【作者】 郭玉琼；

【导师】 吕柳新； 赖钟雄；

【作者基本信息】 福建农林大学 ， 果树学， 2007， 博士

【摘要】 龙眼、荔枝同属无患子科，是我们南方重要热带亚热带特产果树。本研究以龙眼、荔枝胚性培养物为材料，进行龙眼、荔枝玻璃化超低温保存方法研究；同时，以龙眼胚性培养物为材料，进行超低温保存过程中低温预培养的蛋白质组学研究，以进一步探索超低温保存机理。主要研究结果如下：1．龙眼、荔枝胚性培养物的玻璃化超低温保存技术体系以龙眼胚性培养物为供试材料，探讨继代培养时间、化冻方式、不同发育阶段低温预培养对龙眼胚性培养物玻璃化超低温保存的影响。试验结果表明，继代培养12～15d的龙眼胚性愈伤组织用于玻璃化超低温保存，可获得较高的细胞活力；40℃温水浴、25℃左右室温和自来水冲洗3种不同的化冻方式对龙眼胚性愈伤组织玻璃化超低温保存后的存活率影响差异不大；球形胚玻璃化超低温保存后的细胞活力，比正常的胚性愈伤组织玻璃化超低温保存后的细胞活力高。以荔枝胚性愈伤组织为供试材料，探讨低温预培养、化冻方式以及光照条件对荔枝胚性愈伤组织玻璃化超低温保存的影响。结果表明荔枝胚性愈伤组织5℃低温预培养2d再进行超低温保存，采用40℃温水浴化冻和自来水流水冲洗化冻都能明显提高保存后的细胞活力；超低温保存后的培养物置于黑暗中进行为期15d的恢复生长，然后转到正常光照条件下进行培养，能使超低温保存后的荔枝愈伤组织较快地恢复生长，细胞存活率较高。2．龙眼、荔枝胚性愈伤组织玻璃化超低温保存后的体胚发生与植株再生龙眼、荔枝胚性愈伤组织经玻璃化超低温保存后均能进行正常的体细胞胚胎发生，与超低温保存前正常的胚性愈伤组织体胚发生数量接近，并且体胚能完成正常分化，形成形态正常、粗壮的完整再生植株，表明龙眼、荔枝胚性愈伤组织经玻璃化超低温保存后，在体胚发生与再生植株能力方面没有产生明显变化。3．龙眼、荔枝胚性愈伤组织玻璃化超低温保存后遗传稳定性的检测利用RAPD技术从DNA分子水平对玻璃化超低温保存的龙眼、荔枝胚性愈伤组织进行遗传稳定性检测。筛选了10条随机引物，对玻璃化超低温保存前和保存后的胚性愈伤组织基因组DNA进行扩增。检测结果表明，超低温保存的体细胞无性系遗传稳定性基本得到保持，但也发现存在小量的变异现象，有个别引物扩增的谱带，在超低温保存前和超低温保存后存在一些差异。其中，龙眼胚性愈伤组织超低温保存后的变异率为3.0～3.5％，荔枝胚性愈伤组织超低温保存后的变异率为7.48％。玻璃化超低温保存后产生的遗传变异与超低温保存过程中环境胁迫有关。4．低温预培养对龙眼球形胚存活力和蛋白质表达的影响玻璃化超低温保存前的低温预培养对保存后球形胚活力与蛋白质的表达有直接的关系。在5℃预培养4d时，超低温保存后的球形胚活力最高，达82.58％，此时蛋白质表达情况为新诱导4个蛋白点，上调表达1个蛋白点，新诱导与上调表达的蛋白点数最多，共5个蛋白点；在低温预培养6d时，超低温保存后的球形胚活力反而下降，为70.25％，此时蛋白质表达情况为消失4个蛋白点，下调表达153个蛋白点数，消失与下调表达的蛋白点数达157个。新诱导和上调表达的蛋白增强了细胞的抗冻能力，使超低温保存后的存活率提高。消失和下调表达的蛋白导致细胞的抗冻能力减弱，由此引起超低温保存后的存活率下降。表明龙眼体胚超低温保存前的低温预培养以5℃、4d为最佳。5．低温预培养特异表达蛋白的生物质谱鉴定与功能分析对龙眼LC₂球形胚5℃低温预培养过程中出现的特异表达蛋白LLT1、LLT2、LLT3和LLT4，应用基质辅助激光解吸／电离飞行时间质谱（MALDI-TOF MS）进行肽质量指纹分析，匹配分析结果表明：LLT3可能为抗性蛋白，推测是龙眼的抗冻蛋白，对细胞具有直接保护作用，并且由PI演变而来；LLT4可能是龙眼的果糖-1，6-二磷酸醛缩酶，通过改变代谢途径，提高抗冻能力。通过蛋白质的功能分析推测龙眼球形胚5℃低温预培养4d时可能有抗冻蛋白和与物质合成有关的蛋白质的表达。更多还原

【Abstract】 1．Establishment of cryopreservation technique systems of longan and litchi embryogenic cultures by vitrificationIn the experiment, the effects of the factors including the subculturing time, throwing methods and developmental phases on cryopreservation by vetrification of longan embryogenic cultures were investigated. As a result, the cell survival rate after cryopreservation was higher by using longan embryogenic calli after subculturing 12～15 d; the comparison of 3 throwing methods (40℃water bath, room temperature 25℃and tap water)was carried out which indicated that they had the same effect on the survival rate after cryopreservation for longan calli. The survival rate of cryopreservated globular embryoids was higher than that of normal embryogenic calli.By experiment, the effects of low-temperature preculture, throwing methods and illumination condition on cryopreservation of litchi embryogenic calli by vitrification were tested. The results indicated that both 5℃low-temperature preculture for 2 d and using throwing with 40℃warm bath and tap water could all improve the survival rate after cryopreservation by vitrification significantly. In the experiment of recovery growth, we found that if the embryogenic c alli after cryopreservation had been in darkness for 15 d first, and then they were transferred under normal illumination, the rate of recovery growth became faster and the survival rate after cryopreservation was also higher.2．Somatic embryogenesis and plantlets regeneration normally from cryopreserved longan and litchi embryogenic calli by vitrification.Plantlets regenerated normally via somatic embryogenesis after cryopreservation of longan and litchi embryogenic calli. The number of somatic embryos was similar to that of the embryogenic calli before cryopreservation. Through the maturation culture, the somatic embryos could germinate and form plantlets normally. It showed that the ability of somatic embryogenesis and plantlets regeneration didn’t changed after cryopreservation of longan and litchi embryogenic calli.3．Test of hereditary stability after cryopreservation of longan and litchi embryogenic calli by vitrificationThe hereditary stability of cryopreserved embryogenic calli from longan and litchi was tested by randomly amplified polymorphic DNAs (RAPD) technique on molecular level. The results of RAPD by 10 primers proved that somaclonal hereditary stability was almost preserved, only appearing little variation. It was founded that there were some differences between the amplified sites of embryogenic cultures before cryopreservation and after cryopreservation in exceptional primers. In the experiment, the variations of cryopreserved longan and litchi embryogenic calli were 3．0～3．5% and 7．48% respectively. The genetic variation of cryopreservation by vitrification was related to environmental stress. 4．Effects of low-temperature preculture on the survival rate and proteins expression for globular embryoids of longanThe study indicated that there were some direct effects on the survival rate and specific proteins expression after cryopreservation of longan globular embryoids. The survival rate of longan globular embryoids after cryopreservation reached the highest (82．58%) when precultured in 5℃low-temperature for 4 d, while the expression of proteins showed 4 novel protein spots appeared, 1 protein spot was up-regulated.The numbers of protein spots novel and up-regulated were 5, which were the most. The survival rate of longan globular embryoids after cryopreservation decreased to 70．25% when precultured in 5°C low-temperature for 4 d, while the expression of proteins showed 4 protein spots disappeared, 153 protein spots were down-regulated. The numbers of protein spots disappeared and down-regulated were 157, which were the most. All these suggested that the proteins novel and up-regulated strengthened the ability of anti-freezing while the proteins disappeared and down-regulated weakened the ability of anti-freezing.The former increased the survival rate after cryopreservation, and the later decreased the survival rate after cryopreservation. It showed that 5℃low-temperature preculture for 4 d was the best preculture method for cryopreservation of longan globular embryoids.5．Identification of novel proteins induced during 5℃low-temperature preculture by biology MS and analysis of their functionsIn the experiment, by using Matrix-Assisted Laser Desorption/Ionization Time of Flight-Mass Spectrometry (MALDI-TOF MS), the peptide mass fingerprinting (PMF) was analyzed in LLT1, LLT2, LLT3 and LLT4 which were the novel protein spots appeared during 5℃low-temperature preculture. The PMF data of these 4 proteins were used in Mascot search. The results indicated that LLT3 was perhaps resistance protein. It was predicted that LLT3 was an AFPs in longan and derived from PI. Moreover, it was predicted that LLT4 was a FDP-ALD in longan which was related to synthesis of compounds. The analysis of the function of proteins predicted that AFPs and proteins concerning synthesis of compounds expressed when 5℃low-temperature precultured for 4 d of longan globular embryoids.更多还原