【作者】 宋秀珍；

【导师】 黄勤妮； 李艳红；

【作者基本信息】 首都师范大学 ， 植物学， 2003， 硕士

【摘要】 第一部分：立碗藓原生质体分离及培养的研究 基因打靶技术通过利用同源重组可以在一定程度上实现外源基因的定向整合；同时还可通过对某基因的剔除研究基因的功能或通过创造定点突变来研究某种代谢机理。 但基因打靶技术目前主要应用于细菌，酵母和一些丝状真菌，在高等植物中应用极少。原因是高等真核生物中同源重组的发生频率极低。小立碗藓是最近被开发出来的用于研究基因打靶的模式植物，其基因打靶的效率可以与酵母相比。 本论文采用生长在北京地区的与小立碗藓同科立碗藓(Physcomitrium sphaericum)为材料，探讨其无菌培养的条件。由于藓类植物特殊的生物学结构用农杆菌侵染其原丝体或者茎叶体很难实现转化，以原生质体作受体是藓类植物转化的常用途径。本实验摸索得到了影响立碗藓原生质体产量及活力的几项因素。为藓类植物基因打靶的研究奠定了一定的基础。 通过对立碗藓的无菌培养和原生质体操作发现： (1) 立碗藓孢朔接种在无菌MS、Benecke、Knop培养基上，均可萌发产生原丝体，但不久便分化为茎叶体，很难长期保持其原丝体状态，不同培养基条件下原丝体状态有所不同。 (2) 通过不同浓度的激素组合诱导愈伤组织研究发现：当采用6-BA 0.5 mg/l，2,4-D0.5mg/l时，立碗藓原丝体上长出疏松易碎的白绿色愈伤组织。 (3) 采用不同阶段的原丝体和茎叶体进行原生质体的游离发现：15-17天的原丝体是分离原生质体的最佳材料，超过三周的原丝体酶解得到的原生质体数量很少，茎叶体分离原生质体更难。 (4) 通过对原丝体预处理不同时间观察表明酶解前用0.02％Tween-80预处理30min能明显提高原生质体的产量。 (5) 采用不同浓度和种类的酶来游离原生质体效果不同，其中，0.5％纤维素酶+0.4％果胶酶+0.5％离析酶+0.3％半纤维素酶对0,1g的原丝体进行2小时的酶解能够得到5×10~3个/ml原生质体，原生质体保持较高的活力。更多还原

【Abstract】 Gene targeting by homologous recombination can realized targeted integration. At the same time we can knock-out some gene to know its genetic function and create specific mutation to study some kinds of metabolizing mechanism.But gene targeting was mainly applied to such field as bacterium, yeast and some epiphyte at present, seldom used in higher plants because of the lower frequency of homologous recombination. The moss Physcomitrella patens (Funariales Bryophyta) is being developed as a model system for plant biological studies. Its gene targeting efficiency can be compared with that observed in Saccharomyces cerevisiae.In this thesis, Physcomitrium sphaericum growing in the area of Beijing, which belongs to the same section as Physcomitrella patens, is applied as experiment material to explore the condition of the sterile culture. Because of the special biological structure of bryology, it was very difficult to transfer foreign gene into the protonema or gametophyte by Agrobacterium-mediated transformation. Protoplasts as acceptor, using direct DNA transfer methods such as microprojectile bombardment and PEG-mediated transformation is becoming a good way. So how to obtain high output and high energy protoplasts was very important. Here we studied the relationship of various factors and the quality of protoplasts.which maybe could be the basic of moss gene targeting.Results showed:Inoculated the spores onto diferrent kinds of media, such as MS, Benecke and Knop, we found that there was no difference when the spores germinated and differentiated into cauliform soon. It was very difficult to keep protonema longer. The media types had some effect on the state of protonema.The media supplemented with hormone of different types and concentration to induce callus, Only the medium added with 6-BA 0.5 mg/1 + 2,4-D 0.5 mg/1 had good effect on callus formation.Isolation of protoplasts from different stage of protonema and cauliform revealed that 15-17 days protonema was the best material because of the higher protoplasts output. There was less protoplasts when the protonema was over three weeks old. It was much more difficult when we use the cauliform asmaterial.Pretreatment for 30 minutes by 0.02% Tween-80 before digestion can obviously increase output of protoplasts.Using 0.5 % CELLULASE + 0.4% PECTOLYASE Y-23 + 0.5% MACEROZYME +0.3%HEMICELLULASE H-2125 to digest 0.1 g protonema for 2 hours, can abtain 5 +103 protoplasts /ml, which keep more energy.更多还原