留兰香种质材料超低温保存及遗传变异的分析.docx

摘要超低温保存被认为是植物种质资源长期保存最有效的方法。本文以留兰香( Mentha spicata L.) 茎尖为材料,对其玻璃化超低温保存条件进行了研究,另外研究了超低温保存对留兰香材料生理生化反应、基因组稳定性以及 DNA 甲基化的作用。主要研究结果如下: 1对留兰香茎尖玻璃化法超低温保存的程序进行了探索研究,结果表明: 4℃低温锻炼 3-4 周,在添加 2 mol ·L -1甘油的 MS 培养基中预培养 3天,液体预处理培养室温处理 20 min , PVS 20℃脱水 50 min 时,成活率最高,可达 60% 左右。再生植株分化正常。 2留兰香经超低温处理后与对照组相比丙二醛(MDA) 含量显著升高,过氧化物酶(POD) 、过氧化氢酶(CAT) 活性均显著增强。上述生理生化指标说明,超低温处理影响了留兰香的正常生理生化反应,导致丙二醛积累,一些抗氧化酶活性改变。 3运用扩增片段长度多态性(AFLP) 技术对处理再生材料及对照组的基因组 DNA 进行了分析,在处理组与对照组之间未发现有差异片段。表明在超低温保存过程中没有引起保存材料基因组 DNA 序列的变化。 4应用甲基敏感扩增多态性(MSAP) 标记技术分析处理与对照组样品之间的甲基化水平情况。 MSAP 技术对超低温处理前后材料的甲基化情况分析表明:与对照相比, 超低温处理后的再生材料均发生了甲基化状态与水平的变化,全基因组 DNA 胞嘧啶甲基化水平降低,另外甲基化与去甲基化分别为 % % 。关键词:留兰香,超低温保存,遗传变异 I Abstract Cryopreservation is considered to be the most effective way to long-term preservation of plant germplasm resources. In this paper, we used the spearmint ( Mentha spicata L.) tips as the materials to exploratory research the vitrification cryopreservation procedure. And studied the effects of cryopreservation on physiology , genome stability and DNA methylation in Mentha Spicata . The main results as follows: 1. The procedure of spearmint shoot tips vitrification cryopreservation was exploratory studied. The results showed that: the shoot tips were limatized ata low temperature (4℃) for 3-4 weeks; pre-cultured in the MS medium with adding 2 mol .L -1 glycerol after 3 days; treated with liquid pretreatment medium for 20mins at room temperature and dehydrated in PVS2 at0℃ for 50mins, the survival rate was highest up to 60%. Regenerations differentiated normally. 2. The content of malondialdehyde (MDA) of the spearmint treated by Cryopreservation increased pared with controls, the activity of peroxidase (POD) and catalase (CAT) aslo significantly enhanced. The physiological indexes illustrated ultra-low temperature treatment affected the normal physiological and biochemical reactions of spearmint, resulted in MDA accumulation, activity of some antioxidant enzymes changing. 3. The technique of amplified fr