【作者】 郭长禄；

【导师】 陈力耕；

【作者基本信息】 浙江大学 ， 果树学， 2004， 博士

【摘要】 银杏(Ginkgo biloba)又名白果、公孙树，新生代第四纪冰川期的孑遗植物，为我国独存的珍稀名贵树种。我国是银杏的故乡，银杏与大熊猫一样，已被世界各国公认为中国代表。银杏被称为“活化石”，植物学家常把银杏与“恐龙”相提并论，在生物的起源、进化等研究方面提供了理想的试材和有力证据，具有重要的科学研究价值。银杏的经济价值很高，随着银杏产业的发展，银杏在食品、保健、医药、木材、绿化等领域得到广泛的开发利用，然而银杏童期长，实生苗20-30 a才能开花结果，常规育种周期较长，严重影响了银杏的品种改良。目前LEAFY基因被认为是诱导植物开花的枢纽基因，其过量表达可以促进植物提早开花。研究银杏LEAFY同源基因的时空表达模式有助于我们初步认识LEAFY同源基因的功能。通过组织培养、遗传转化等方法从分子水平对其进行改良，可以开辟银杏育种新途径。 我们通过实验获得了以下研究成果： 1．裸子植物银杏组织培养分化特别困难，分别以银杏胚乳、胚、胚轴、子叶以及不同采收期的未成熟胚为外植体，研究了在不同培养条件下愈伤组织和胚状体的发生条件，建立了MK新型培养基配方，结果显示，只有未成熟胚及其胚轴、子叶能够产生胚状体。大于3mm的未成熟胚，在MK+NAA 1 mg/L+BA 1 mg／L培养基上胚状体诱导率最高，达到53.6％，最多的一块愈伤组织形成多达38个胚状体；胚轴最佳诱导条件为：MK+NAA 1 mg／L+BA 1 mg/L，诱导率为45.3％；子叶最佳诱导条件为：MK+NAA 1.5 mg／L+BA 1 mg／L，诱导率为12.9％。暗培养不利于胚状体的发生。不同采收期的未成熟胚愈伤组织和胚状体的诱导率不同。 2．在国内外首次把胚状体培育成苗，建立了银杏组织培养再生体系。利用银杏未成熟胚诱导的胚状体在无菌的条件下，接种在添加不同浓度的活性炭、蔗糖、椰汁的MK培养基上，胚状体可以发育成苗。其中在MK培养基上添加3 g／L活性炭，有10％胚状体生长成苗。在MK培养基上添加40 g／L蔗糖，有19.2％胚状体生长成苗。在MK培养基上添加10%椰汁对胚状体生长发育的促进效果最好，有34.5%的胚状体生长成苗。 3.通过对传统CTAB提取RNA的方法进行适当改良，建立了银杏等木本裸子植物RNA的提取方法。提高提取缓冲液中的p一琉基乙醇的含量，有效地去除了多酚类物质对银杏RNA提取的影响;在研磨过程中，适当加入聚乙烯毗咯烷酮 (PVP)，防止了酚类物质被氧化，从而获得了高质量的银杏RNA样品。经紫外分光光度计、琼脂糖凝胶电泳分析，RNA完整性好、不降解、杂质少、纯度高，同时也适用于反转录、、RT一PCR和Northern杂交等研究，为银杏的分子生物学研究奠定了基础。 4.LEA那基因是调控植物开花的关键基因，裸子植物银杏童期长，也许是由LEAFy同源基因所决定的，我们利用同位素标记，制备Gin妙和Gin从梅两个特异探针，进行Northern分子杂交，对银杏还没有开花功能的幼树、已开过花的银杏成年雄株和雌株的根、茎、叶以及幼果、不同时期的雌花芽、雄花芽进行了LEA那同源基因的表达分析，发现银杏乙州Fy同源基因Gin势在各部位都有表达，属组成型表达，而另一个同源基因GinNdlJ笋只在银杏雌花芽、雄花芽和叶片中表达，属特异性表达。由此可知，银杏双拷贝乙别那同源基因中的GinN’lly基因与银杏开花关系更为密切，同时也与叶片的发育有关，这也许是银杏长期进化的结果。 5.进行了胚愈伤组织遗传转化的研究。通过卡那霉素筛选、PCR鉴定、Southern杂交分析，证明乙EAFy基因己成功的整合到银杏胚愈伤组织基因组中，进一步证明裸子植物银杏可以利用根癌农杆菌介导法进行遗传转化。研究了影响根癌农杆菌转化效率的因素。其中侵染的根癌农杆菌菌株、菌液浓度、共培养时间、抽真空时间以及添加乙酞丁香酮等，对愈伤组织转化效率都有影响。根癌农杆菌菌株EHA 1 05对愈伤组织的转化效率高于GV3lOI;根癌农杆菌菌株EHAIOS培养到对数生长期后，稀释10倍使用，共培养3天，可以获得较好的转化效果，转化率可达38%;加入300 p mol乙酞丁香酮，转化率可以升高到48%;而且浸泡5 min后，抽真空10min，再浸泡5 min比单纯浸泡20 min的处理转化效率局。更多还原

【Abstract】 Ginkgo Biloba L, with a common name of white fruit is a primitive species of China. Longago grown in China, Ginkgo is considered as symbol of the country in tree plants as that giant panda in animals. Ginkgo Biloba L. is considered as a living fossil. This tree is one of the remaining evidence of biologic origin and evolutionthe.It is thought important as dinosaur. It has many important uses including food, medicine, wood and ornamental with industrial development of Ginkgo. It has a long juvenile period and seedlings come into flowering after twenty years. Long juvenile period severely restricts the progress of its breeding. To shorten the juvenile period, LEAFY gene is thought as key gene in the early flower formation and its superfluous expression may tend the plants flower early. Studies on expression of homologous LEAFY gene can help to learn function of LEAFY gene in Ginkgo. Breeding through tissue culture and gene transformation techniques is a new method for the improvement of Ginkgo Biloba L.1. Being a gymnosperm Ginkgo Biloba L.is very difficult to differentiate in tissue culture. Embryonic axis, cotyledon,endosperm,different size of immature and mature embryos and different stages of immature embryos were induced as explants to study callus formation and embryogenesis in different culture condition in Ginkgo biloba. A kind of new culture medium-MK is made. Results showed that somatic embryos could be induced only through embryo,embryonic axis and cotyledon of immature embryos. MK + NAA 1 mg . L-1 + BA 1 mg . L-1 was the best medium for somatic embryogenesis from embryonic axis, and somatic embryogenesis rate was 45.3%. MK + NAA 1.5 mg .L-1 + BA 1 mg . L-1 was the best medium for somatic embryogenesis from cotyledon, and somatic embryogenesis rate was 12.9%. Highest rate (53.6%) of somatic embryogenesis was obtained on MK medium containing NAA 1.0 mg.L-1+ BA 1.0 mg mg.L-1.Maximum number of embryoids observed on callus was 38 with 3 mm plus embryos. Darkness is not suitable for somatic embryogenesis. Induction frequency of callus and embryogenesis is different in different stages.2. Embryoids were cultured into plants, which is the first report and Ginkgo Biloba L regeneration system was established. Embryoids stop growing after growing into some phase in the callus. Embryoids is taken off from callus in the sterile condition and is inoculated in MK culture medium with active carbon, sucrose and coconut milk in different concentration. The growth and development of somatic embryos were promoted. When MK culture medium is addedby active 3 g/L carbon, 10% of embryoids can develop into plants,When MK culture medium is added by 40 g/L sucrose, 19.2% of embryoids can develop into plants. When MK culture medium is added by 10% coconut ,34.5% of embryoids can develop into plants.3. A rapid method of isolation of total RNA from gymnospermic plant was developed by improving method of routine CTAB. Concentration of p-mercapto ethanol was added in the extract solution to separate the phenolic compounds from the total RNA .A proper amount of PVP is added during grinding to prevent oxidation of phenolic compounds. Using this technique, high quality RNA was seprepared from Ginkgo, Torrega and Cerdar species. The results of ultraviolet spectrophotometer and agarose gel electrophoresis analysis show that the RNA obtained has no obvious degradation and a good purity sufficient for RT-PCR and Northern blotting. The method can be used for the isolation of total RNA from gymnosperm such as Ginkgo Biloba L and it would make a good foundation for molecular studies of Ginkgo Biloba L.4. LEAFY gene is thought as key gene that controls flower formation and initiation. Longjuvenile period of Ginkgo Biloba L. being a gymnosperm perhaps is controlled by LEAFYhomologous genes. By Ginljy and GinNdly probes were labeled with [32P]-dCTP, Northern blotting was done to study expression of Ginljy, GinNdly gene from root, stem, leaf of juvenile and male and female trees, immature更多还原