【作者】 朱文涛；

【导师】 王子成；

【作者基本信息】 河南大学 ， 遗传学， 2013， 硕士

【摘要】 超低温保存技术不仅能作为野生植物种质资源长期保存的有效方式，也能成为一种高效的脱毒技术——低温疗法。本文以野生资源五叶草莓为材料，研究了其最佳的超低温保存条件，然后分析了超低温保存后五叶草莓基因组DNA序列的稳定性以及基因组DNA甲基化的变化水平；同时以携带草莓轻型黄边病毒的全明星草莓为材料，研究了玻璃化法的低温疗法脱毒的最佳条件，探讨了低温疗法脱除草莓轻型黄边病毒的效率，分析了低温疗法后全明星草莓基因组DNA序列的稳定性以及基因组DNA甲基化的变化水平。主要研究成果如下：1.探索五叶草莓组织培养茎尖离体玻璃化法超低温保存的最佳条件，比较后发现：草莓组织培养材料在4℃条件下低温炼苗3周，切取2³mm的离体茎尖，在固体预处理培养基(0.4mol·L^-1蔗糖+MS+7g·L^-1琼脂，pH=5.8)中4℃处理3天，液体预培养液(0.4mol·L^-1蔗糖+2mol·L^-1甘油+MS，pH=5.8)25℃培养30min，玻璃化保护溶液PVS₂(0.4mol·L^-1蔗糖+15%（w/v） DMSO+15%（w/v）乙二醇+30%（w/v）甘油+MS，pH=5.8)0℃处理1h时，能达到最佳条件使成活率达到79.7%，且草莓再生植株直接分化成草莓植株。2.研究携带草莓轻型黄边病毒的全明星草莓玻璃化超低温保存最佳条件，与五叶草莓的玻璃化法超低温条件比较发现：全明星草莓离体茎尖在在4℃条件下低温炼苗4周，固体预处理培养基处理3天，液体预处理培养液培养30min，玻璃化保护溶液PVS₂处理1h时能达到最佳条件，然而成活率只有64.3%，植物材料不同玻璃化超低温保存程序会发生改变。利用常规RT-PCR技术检测低温疗法后的植株病毒后，草莓轻型黄边病毒的脱除率达到了95%。3.采用扩增片段长度多态性（AFLP）技术分析了超低温前后的五叶草莓和全明星草莓的基因组DNA序列，没有发现多态性差异条带。说明经过超低温之后的再生五叶草莓和全明星草莓植株基因组DNA序列没有发生改变，基因组DNA序列的稳定。4运用甲基敏感扩增多态性（MSAP）标记技术对超低温或者低温疗法前后的五叶草莓和全明星草莓的基因组DNA甲基化水平进行了研究。统计和分析了聚丙烯酰胺变性凝胶电泳的多态性条带之后，发现超低温过程促使五叶草莓基因组DNA甲基化水平降低了6.73%，且去甲基化水平变化率和甲基化水平变化率分别为12.43%和5.70%；同样，经过低温疗法脱毒之后的全明星草莓基因组DNA甲基化水平也出现了降低的状况，仅为3.83%，且去甲基化水平变化率和甲基化水平变化率分别为12.44%和8.61%。更多还原

【Abstract】 Cryopreservation is not only considered to be the most effective way to long-term preservation ofwild plant germplasm resources,but also a highly efficient virus-free technology,“Cryotherapy”.In thispaper, we studied the procedure of Fragaria pentaphylla and “All-Star”strawberry shoot tips vitrificationcryopreservation, and “All-Star”strawberry materials were infected theStrawberry mild yellow edge virus（SMYEV）, we detected virus-free rate after Cryotherapy. Then, we detected the genome sequences DNAand analyzed the variation of DNA methylation level of samples between the treatment and the control.The main results below:1.The procedure of Fragaria pentaphylla shoot tips vitrification cryopreservation wasexplorato-ry studied. The results showed that: the shoot tips were cryoacclimatized at a low temperature （4℃） for3weeks; pre-cultured in the Pretreatment of solid culture medium (0.4mol.L^-1sucrose+7g.L^-1agar+MS, pH=5.8) after3days; treated with liquid pretreatment medium (0.4mol.L^-1sucrose+2mol.L^-1glycerin+MS, pH=5.8) for30mins at room temperature and treated in PVS₂(0.4mol.L^-1sucrose+15%（w/v） DMSO+15%（w/v） ethylene glycol+30%（w/v） glycerin+MS, pH=5.8)at0℃for60mins, thesurv-ival rate was highest up to79.7%. Regenerations differentiated normally.2. Distinction of the best vitrification cryopreservation procedure between “All-Star”strawberryand Fragaria pentaphylla were found that: the shoot tips of “All-Star”strawberry were cryoacclimatized at alow temperature （4℃） for4weeks; pre-cultured in the Pretreatment of solid culture mediumafter3days;treated with liquid pretreatment medium for30mins at room temperature and treated in PVS₂at0℃for60mins, the survival rate was highest up to64.3%, different plant material have different cryopreservationprocedure. Then, SMYEV weredetected by RT-PCR technology,wefound that virus-free rate was highestup to95%.3. The technique of amplified fragment length polymorphism （AFLP） was used to analyze thegenome sequences DNA of the treatments and controls, no variation of bands was found, which indicated that there were no changes in the genomic DNA sequences of Fragaria pentaphyllaand“All-Star”straw-berry after cryopreservation.4. The methyl-sensitive amplified polymorphism （MSAP） markers techlologe was used toanaly-sis the variation of DNA methylation level of samples between the treatment and the control. Theresults indicated that DNA methylation states of treatment materials reduced compared with controls, DNAmeth-ylation state ofFragariapentaphylla and “All-Star”strawberrywerereduced6.73%and3.83%.Methyla-tion and demethylation of Fragaria pentaphylla DNA were5.70%and12.43%,but, Methylationand dime-thylation of “All-Star”strawberry DNA were8.61%and12.44%.更多还原