【作者】 徐瑾；

【导师】 刘燕；

【作者基本信息】 北京林业大学 ， 园林植物与观赏园艺， 2014， 博士

【摘要】 超低温保存是一项广泛应用于水产养殖、资源保护和医药等领域的生物技术,在理论上具有“永久性”保存生物材料的特点。尽管技术研究的发展大大提高了保存后生物材料的生活力,但冷冻和解冻仍然会对许多材料的原始功能产生负面影响,甚至会引起细胞广泛的致死或亚致死。技术的突破依赖于机制理论研究的成果,超低温保存的机制不清已成为其技术发展的主要瓶颈。超低温保存是花粉长期保存的适宜方式,并且已经成功地应用于多种物种的花粉保存。作为少数几种可以无需经过复杂的脱水和冷冻保护剂的参与即可直接投入液氮成功实现超低温保存的单细胞材料,花粉无疑是超低温保存机制研究的理想模板。本文以玉兰花粉为主要的试验材料,研究了超低温保存对其生理和分子层面所造成的影响,在此基础上,探讨花粉超低温保存的相关机制。其主要研究结果如下：1)扩建了课题组前期建立的园林植物超低温保存花粉库,成功实现了59种／品种植物花粉长达1年的液氮超低温保存,共涉及了17个科44个属的木本和草本的园林植物。2)选择超低温保存前后生活力没有显著变化的玉兰花粉作为试验材料,对超低温保存前后与氧化应激和细胞凋亡相关的指标进行测定,其中活性氧水平和丙二醛含量在花粉超低温保存前后没有显著变化,而质膜相对透性在超低温保存后显著提高,说明脂质过氧化不是造成细胞膜通透性增强的直接原因；超低温保存后过氧化氢酶活性显著升高,而抗坏血酸含量显著下降,说明超低温保存引起了玉兰花粉细胞内抗氧化防御系统的显著变化,细胞通过氧化／抗氧化系统的自我调节取得了新的平衡,因而使活性氧的水平在保存前后维持了稳定,并未诱导氧化应激的发生；对磷脂酰丝氨酸的外化水平和DNA片段化程度的测定表明,超低温保存并未引起细胞凋亡现象的发生。3)采用蛋白质组学技术研究玉兰花粉超低温保存前、中、后可溶性蛋白质的变化,共获得丰度变化在1.5倍以上的差异表达蛋白质点88个；选择三组比较中两组差异均显著的蛋白质点11个和表达量变化大于5倍的点6个进行质谱检测,获得有效鉴定8个,分别是：内质网网眼结合蛋白5、丙酮酸脱氢酶、过氧化氢酶同工酶3、假定脱氢酶、烯酰(基)ACP还原酶、天门冬氨酸氨基转移酶、无机焦磷酸酶和线粒体苹果酸脱氢酶,功能涉及了蛋白质折叠、抗氧化作用、三羧酸循环、嘧啶降解、脂肪酸合成、苹果酸-天冬氨酸转运、无机焦磷酸的利用等多个细胞生命过程。4)对8个差异蛋白的转录水平变化进行研究,获得其中2个——假定脱氢酶和烯酰(基)ACP还原酶——在超低温保存前、中、后的基因转录水平变化,对比其蛋白质水平的变化表明,两者之间存在不一致性,认为蛋白质的表达变化可能更多地来自于基因的翻译水平和翻译后水平调控,而不是基因的转录水平调控。5)为了验证差异蛋白质在超低温保存中的功能,选择其中的过氧化氢酶和苹果酸脱氢酶,将其同源替代物应用于扶芳藤和卷丹茎尖的玻璃化超低温保存,其结果表明：在适宜的阶段,将适宜浓度的过氧化氢酶或苹果酸脱氢酶添加于玻璃化超低温保存使用的溶液中能提高保存后茎尖的存活率和再生率。这在一定程度上证明了玉兰花粉超低温保存所鉴定的差异蛋白质在超低温保存进程中发挥着积极的保护作用。本研究的主要贡献在于扩建了课题组前期建立的园林植物超低温保存花粉库；证实了保存前后生活力没有显著变化的玉兰花粉在超低温保存过程中不存在氧化应激和细胞凋亡；获得玉兰花粉超低温保存前、中、后8个差异表达的蛋白质,并验证了其中2个蛋白质与超低温保存相关。更多还原

【Abstract】 Cryopreservation is a biotechnology widely used in the fields of aquaculture, conservation and biomedicine. Theoretically, the material can be stored without alteration or modification for an unlimited period of time. However, although highly optimized protocols improve cell viability, the extreme stress of freezing and thawing treatments can negatively affect the original functions of the cells, even cause extensive lethal and sub-lethal cryoinjuries. Technological breakthrough relies on the results of mechanism researches. The unclear mechanism of cryopreservation has become a major bottleneck in the development of its technology.Cryopreservation is an appropriate strategy for long-term pollen preservation and successful pollen cryopreservation has been achieved in many species. Pollen is one of the minority materials which can be cryopreserved without any pretreatment, excluding effects of osmotic shock during dehydration and the toxicity of the cryoprotectant agents, so it is an ideal material to study cryopreservation mechanism.In this study, Magnolia denudata Desr. pollen was taken as the main material and effects of cryopreservation on it at physiological and molecular level was studied to explore the mechanism of pollen cryopreservation. The main results and conclusions are as follows:1) The species/cultivars of the ornamental plants pollen cryopreservation bank were enriched. Pollen from59species/cultivars was viable after one year of storage in liquid nitrogen, involving a total of17families44genera of woody and herbaceous ornamental plants.2) M. denudata pollen from the pollen bank with nonsignificant change in germination before and after cryopreservation was chosen for a further study. Oxidative stress and apoptosis-related indicators were measured. There was no significant difference on reactive oxygen species generation and malonic dialdehyde content, whereas significantly increase was observed on relative conductivity, which indicate that lipid peroxidation is not a direct cause for enhanced membrane permeability. Significant increase on catalase activity and obvious decrease on ascorbic acid content were discovered, which suggest that cryopreservation has caused a significant change in antioxidant defense system of the pollen cell and a new balance has achieved via self-regulation of oxidant/antioxidants system, resulted in nonsignificant change on reactive oxygen species generation and no oxidative stress occurred. Results on phosphatidylserine externalization and DNA ladder measurements revel that apoptosis does not exist in M. denudata pollen cryopreservation. 3) A proteomic study was carried out on cryopreservation of M. denudata pollen from fresh, cryopreserving, and cryopreserved pollen. Comparative analysis showed in total88differentially displayed protein spots whose abundance was altered by at least1.5-fold between two pollen samples.8of17differential expression protein spots (including11protein spots which were changed significant among3profiles and6protein spots whose abundance was altered by at least5-fold between2profiles) were successfully identified as a luminal-binding protein5, pyruvate dehydrogenase, catalase isozyme3, putative dehydrogenase, enoyl-ACP reductase, aspartate aminotransferase, inorganic pyrophosphatase and mitochondrial malate dehydrogenase. Functional analysis of these proteins indicated that they are involved in many cell processes, such as protein folding, anti-oxidative defense, TCA cycle, pyrimidine degradation, fatty acid synthesis, malate-aspartate shuttle and utilization of inorganic pyrophosphate.4) Changes in transcript levels in samples during-LN and post-LN with reference to pre-LN samples for8differential expression proteins were studied, of which enoyl-ACP reductase and putative dehydrogenase were achieved. It shows that gene expression at transcript level was not always corresponded with the changes at protein level. It indicates that the changes at protein level may come from translational control or post-translational control.5) To verify functions of differential expression proteins on cryopreservation, catalase and malate dehydrogenase were added separately to solutions used in vitrification of Euonymus fortunei and Liliun lancifolium shoot tips. It shows that at a suitable stage, addition of these two enzymes with an appropriate concentration can improve the survival and regeneration of shoot tips after cryopreservation, which suggests that these differentially expressed proteins probably play a protective role on M. denudata pollen following cryopreservation.The main contributions of this study is that:the species/cultivars of the ornamental plants pollen cryopreservation bank were enriched; oxidative stress and apoptosis were confirmed not exist in M. denudata pollen which has nonsignificant change in viability after cryopreservation;8differential expression proteins were successfully identified from fresh, cryopreserving, and cryopreserved M. denudata pollen and2of them were verified related to cryopreservation.更多还原