【作者】 黄浩；

【导师】 陈崇顺； 柳李旺；

【作者基本信息】 南京师范大学 ， 细胞生物学， 2004， 硕士

【摘要】 萝卜是世界上最重要的园艺作物之一。萝卜杂种优势十分明显，可有效提高产品品质与抗病、抗逆性。利用胞质雄性不育的三系配套制种方式具有省工、方便、种子纯度高、成本较低等优点，将成为萝卜商业化制种最有效途径之一。本研究利用多个CMS与保持系配制的子代群体，综合利用光镜、电镜和mRNA差异显示技术，对中国萝卜的CMS这一性状进行了初步研究。 研究了萝卜胞质雄性不育系A₂、A₄及其相应保持系B₂、B₄的小孢子发生与花药壁发育的细胞学特征。结果表明：不育系A₂的绒毡层细胞在四分体时期出现异常，小液泡增多，至单核期汇合形成大液泡，绒毡层细胞异常膨大；小孢子外壁染色浅，细胞壁受到破坏，最后与绒毡层一同降解。不育系A₄在减数分裂期即表现出异常，绒毡层异常肥大；至花药发育后期，小孢子外壁亦染色较浅，细胞壁受到破坏；绒毡层细胞融合形成细胞团块侵入药室挤压小孢子，两者一同降解。 运用光镜和透射电镜方法对萝卜胞质雄性不育系A₁₄及其保持系B₁₄小孢子发生过程进行观察。结果表明，光镜下不育系A₁₄在单核早期小孢子开始出现败育迹象；在电镜下观察，处在减数分裂期的花粉母细胞已有异常表现，细胞器降解只剩下空壳，核膜不完整。可见核仁；此时绒毡层细胞发育状况与保持系相似。随着花药的进一步发育，绒毡层出现异常膨大等异常现象，不育系小孢子发育与保持系相比始终表现出明显差别。推测小孢子败育可能是由于育性相关基因突变造成，随之导致绒毡层异常及其超微结构异常。 从萝卜嫩叶中提取总DNA，针对orf138基因区域，通过设计两套引物进行PCR反应，得到长度约为780bp与280bp的扩增产物，两种产物扩增结果在供试材料中表现基本一致。将780bp的扩增产物回收并纯化、测序。结果表明：所得扩增产物为orf138基因，经测序得到有效碱基705bp，其中orf138基因编码区序列长417bp，包括起始密码和终止密码，是完整的功能单位；所测序列与资料报道序列比较，其编码序列完全一致，同源性为100％；恢复基因标记分析表明，引物STS-R和STS-F在供试材料中扩增出预期结果，表明这些标记可以应用于标记辅助选择，所得材料遗传背景还有待于田间试验验证以确定其是否携带恢复基因。从96个引物中筛选出雄性不育与可育材料有差异的引物，并利用其对雄性不育与可育材料进行初步鉴定。扩增结果显示硕士研究论文萝卜细胞质雄性不育细胞学与分子标记的初步研究条带丰富，并且有差异条带出现，表明可以利用RAPD法鉴定与育性恢复基因连锁的遗传标记。 以萝卜幼小花药与叶片为材料，初步建立了mRNA差异显示技术体系。4种提取总RNA方法中，改进的SDs/酸酚法比CTABZ酸酚法和SDS尹碱酚法更合适:改进的热翻酸法（HB）提取的RNA质量也较高，但所需时间较长，成本较高。以改进的SDS破酚法获得的RNA进行纯化后，其0 DZ耐0 D280在2.0².2之间，说明 RNA样品蛋白或杂质较少;总RNA进行反转录与差异显示分析表明，高分辨力的cDNA片段在100一38obP之间。 运用DO一RT一PCR技术对萝卜CMS不育系与可育材料间的mRNA差异进行了初步的显示分析，结果表明不育系中展示的带型与可育材料间并无明显数量上差异。在花序中，可育系有许多特异表达的条带，在叶片中也获得类似表达特征的。DNA条带;有的cDNA条带在可育系中强表达，在不育系中弱表达，存在一定的基因剂量效应。这些差异表达的片段，可能是发育前期异常小抱子发育过程中重要基因的表达产物，可能与小饱子败育的决定因素有关。更多还原

【Abstract】 The heterosis in radish is much significant, especially in increasing yield and fiber quality. Comparing with hand-emasculation and pollination, and genetic male sterile lines, utilization of CMS lines is much more effective and economical in producing commercially hybrid seeds. In this study, light-and electro-microscopy, DD-RT-PCR was performed to detect the mechanism controlling the male cytoplasmic sterility in Chinese radish.The cytology of the microsporogenesis and development of anther wall of cytoplasmic male-sterile (CMS) A2, A4 and its maintainer lines B2, B4 was studied. The results revealed that the tapetal cells of the male sterile A2 developed abnormally at tetrad stage, and they became vacuolated and numerous small vacuoles are present. Exine of microspore was light stained, and cell wall was destructed. Finallywith degeneration of the tapetal cells, the pollen aborted. The tapetal cells of CMS line A4 enlarged abnormally at meiotic phase, pressing microsporocyte seriously. Lately, their cell walls had not been observed, and the cell contents fused to form big lump invading the loculi, and finally leading to pollen grain abortion.The cytological development of male cells and the tapetum of male fertile and combined cytoplasmic-genetic male sterile line A)4 of radish was studied using light, and transmission electron microscopy. The cytoplasmic-genetic male sterile line began to abort when microspores had just released from tetrads by light microscopy investigation, while abnormal behavior of pollen mother cells were observed during meiotic stage on its ultra-structural study, including degeneration of organelles, irregularity of nuclear membrane etc. At the same time, development of tapetal cells was similar to that of the normal anther. With the development of the anther, the tapetal cells of CMS line showed an abnormal increase in size and other appearance, microspores were always distinguishable from the maintainer line. It is inferred that abortion of microspores contributed to mutation of genes controlling male sterility, which further leading to hypertrophy of tapetum and abnormal changes of ultra structure. The total DNA was isolated from young radish leaf. Two DNA fragments about 780bp and 280bp were obtained with PCR. After recovering the fragment about 780bp, the PCR product was sequenced and 705bp was obtained. Blast results shows that the full -length or f138 coding sequence is 417bp and the sequence showed 100% homology with the reported orf1!38 nucleotide sequence. Of the 96 arbitrary primers used in the experiment, the primers which can produce the polymorphism were obtained was used to test the fertility of genetic stocks successfully, which shows the RAPD technique determine the fertility of land races rapidly and precisely to back up seed production strongly.Using leaf and young anther as materials, a highly efficient method system for radish, including total RNA extraction and purification, Reverse Transcription Differential Display-PCR (DD-RT-PCR), PAGE and fast-sensitive silver staining was founded. In order to extract high quality RNA from radish, four protocols were applied. The results indicated that the modified SDS-acidic phenolic method was more suitable for total RNA extraction than CTAB-acidic phenolic method and SDS-alkaline phenolic method. High quality RNA was also obtained with the modified hot borate (HB) procedure that needs more time and higher cost. Total RNA with modified SDS-acidic phenolic method was subjected to purification with DNase 1 and the OD260/OD280 ranged from 2.0 to 2.2. The cDNA bands with higher discrimination were between 100 and 380 bp with DD-PCR.Transcription Differential Display-PCR (DD-RT-PCR) was conducted in fertile and male sterile radishes. The results indicated that cDNA bands of fertile and male sterile materials didn’t differ with quantity. Special expressed patterns of flowers were detected in fertile materials, so did leaves. In addition, thereis some extents of dose effect, which are due to some of them, were expressed更多还原