低能离子束介导的遗传转化研究进展

低能离子束注入对细胞的刻蚀作用提供了外源遗传物质进入细胞的途径,离子束介导的遗传转化技术已在水稻,小麦,烟草,棉花等多种植物上取得成功,对离子束介导遗传转化的原理和最新进展进行了评述。．     

建立低能离子束介导小麦转基因方法并获得转GUS基因植株

研究了注入离子种类、能量、剂量等参数对于低能离子束介导的遗传转化的影响,建立了适于小麦成熟胚转化的组培条件和筛选程序。以携带ＧＵＳ基因的质粒为供体,进行了报告基因转化研究。分子生物学证据表明ＧＵＳ基因已整合到小麦基因组中。３个小麦品种的抗性愈伤转化率分别为９．５％、１０．８％、１１．２％,再生植株转化率分别为１．４％、３．４％、１．７％,首次证明了离子束介导小麦遗传转化是可行的,为离子束介导小麦遗     

低能离子束介导水稻遗传转化的研究进展

介绍了低能离子束介导遗传转化的原理,比较了离子束介导法与其他遗传转化方法的优缺点,重点对离子束介导水稻遗传转化的最新研究进展进行了评述,同时提出了问题和展望.     

低能离子束在生物技术中的应用研究

自从发现离子注入生物效应后,低能离子与生物体系相互作用研究在我国率先兴起,并很快投入应用。简要介绍低能离子注入生物效应的机理研究和应用研究的进展状况,并展望未来 。     

低能离子束介导外源基因转化烟草的研究

以烟草ＮＣ－８９种子为材料,用显微扫描电镜（ＥＳＭ）和电子自旋共振（ＥＳＲ）波谱仪研究氮离子束对烟草种子表面的刻蚀作用及能量沉积产生自由基的间接效应,为离子束介导转移外源基因提供了形态结构依据。将烟草种子用２０Ｋｅｖ的氮离子束处理后,浸入含有ＰＢⅠ１２１质粒的缓冲介质中,在含有卡那霉素１００ｍｇ／Ｌ的ＭＳ０培养基上继代筛选,得到３株抗性植株。取抗性植株的叶片,经组织培养后得到再生抗性植株。经过ＰＣＲ及ｓｏｕｔｈｅｒｎ杂交分析,证明外源基因已转入烟草。     

低能离子束辐照植物样品质量损失研究

采用50 keV的N+对100 滋m厚的芸豆切片和75 滋m厚的Mylar(聚乙烯对苯二酸酯)高分子膜辐照,辐照剂量从1×1015 ions/cm2到1×1017 ions/cm2。利用高灵敏度的天平测量样品辐照前后的质量,得到了辐照后样品的质量变化。结果表明,当离子剂量大于3×1016 ions/cm2时,切片样品的质量明显减少,并且质量损失随辐照剂量增加而增加;但Mylar膜样品在辐照剂量达到7×1016 ions/cm2时仍没有测量出明显的质量变化。由测量的质量损失计算出50 keV的N离子轰击切片样品时溅射产额约为560 atoms/ion。结合植物样品的结构和分子组成特性对这种辐照引起的高溅射产额现象作了分析。     

低能离子束生物工程的发展趋势

简述了低能离子束生物技术在6个主要方面的研究成果。随着离子束生物技术不断发展,迫切需要建立一个更加完善的物理学平台,形成一个更加完整的研究体系,借助于其它学科的新技术或新工艺形成一个相互补充的技术集成体系,更应该将离子束生物技术的实用性和普遍性的范围不断地拓宽。明确提出了低能离子束生物工程的技术思路,即立足于低能离子束这一物理学技术平台,瞄准2个发展方向,促进3个有效转变,分清4个研究层次,注重5大生物学特性。     

低能离子束生物技术的应用

自从我国科学家发现离子注入生物学效应后,低能离子束生物技术的研究就在我国率先兴起。随后,越来越多的科学家基于低能离子与生物体之间存在的能量沉积、动量传递、质量沉积及电荷中和与交换的相互作用,对生物体内的遗传物质进行加工、修饰、重组,开辟了农作物和微生物等遗传改良及转基因的新方法。本文简要介绍了低能离子束生物技术产生的背景、低能离子束与生物体之间相互作用的机理和特点以及目前低能离子束在诱变育种和转基因等生物技术领域的研究进展,并展望了离子束技术在藻类基因工程方面的发展潜力。     

超低能离子束注入后番茄的生物学效应

利用超低能氮离子束注入河南四号番茄干种子,观察发芽率及田间农艺性状的表现,采用聚丙稀酰胺凝胶电泳的方法分析M0代、M1代幼苗期过氧化物酶和酯酶同工酶的变化。结果表明,不同剂量间芽率的变化幅度较小,其总趋势是先升高后略微下降,呈现“马鞍型”曲线。但在幼苗期群体内某些单株的子叶呈镰刀型,其刺激效应十分明显,并且在7×1017N /cm2剂量下M1材料中发现5株突出的变异单株,呈细叶型、长花瓣,株型、果形已发生明显得变异,并且已经稳定遗传到第四代。同工酶分析显示:超低能离子束注入河南四号番茄后不同剂量间POD酶活性有差异,比对照酶活性增强,M1代突变体的POD酶活增强更显著;EST同工酶的活性表现不同,M1-1酶谱的带数比对照少四条,M1-2比对照多出四条,且主酶带比对照颜色深。由此认为,超低能离子注入技术对番茄的遗传改良有一定效果。     

低能离子束诱发大肠杆菌甲基化效应研究

表观遗传学是指在DNA序列未发生变化的情况下,基因表达发生可遗传的变化,而甲基化水平的变化是表观遗传效应的重要指标。在构建一种新的敏感大肠杆菌Dam(GATC)位点甲基化检测方法的基础上,探讨了不同种类及剂量低能离子注入甲基缺陷型菌株GM2929前后甲基化水平的变化。结果表明:能量为10keV,剂量为3.9×1015ions/cm2、4.68×1015ions/cm2的氮离子以及能量为10 keV,剂量为4.68×1015ions/cm2的氩离子都不能诱发大肠杆菌甲基化,产生表观遗传效应,可遗传的变异与活细胞甲基化没有关系。     

Radiobiological effects of a low-energy ion beam on wheat

The radiobiological effects of a low-energy nitrogen ion (N⁺) beam on wheat were studied, particularly with regard to the induction of chromosome aberrations. The results demonstrated that the three test varieties showed different sensitivities to ion implantation, and a higher dose of ion implantation had a marked effect on the germination and survival rate of the seeds exposed. The germination rate and survival rate curve basically followed a similar trend in the same variety. Cytological analysis indicated that ion beams were effective in producing chromosome aberrations. The frequencies of mitotic or meiotic cells with chromosome aberrations increased linearly with increasing doses. The aberration types included, for example, acentric fragments, chromosome deletions, lagging chromosomes, chromosome bridges and micronuclei. In the root tip cells, aberrations chiefly consisted of acentric fragments and deletions. Chromosome bridges and lagging chromosomes were the main aberration phenomena observed in the pollen mother cells. The highest frequencies of root tip cells and pollen mother cells with chromosome aberrations were 15.2% and 39.8%, respectively. Changes in morphology and mutant were also observed in the plants derived from exposed seeds. Received: 10 April 2000 / Accepted: 10 October 2000     

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.     

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.     

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.     

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.     

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.