【作者】 李炫丽；

【导师】 龙鸿；

【作者基本信息】 华中农业大学 ， 细胞生物学， 2010， 硕士

【摘要】 雄性不育是高等植物一种常见的遗传学现象。从被子植物雄蕊原基分化到花粉成熟,要经历一系列形态、生理生化等方面的变化,如果这些变化任一过程出现异常,将导致雄性不育个体的产生。植物花粉的产生需要众多基因按照一定的时间、空间精细表达,是一个精准的基因调控过程。研究不同发育阶段花药和花粉特异基因的表达量可为花药基因的时空表达提供非常有价值的信息(Scott et al,1991)。萝卜-芥蓝异源四倍体是以萝卜为母本,芥蓝为父本,配制杂交组合,F1代经秋水仙素处理后经过连续自交得到稳定遗传的F10代,可以作为育种的桥梁作物以及饲料作物、油料作物,具有重要的研究价值。杂种F1代不育,在F10代以上育性恢复(Chen et al,2008),但仍有不育株出现,不育机制不详。本研究以萝卜-芥蓝异源四倍体(Raphanus sativus×Brassica alboglabra allopolyploids)F10代可育株和不育株花蕾为材料,通过半薄切片和石蜡切片,探讨此不育系花药败育时期及败育特点。该种不育材料属于单核花粉败育型不育,败育特点为：在四分体时期和单核花粉粒晚期绒毡层异常,出现液泡化现象。随着花粉粒的继续发育,液泡化现象有逐渐加剧,绒毡层细胞纵向伸长,侵占药室内部空间；形成的花粉粒凝集,与绒毡层一起解体,最终形成空的花粉囊,不能产生可见花粉粒。通过提取减数分裂期和单核期的花粉RNA,反转录为cDNA后以此为模板扩增育性相关基因ms2Bnap同源序列,结果仅减数分裂期能扩出产物。半定量RT-PCR实验表明ms2Bnap同源序列在花粉减数分裂时期可育株中的表达量高于不育株。另外,通过提取减数分裂时期的可育株、不育株花蕾DNA,对ms2Bnap同源序列进行克隆、测序,并将测序结果进行序列比对,比对结果表明：(1)从萝卜-芥兰异源四倍体中克隆的ms2Bnap同源序列mk和mb与ms2-gDNA、ms2Bnap-gDNA的同源性分别为82%和96%。(2)将mk和mb与ms2Bnap-gDNA进行序列比较分析显示：mk和mb有7个外显子、6个内含子,其中外显子大小为1961 bp,内含子大小为649 bp。每个内含子两端均存在典型的GT/AG双核苷酸结构。(3)将mk和mb的外显子序列进行比对,两者的同源性为99%。mk和mb外显子序列间有4个核苷酸存在差异。涉及到4个密码子,其中有两个密码子(AGG/AGA、ACA/ACC)突变为同义突变。另两个密码子(GCA/ACA、GAC/GAA)的突变为错义突变。本研究利用亚硫酸氢钠结合PCR(BSP)技术,选择ms2Bnap基因同源基因外显子中C+G含量高于40%的5个序列作为研究对象,初步检测减数分裂期ms2Bnap基因同源基因在F10可育、不育植株中的甲基化变异情况。F10可育株和不育株中f1-f5片段序列甲基化程度分别为2.5%和10.4%。可育和不育株中ms2Bnap基因同源序列的甲基化水平的差异可能与植物的育性相关。更多还原

【Abstract】 Male sterility is an inheritable trait characterized by the inability of a plant to produce functional pollen. Pollen development is a complicated process and needs a lot of genes to express at certain time and space. In the process of pollen development in angiosperms, from stamens primordia differentiation to mature pollen, a series of morphological, physiological and biochemical changes have taken placed and if any abnormal change occurs, it will lead to male sterility. Data of different amount of gene expression can provide valuable information for anther development.F1 plants from intergeneric hybrids between Raphanus sativus L. (2n=18,RR) and Brassica alboglabra Bailey(2n=18,CC) were obtained by hand cross and through continuous selfing we get the stable genetic F10 generation. This new material has good prospects for the breeding of fodder crops or being used as a genetic bridge for intergenetic gene transfer. However, the lower seed fertility limited its practical utilization. Compared to F1 generation with complete sterility, F10 plants exhibited good fertility but there still had some sterile plants.The mechanisms of infertility are largely unknown.In present paper, paraffin and semi-thin sectioning were used to investigate the time of microspore abortion and characteristics of fertility alteration. Results showed that the male sterile line was abortive completely. The key stage of male sterility was from microsporocyte to microspores.The main characteristic was large vacuoles in tapetum. The tapetum of male sterile anther enlarged radial with a number of larger vacuoles; the surface of male sterile microspore was smooth without exine formation. Till the stage of later microspore, tapetum and microspores were disaggregated to be an empty pollen sac.RT-PCR was used to indentify the expressive stage of the gene homologous to ms2Bnap and the gene expression differences in F10 generation of Raphanus sativus×Brassica alboglabra allopolyploids male sterility and male fertility. The results showed that fragment homologous to ms2Bnap was steadily amplified from meiosis stage, while there was no PCR products using cDNA of single nucleus stage as template. These indicated that the gene homologous to ms2Bnap was not expressed in single nucleus stage. The gene expression amount in male fertility was higher than male sterility. Cloning and analyzing of sequence of gene homologous to ms2Bnap showed that there were four different nucleotides dispersed in the code region of the male sterility and male fertility, with two sites of synonymous mutations and two sites of missense mutation. This changes maybe related to sterility of F10 generation of Raphanus sativus×Brassica alboglabra allopolyploids.Bisulfite sequencing PCR was used to reveal DNA methylation variation of the gene homologous to ms2Bnap between male sterility and male fertility. The methylation ratio of fl-f5 fragments in sterile plants and fertile plants was 10.4% and 2.5%,respectively. This result indicate that the different methylation ratio between fertility and sterility may related to sterility of F10 generation of Raphanus sativus X Brassica alboglabra allopolyploids.更多还原