离子作为诱变源,以35keV的能量,分别以20次、40次、50次、60次脉冲,注入剂量2×1016N+/cm2、4×1016N+/cm2、6×1016N+/cm2、10×1016N+/cm2进行激发诱变,变异显著.表现为出苗率降低,座果提前.在果实的品质性状变化不大情况下,座果数明显增加,部分处理材料的产量明显提高.

紫外线与N^+注入复合诱变选育曲酸高产菌株

为得到一株曲酸的高产菌株,从5种实验菌株中筛选得到1株米曲霉CICC-2336菌株,以其作为出发菌株,经过紫外诱变和氮离子注入诱变两种复合诱变,得到1株曲酸高产菌株,将其命名为Aspergillus oryzaeN11。该菌株经过传代6次后,性状稳定,产酸量基本不变。在接种量15%、30℃恒温、摇床180r/min条件下发酵培养7d后,其产酸量达到24.12g/L,比出发菌株提高101%。

N^+离子注入对Aspergillus sp.产原果胶酶的诱变效应

原果胶酶是一种具有广泛应用前景的工业酶类,为获得高活性原果胶酶的菌株,用能量为15keV、注量为20×2.6×1013—180×2.6×1013cm-2的N+注入原果胶酶产生菌黑曲霉Aspergillussp.XZ-131的成熟孢子,其存活率曲线为典型的“马鞍型”剂量-效应曲线。经筛选确定高产诱变菌株Z-25,经传代培养,高产性状稳定遗传。发酵过程中比原始菌株提前24h进入生长旺盛期,随后在稳定期期间大量产酶,发酵至72h时酶活性达到最高,比原始菌株提高了179%,每克干细胞性比原始菌株提高了84%。

N^+离子注入诱变选育中性蛋白酶高产菌株及发酵条件的研究

对产中性蛋白酶的枯草芽孢杆菌(Bacillus subtilis)AS1.398进行离子注入诱变,从正突变率较高的注入剂量30~50×1014ions/cm2范围内,筛选出一株高产菌株ZC-7。该菌株在优化了的摇瓶培养基中,培养42h,产酶可达16900U/mL。在7L发酵罐上控制pH6.0~7.0,溶氧10%~20%,以32℃~40℃~30℃变温发酵42h,酶活力最高可达19680U/mL,为初始菌株的2.1倍。

N^＋注入选育漆酶高产菌株及其产酶优化研究

以糙皮侧耳Pleurotus ostreatus WY01为出发菌株,通过N+注入诱变处理担孢子、RBBR-PDA平板变色法初筛、ATBS法测定漆酶活性复筛,获得1株漆酶高产诱变菌株ADW-08。用高碳低氮无机盐制备的油菜秸固体培养基(SM)培养,其峰值酶活力比出发菌株高出2.80倍,达7.78U/g,且产酶稳定。对ADW-08固体培养产酶条件的研究表明,以葡萄糖为碳源明显优于蔗糖、麦芽糖、麸皮和可溶性淀粉;酒石酸铵较有利于ADW-08漆酶的分泌;适宜初始pH为5.0或6.0;ABTS和藜芦醇对产酶均有明显的诱导作用,而吐温-80对产酶有一定的抑制作用;正交试验结果表明,葡萄糖、酒石酸铵和pH最佳参数分别为15.0g/L、0.2g/L和5.2,酶活峰值为8.33U/g。