【作者】 宁国贵；

【导师】 包满珠；

【作者基本信息】 华中农业大学 ， 园林植物与观赏园艺， 2006， 博士

【摘要】 近十多年来，通过对模式植物拟南芥、金鱼草和矮牵牛等花发育突变体的不断研究，科学家们对花发育提出了不同的模型。然而，对花发育的认识因为突变体的数量及类型有限而受到限制，对有关花瓣数目增多以及花瓣花药化的研究还很不深入。对矮牵牛的研究，到目前为止，发现的花器官自然突变体不多，局限于dol、blind、gb与phoenix等，而且在后来的研究中一直被人们用其来研究基因的互作。人们对矮牵牛的部分花发育相关的MADS-BOX基因克隆并通过转基因方法获得一些人工花器官突变体的研究，丰富了花发育的理论。另外，人们虽然对矮牵牛的B类基因研究较多，但很少涉及到花瓣增多的研究。矮牵牛既是一种研究花发育的模式植物，又是一种在园林中广泛应用的花卉植物，对其花器官突变体的研究以及通过转基因获得变异植株，无论对植物花器官发育研究还是对植物育种均有重要意义。本研究以重瓣矮牵牛、新发现的blind-like矮牵牛花器官突变体为切入点做了部分研究，获得了以下主要结果：1．通过对许多矮牵牛种质资源进行连续的自交，从一自交系中发现了blind-like突变体植株，并对该植株进行组培及扦插繁殖，确认该突变体性状保持稳定，主要表现为花数增多，花冠退化，花瓣顶端出现花药状结构。通过体细胞突变体诱导和染色体加倍等方法，得到了大量的与重瓣性相关的变异植株，重瓣矮牵牛具有无限增殖的雄蕊与增多的花瓣，并且对重瓣矮牵牛叶片诱导的愈伤组织进行长期离体培养获得了大量遗传变异植株。2．对单瓣、blind-like突变体和重瓣矮牵牛解剖结构及扫描电镜的比较观察分析表明，它们的雄蕊与花瓣之间相互具有对方的组织器官，重瓣起源与别的物种存在差异，发现重瓣矮牵牛的内层部分花瓣明显具有雄蕊特征。blind-like突变体的花瓣上的花药化结构在雄蕊原基的形成时就已经出现．3．构建了重瓣矮牵牛花发育各个时期的均一化cDNA文库，通过克隆与初步分析了PHCYP51基因与及其它基因检测了文库质量。构建了单重瓣花发育的cDNA差减文库，完成了初步筛选并得到了一批差异cDNA片段。利用文库与合成第一链cDNA，重复的通过PCR克隆了10多个重瓣矮牵牛MADS-BOX基因的读码框，并进行了与单瓣的比较分析，表明了单重瓣之间的部分MADS-BOX基因内读码框碱基存在非同义突变。4．对blind-like突变体和重瓣矮牵牛进行了初步的遗传分析，blind-like突变体可能是由转座子插入引起突变，而矮牵牛的单瓣为隐性形状。利用RT-PCR对矮牵牛多个MADS-BOX基因在单瓣、重瓣与blind-like突变体矮牵牛各个花器官上的表达特性的研究结果表明，部分基因在单瓣、重瓣与blind-like突变体矮牵牛各个花器官上的表达模式在花器官之间存在差异。以PHAP2A与PMADS3为探针对花器官组织原位杂交表明，PHAP2A在重瓣的表达模式跟单瓣之间没差异，PMADS3仅在重瓣矮牵牛的内轮花瓣、雄蕊及心皮中表达，而在blind-like突变体上其表达则扩展到每一轮花器官。5．对由cDNA文库与差减文库克隆到的cDNA大片段和部分MADS-BOX基因进行了正义与RNAi的植物表达载体构建，部分已经转化到矮牵牛、烟草植株中，并在某些转化体上改变了花型。其中花瓣增多的烟草转基因植株是由S3基因的cDNA片段以RNAi的形式单转化的引起的，在一些转化植株中，有的花药形状发生变化，有的雄蕊完全瓣化，看到的均为花瓣，而且对烟草植株的结实具有比较明显的影响。对这些转基因植株的分子分析正在进行中根据以上实验结果，按照blind-like突变体与重瓣矮牵牛表型与基因表达的相似性，我们初步推测出以下结论，blind-like突变体的形成是矮牵牛C类基因在第二轮花器官异位表达所致；鉴于从矮牵牛上分离的基因以RNAi作用的形式导入烟草得到大部分转基因烟草植株雄蕊瓣化、花瓣增多等表型的现象，结合对重瓣矮牵牛的解剖与MADS-BOX的表达分析，重瓣矮牵牛的形成有两方面原因：一方面某类基因的表达或失活使得雄蕊增多，另一方面部分基因功能的缺失，使雄蕊发生瓣化。更多还原

【Abstract】 In the past decades, genetic studies in several floral organ mutants of Arabidopsis, Antirrhinum and Petunia have led to many models established for the determination of floral organ respectively, such as ABC, ABCD and ABCDE. The understanding of human beings to flower development, floral organs formation was restrained to some extents due to the limited flower or floral mutants, furthermore very few were known to the antheroid petal and multi-floral organs. Up to date, only dol, blind, gb and phoenix floral organ spontaneous mutants were discovered in petunia and they were usually used to investigate the interaction of those floral organ identification genes. The theory of flower development was improved by the studies in petunia, in which some man-made floral mutants were constructed through isolating many MADS-BOX genes transferring to the wild type or spontaneous mutant or insert mutagenesis. Though much more studies were concentrated on the class B genes, there were quite few literatures about petal number to be referred. Petunia hybrida, either as a model plant for plant molecular biology research or as a widely used garden flower, plays an important role in scientists’ studies and people’s life. Using blind-like floral mutant and the double flower petunia plants constructed by us, the structural and molecular analysis were conducted and the main results were as follow:1. The blind-like mutant was discovered among the inbred lines and followed by propagation of cutting and in vitro micro-propagation, the traits were found to be stable. The main varied morphological characteristic of blind-like mutant is multi-flower and exhibiting antheroid structure on the top of petals. Lots of double flower petunia plants were obtained by in vitro macro-propagation and in vitro culture, chromosome doubling. Numerous stamens and many petals can be seen in double petunia flower. There was dramatic genetic variation among somatic variants regenerated from callus of double petunia after long-term subculture.2. Paraffin section microscopy and Scanning electron microscopy analysis indicated that there are antheroild structure in the petal of blind-like mutant and the inner petals of double petunia. There are difference in the formation between double petunia and Prunus mume, some of the excessive inner petals have the clinandrium-like structure. The antheroid structure on the top of petal of blind-like mutants was formed as soon as the stamen initiating.3. A normalization cDNA library was constructed using RNA extracted from flower during different developmental stages of double petunia. The cDNA library quality was tested by isolation and analysis of a novel gene PHCYP51. A subtractive library between single and double petunia was constructed, and some candidate cDNA fragments were obtained through anti-northern blot. Utilizing the cDNA library and the first strain cDNA, we isolated ten MADS-BOX genes’ coding sequence and compared with that of the single petunia, which indicated some of them exhibit sense mutant and nonsense mutant between them.5. The primary genetic analysis of the blind-like mutant and double petunia indicated that the blind-like mutant maybe resulted from transposon insert mutagenesis and the recessive allele is responsible for the single-flower phenotype. RT-PCR analysis according to the ten MADS-BOX genes among the floral organs from single, double and blind-like petunia indicated that the expression pattern of some of them is different. Through tissue in situ hybridization analysis, we discovered that there was no difference in PHAP2A expression pattern among wild type, double and blind-like petunia from the flower meristem to the floral organ formation, on the other hand, PMADS3 was differentially expressed in all the floral organs of blind-like mutant, in stamen and pistil of wild type and in the inner petals, stamen and pistil of double petunia.6. The sense and RNAi vectors of the genes that isolated from cDNA library combined with RACE method according to subtractive cDNA and some MADS-BOX genes were constructed. Some of them have been transferred to petunia and tobacco plants. Through investigating the phenotype of those transgenic plants, we discovered that double flower transgenic tobacco plants, which carded RNAi structure of S3 fragments. Further investigation demonstrated that the excessive petals were transformed from stamen. Comparing with wild type plants, the transgenic plants showed various shapes in anther and some of the transgenic plants with excessive petal in one solitary flower, moreover, the plants in which all the stamen transformed into petals were complete male sterile.Aaccording to the similarity between antheroid structure of blind-like mutant, inner petal anatomical observation and MADS-BOX genes expression pattern of double flower petunia, we proposed that the blind-like mutant was resulted from ecotopic expression of class C genes in petal. Since the formation of double flower tobacco attribute to the RNAi function of gene isolated from petunia, and the transgenic plants with severe phenotypes have much more excessive petal, which is same as to double flower petunia, we think that the possible formation of double flower petunia resulted from (1) the numerous proliferation of stamen number and (2) the loss of function of petal identified genes in the part of stamen and those stamen transformed into petals.更多还原