【作者】 李永亮；

【导师】 霍裕平；

【作者基本信息】 郑州大学 ， 凝聚态物理， 2009， 博士

【摘要】 20世纪80年代起,余增亮等发现了低能离子束生物诱变效应。此后引发了离子束生物诱变效应的广泛研究,学者们发现了多种多样的诱变效应,包括生物表型性状、细胞学效应、生化效应、遗传效应及分子生物学效应等,其中,通过突变体选择选育了一大批包括水稻、小麦、微生物等在内的优良农作物新品种。在机理机制方面,围绕细胞刻蚀穿孔、物质沉积、能量沉积、电荷沉积、近旁效应等做了大量的研究,做出了很好的尝试,值得深入研究。但是,一项新兴的研究领域,离子束生物技术还存在处理方法有待拓展、处理设备有待改进、处理群体小、诱变机理有待阐明等诸多挑战。为更深入开展离子束生物诱变研究,我们选择玉米作为研究材料,开展机理及应用研究,以期获得新的进展。通过玉米离子束诱变研究,我们获得了如下结论：①研究发现,玉米作为离子束诱变研究的对象,具有独特的优势：首先,作为二倍体,其基因组较小麦为简单；第二,玉米种子较大,易于剥除种皮从而可免除种皮对离子束注入的限制,使生长点可直接被辐照诱变；第三,相对小麦与水稻,玉米具有较大的胚,可以提供更多的离子束辐照表面积以接受更多离子束辐照：第四,玉米100天左右的较短生育期,可以在海南加代繁殖,实现一年两到三代的诱变世代,为扩大选育突变体群体,缩短研究周期,提供了极大的便利；第五,最值得关注的是,相对小麦与水稻,玉米花粉具有较强的生活力和较长的存活时间,使花粉诱变育种成为可能,可以进行花粉诱变创造极大地突变体群体,为选育突变体提供了宝贵的机会。②通过花粉诱变效应探索,克服了花粉诱变操作中存在的花粉抽真空逸失、活力测定方法选择、高死亡率、快速授粉、加代快速选育突变体、花粉处理方法及剂量选择等诸多困难,初步建立了较为完整玉米花粉诱变方法,获得了花粉囊突变体、籽粒致死突变、籽粒颜色突变、粒形突变、白化苗突变等丰富的突变类型,初步证明玉米花粉离子束诱变是一种高效快速的诱变方法。③探索建立了玉米种子胚蛋白质组学研究方法,克服了蛋白质提取、双向电泳除盐升压、高效快速染色等研究难点,获得了稳定有效的实验操作方法,高质量的电泳图谱的获得,通过大量实验探索改进的改良TCA/丙酮蛋白质提取、改良电泳程序、改良胶条平衡及改良胶体考染程序等方法的建立,为进一步开展离子束诱变效应蛋白质组学研究打下了良好基础。④离子束生物诱变效应研究的创新。在吸取前人研究成果的基础上,率先开展了离子束诱变效应的蛋白质组学研究。以前人关于离子束辐照引起自由基产生、引起同工酶谱变化为启发,考虑结合最为先进的前沿生物学研究技术——后基因组学研究技术,使低能离子束生物诱变与以双向电泳及质谱鉴定技术为核心的蛋白质组学研究技术相结合,从整个蛋白质组的角度探索离子束辐照诱变处理引起机体的蛋白质类型群体变化出发,综合研究其效应。关键酶蛋白质的变化,即是诱变效应的本身,又是更多表型效应产生的内部机制,蛋白质变化研究,集效应与机制研究于一体,为离子束生物效应研究提供了很好的平台,值得深入研究。结果表明,离子注入机产生的真空环境即可引起玉米胚蛋白质组发生变化,一些胁迫应激酶产生了响应。离子束注入玉米种子后,取其萌发胚进行研究,发现了诸多耐人寻味的变化：分子伴侣smHSP(如HSP16.9、HSP17.4)、HSP70、LEA3、DHN1等明显上调表达,可能与离子胁迫保护有关；多种抗氧化酶大量上调表达,如MnSOD、GST21、peroxidase等均出现表达量增加,可能与抵抗离子束引起的自由基增加有关；蛋白降解体系变化,如26S蛋白酶体等上调表达,可能与降解离子束辐照引起的变性蛋白有关；大量调控因子出现、消失、上调或下调表达,如Ran蛋白、MAPK因子、PP2A、14-3-3蛋白等,可能与机体应激调控有关；糖、蛋白质、核酸等代谢酶下调,可能与离子束胁迫引起损伤有关,也可能是离子束辐照引起种子萌发降低,生长缓慢等形成的原因；一些基因表达相关蛋白或核酸结合蛋白的变化可能引起突变效应的产生。⑤玉米杂种优势在蛋白质组上有突出表现。研究发现玉米杂交种与其亲本自交系对离子束辐照的反应不同,突出表现在杂交种能耐受更大剂量的离子束辐照,同样的辐照剂量下,杂交种受损伤小。相应蛋白质组上的表现则为：杂交种对抗离子束辐照的应激更为积极和复杂。如产生更多的分子伴侣,分子伴侣的种类更为复杂可能更为有效的消除变性蛋白质；抗氧化酶种类更多,表达量较亲本增加；调控因子种类更多,大量调控因子出现或增加可能有利高效调控。杂交种代谢酶类表达下调的幅度较亲本自交系为小,可能是杂交种对辐射表现耐性形成的原因之一。更多还原

【Abstract】 Ion beam mutation study using high-energy ion beams began in the 1920s. However, at that time, mutations resulting from low-energy ion beams were not recognized. It was not until decades later about 1980s that mutation effects on rice using low-energy ion beams were reported by Yu Zengliang et al.. During the past 20 years, substantial progress has been made in this field of research. Many studies have focused on changes to morphological and cytological traits, isozymes and DNA originating from different mutation materials, and some cultivars in different crops have been generated by exposure to low-energy ion beams, including Wanjing D9055 and S9042 in rice, Wanmai32 and Wanmai42 in wheat and so on. On the mechanism, many research were done including cell sculpting, matter aggradation, energy aggradation, bystander effects and the like. But as a new research realm, a good many challenges were confronted by ion beam bio-engineering, such as improving of treatment methods and equipments, low impact, small treatment colony in one radiation operation and the puzzledom of mechanism etc.. We selected maize as the materials for the application research and mechanism exploring and some results were described as that:①As the highest output food supplies corp, maize is of great importance in the the application and background research realm and fit very much for the research of low energy ion beam implantation because of the five characters found or proved in our experiments:firstly, being diploid plant, maize is smaller and simpler in genome comparing with some other plant such as wheat and so on. Secondly, maize can be free of the seed capsule obstacle to the penetration of ion beam by shucked out of the seed capsule easily than other plant. And so the growth point of maize embryos can be implantated directly and effectively by ion beam for free of seed capsule obstacle. Thirdly, comparing to wheat, rice and other plant, the larger embryo of maize can absorb more ion beam because of its larger surface area. Fourthly, because of its shorter growth period(about 100 days) and the increasing generation technology in Hainan province, that make two or three mutation selection generations in one year possible, maize as the experimental materials of ion beam radiation is efficient that can provide large advantage to enlarge mutant chosen cology and short research period. Fifthly, its strong pollen viability and long existence time, comparing to wheat and rice, made maize pollen mutation by ion beam possible.②A mensuration method to evaluate pollen viability with low energy in maize was established. Several challenges experienced during ion implantation pollen mutation research were overcome, including pollen loss from run-off during vacuum pumping, and high pollen mortality rates. The data showed that a half lethal dose of 1 X 1014N+/cm2 at low-energy 30 keV was most effective for treating maize pollen. Three elite inbred lines were treated in this study. Some mutants such as white anther color, red seed capsules, lethal seed and albino seedling mutants were observed in the MO and M1 populations. The results demonstrated that the pollen treatment with low-energy ion beam was an effectual method in maize mutant breeding.③A method of maize seed embryo proteomics research were established that overcome protein distilled,2-D salt-removed and voltage-hoisted, coloration and so on. The amended method acquired high quality 2-D picture and established good base for the proteomics research of ion beam radiation effects.④Innovation of ion beam radiation effects research. Based on the former research, we studied proteomics research of ion beam radiation effects. Through ion beam radiating treatment of maize seed and proteomics analyzing of treatment seed embryos, some absorbing results were acquired. The vacuum, high temperature and ion of ion beam radiation equipment affected the proteome of the treatment maize seed embryos, and so some protein enzymes answered to the pressure of environment such as smHSP (HSP16.9、HSP17.4)、HSP70、LEA3、DHN1 and so on that up-regulated in output and probably related to defence of ion pressure. Some kinds of antioxidant protein enzymes were up-regulated in output such as MnSOD, GST21, peroxidase and so on that probably has been associated with stress tolerance in plants because it neutralizes the reactivity of O-2, which is overproduced under oxidative Reactive Oxygen Species(ROS). The protein degradation enzymes such as 26S-ubiquitin proteosome pathway conjugating enzymes were up-regulated in output probably related to the degradation of denaturalization protein produced by ion beam radiation. Many regulator protein such as Ran、MAPK、PP2A、14-3-3 were up-regulated or down-regulated related to regulation of organism response. Some metabolize enzyme related to sugar, protein, nucleic acid and so on were down-regulated probably related to the damnification of ion beam radiation or seed bourgeon drawback, slow growth and so on. Some gene expression correlative protein or nucleic acid conjunction protein probably related to gene mutation.⑤The heterosis behaved well in maize proteome, especialy in the proteomic induced by ion beam radiation. More differences were discovered in the response of ion beam radiation among the parents and hybrid. The hybrid can endure stronger ion beam radiation than the parents. In proteome, the response of hybrid became more complex and active than the parents. Such as more kinds and quantity of molecule cheapore as HSPs, LEA3、DHN1 and so on so that clear up denature protein efficiently, more kinds more kinds and quantity of antioxidant protein enzymes to neutralize the reactivity of O-2 and so on, more kinds more kinds and quantity of regulator for regulating efficiently. In addition, more little in down-regulated extent in the hybrid metabolize enzymes than the parents probably related to the strong endurance to ion beam radiation.更多还原