【作者】 万丽丽；

【导师】 杨光圣；

【作者基本信息】 华中农业大学 ， 作物生物技术， 2010， 博士

【摘要】 甘蓝型油菜细胞核雄性不育是油菜杂种优势利用的重要途径,它的优点表现在相对于细胞质雄性不育更为稳定彻底的不育性状,不受恢保关系限制而易获得强优势组合。虽然细胞核雄性不育已经得到广泛的应用,但其雄性不育基因作用的机理还没有被揭示。雄性不育性作为一个复杂的性状关系到雄蕊形态建成、花粉囊中孢子体组织和配子体组织分化过程的基因调控。在这个过程中任何一个基因突变都会影响花药正常发育。随着模式植物拟南芥中与育性相关基因功能注释信息的公布,我们能从生理生化、细胞形态和分子调控等方面更好地理解这些基因在控制育性网络中的作用和相互之间的关系。从而能够从细胞学和分子作用水平深入探索甘蓝型油菜细胞核雄性不育的花粉败育机理,为最终实现人工雄性不育垫定基础。基于以上需要,本研究开展了以下3个方面的研究工作：1.利用光学显微镜(石蜡切片,半薄切片)和电子显微镜(扫描电镜,透射电镜)分别对显性细胞核雄性不育Rs1046A、隐性细胞核雄性不育9012A以及正常可育植株花药的外部形态和切片进行观察,从而确定败育时期和败育特征。2.从Rs1046可育和不育的花药构建的差减文库中筛选到差异表达的基因BnQRT3和BnATA20,在甘蓝型油菜中获得它们的全长序列,并且利用生物信息学软件分析基因的序列。通过RT-PCR和原位杂交,在Rs1046A和Rs1046B的各个组织中研究这两个基因的表达时空特征。3.通过基因敲除的方法研究BnQRT3和BnATA20在甘蓝型油菜花粉囊发育过程中的作用。同时在甘蓝型油菜的各个组织中检测这两个基因的启动子的表达状况。主要结论如下：1.细胞学研究确定了这两种细胞核雄性不育的主要败育时期和特征：显性细胞核雄性不育发生败育在花粉母细胞时期,表型为花粉母细胞不能进行正常的减数分裂,不能进一步地发育成四分体,而是继续发育形成“拟小孢子”。另外不育材料的绒毡层细胞是以“逐渐紧缩”的方式瓦解,不同于正常可育的绒毡层细胞的“均匀网状式”降解的模式。隐性细胞核雄性不育败育发生于减数分裂后形成四分体时期,特征为绒毡层细胞过度生长,液泡巨噬化,挤压药室,同时不能分泌出胼胝质酶来降解包裹四分体的胼胝质,最终四分体逐渐瓦解。2.从Rs1046A和Rs1046B构建的差减文库中筛选到两个EST:2-C15 (GenBank:EE392320)和1-H16 (GenBank:EE392282)。根据已知序列通过5’和3’RACE和Genome Walking的方法获得BnQRT3(2-C15)的cDNA和基因组序列,BnQRT3基因最大开放阅读框为1431bp编码了476aa。TargetP1.1预测BnQRT3蛋白为分泌蛋白。将BnQRT3基因编码的蛋白与已知的植物PGs做树形图分析进化关系,发现其编码的蛋白属于花蕾中表达的基因编码的PGs。通过电子克隆和Genome Walking相结合的方法获得BnATA20 (1-H16)基因的1559bp cDNA和2907bp的基因组序列。BnATA20基因编码的蛋白质中富含甘氨酸重复序列(GXGX)n,所以被称为甘氨酸富集蛋白。3.对BnQRT3在Rs1046AB中的表达模式研究表明它在花粉母细胞时期至二核花粉粒时期的绒毡层细胞中表达,另外在可育材料的四分体、小孢子和成熟花粉粒中表达。BnATA20基因只在可育材料的四分体时期到二核花粉粒时期的绒毡层细胞中表达。4.对BnQRT3反义抑制载体和RNAi载体转化获得的抗性植株中的不育单株进行DNA、外观形态、细胞学和基因表达水平的检测。反义抑制后所得拟南芥的8个阳性单株中有2个单株表现为雄蕊彻底败育。RNAi转化的9个阳性单株中有3株的大多数花中的雄蕊彻底败育,只有少数花中的雄蕊中存在微粉,但是花粉的外壁结构异常,人工授粉后发现不结实。在抑制和干涉载体转化的甘蓝型油菜的不育株中,BnQRT3在花药中表达水平下降。反义抑制转化后阳性植株中有12个单株的所有雄蕊表现为瘦小干瘪,不能产生花粉。而RNAi转化后阳性单株中有4个单株出现不结实的现象。观察发现其中3个单株的所有花中雄蕊彻底败育,只有一个单株出现少数的花中的雄蕊能够散粉,将其授予可育材料15天后,与正常可育单株相比角果短小。光学显微镜观察得出,BnQRT3RNAi所得的彻底败育的花粉囊与BnQRT3反义抑制后出现的败育表型相同,只是能够产生花粉的花药经过醋酸洋红染色,以及花粉管萌发实验证明极少数的花粉能够着色,并且延迟萌发。BnQRT3抑制和RNAi得到的不育单株的子房发育都是正常的。通过石蜡切片和透射电镜观察彻底败育的花粉囊发现绒毡层细胞发育严重受阻,没有孢粉素和脂类物质沉积在小孢子的外壁上,小孢子内部的胞质收缩,外观形态上严重变形,小孢子聚集紧缩在一起直至瓦解。5.在BnATA20反义抑制载体转化甘蓝型油菜得到的阳性不育单株的雄蕊中发现,BnATA20基因在转录水平上受到明显抑制。不育花蕾中的花药皱缩无粉。细胞学观察发现绒毡层细胞在四分体后期就已经开始瓦解,其胞质中没有脂类物质的合成,小孢子被变形的绒毡层包围不能再进一步地发育成花粉粒。6.分析基因BnQRT3和BnATA20基因5’端上游非编码序列,发现其中含有大量的调控元件。GUS染色结果表明BnQRT3-Promoter是花器官中特异表达的启动子,转化植株的雄蕊、子房、花萼、花瓣和花丝中GUS表达很强;BnATA20-Promoter为花药中特异表达的启动子。并且这两个基因的启动子都是与维管组织相关的受伤诱导型的,当植株中各个组织受到外界机械损伤时,在受伤的部位如叶片支脉和主脉,茎,花序与主茎分支处都能检测到GUS的表达活性。在BnATA20基因的启动子区域存在着维管组织中特异表达基因的受伤诱导型调控序列如W-box,W1-box和GCbox。但是在BnQRT3基因的启动子区域却不存在已知的维管组织特异表达的调控序列。更多还原

【Abstract】 Genic male sterility (GMS) systems are regarded as a promising alternative to CMS, due to the stable and complete male sterility and no negative cytoplasmic effect on yield. As an effective and economical pollination control system, GMS are convenient for the production of hybrid seeds. Nevertheless, the mechanism of male sterility has not been completely discovered. Over recent years, significant progress has been made towards understanding the process of pollen development, and much of this has come from the use of male sterile mutants in model system Arabidopsis. The advent of the genome availability and the development of tools for analysis of gene function mean that such methods can now be carried out in Brassica napus.In the present research, light microscopy and transmission electron microscopy techniques were performed to investigate the defects in genic male sterile plant Rs1046A and 9012A from microsporogenesis stage to male gametogenesis stage.Two differentially expressed ESTs from suppression substractive hybridization (SSH) and cDNA microarray between Rs1046A and B were used to obtain full length cDNA and genomic DNA by 5’/3’RACE technology and Genome Walking. RT-PCR and in situ hybridization were conducted to analyze the spatially and temporally expression pattern of these genes. In addition, antisense suppression vector of BnQRT3 and BnATA20 gene were transformed into Brassica napus. The functions of both genes in anther development were studied in detail. In the meanwhile, we analyzed the cis-elements from the promoters of both genes, and detected the activities of them according to stable expression systems. Main conclusions are listed as follows:1. The main sterile stages and characteristics of two male sterile plants were clarified by cytological observations. Rs1046A (DGMS) occurred from premeiosis stage to meiosis stage. Sporogenous cells did not undergo meiosis and become "cotton-clump" structure denoted by abnormal microspore. Additionally, the degeneration pattern of tapetal cell in sterility appeared very differently from fertility. As for 9012A (RGMS), the abortion initiated at the early tetrad stage. The sterile tapetal cells swelled with expanded vacuoles and finally filled the center of the locules where a majority of tetrads collapsed and degraded.2. On the basis of differential expressed EST:2-C15 (GenBank:EE392320) and 1-H16 (GenBank:EE392282), the cDNA sequence and genomic DNA of BnQRT3 (2-C15) were obtained by 5’RACE and 3’RACE and Genome Walking. The major open reading frame of BnQRT3 transcript was 1428 bp, encoding a 476 amino acid protein. Target P 1.1 predicted BnQRT3 was in a secretory pathway. Based on the comparision with plant PGs that functional assays have been reported, BnQRT3 belongs to which is thought to encode polygalacturonase activity and derived from genes expressed mainlv in flower buds. The 1559bp cDNA and 2907 bp genomic DNA of BnATA20 (1-H16)were isolated by in silico cloning and genome walking. Because the 445 amino acid encoded by BnATA20 contained (GXGX)n glycine-rich repeat, we regarded it as GRP (glycine-rich protein).3. RT-PCR analysis indicated that the BnQRT3 mRNA was found in stamens, ovaries, sepals and petals in both fertile and sterile plants, but not found in leaves or in tender stems. In situ hybridization analysis demonstrated the transcription of BnQRT3 in the anther occurred in the tapetum from the microsporocyte stage to binucleated pollen stage, and also can be detected in microspore mother cells, tetrads masses and mature pollen grains. BnATA20 transcripts could be exclusively detected in the tapetal cells from the tetrad stage to binucleated pollen stage by in situ hybridization. RT-PCR analysis showed that no mRNA was found in the other tissues except the stamens.4. The antisense vector and RNAi vector of BnQRT3 gene were transferred into Arabidopsis Columbia and Brassica napus Huashang 5 individually. Male sterility were observed in T1 progeny of BnQRT3 inhibited plants in both species. BnQRT3 expression was inhibited in these male sterile transgenic plants. Under light microscopy and scanning electron microscopy, there are two different phenotypes of sterility between antisense suppression and RNAi:1. A minority stamens of BnQRT3 RNAi transgenic Brassica napus and Arabidopsis contained numerous shriveled pollen grains with deep invaginations, resulting in the poor pollen vitality. In vivo germination test suggested pollen tubes extension were delayed in the style.2. The stamens from 12 antisense suppression plants and 3 BnQRT3 RNAi plants were completely aborted. According to the observation of transverse-section from sterile anthers, we found BnQRT3 gene, when disrupted, caused premature tapetal degeneration and complete microspore abortion, as well as a reduction in filament elongation.5. The BnATA20 antisense suppression vector was transformed to Brassica napus according to the Agrobacterium-mediated method. In T1 progeny, the sterile plants which do not produce pollen could be detected. In contrast with the fertile stamens, no BnATA20 transcript expressed in the transgenic sterile stamens. In the light of cytological analysis, we could find aberrations in the tapetum when BnATA20 was knocked-out, further leading to microspore cell death. BnATA20 was predicted to specifically involve in the tapetum proliferation.6.992 bp region upstream of the BnQRT3 transcript start sites was further analyzed by searching for putative regulatory motifs using PlantCARE and PLACE. A number of motifs involved in pollen-specific expression. The activity of the BnQRT3 promoter was detected by histochemical staining of GUS activity in BnQRT3Promoter-GUS transgenic Arabidopsis and Brassica napus plants. Strong GUS expression was observed in floral organs including the a series of developing florets from the pollen mother cell stage to anther dehiscence stage, stigma, vascular tissue of filaments, veins in sepals and petals, branch connective and flower abscission zone.1149bp BnATA20 promoter-GUS transgenic plants showed GUS expression exclusively in stamens. The two promoters BnQRT3P and BnATA20P conferred an identical GUS expression pattern on different regions associated with excision and wounding, suggesting that both of them are responsive to wounding. Additionally, we noted the localization of both promoter-GUS expression correlated tightly with the aerial vascular systems including petioles, midribs and stem either by compression with forceps or excision. In the region of BnATA20 promoter, we could find several putative wound-responsive elements specifically expressed in vascular systems including the W-box, W1-box and GC box. However, these elements did not exist in the BnQRT3 promoter.更多还原