【作者】 黄鹂；

【导师】 曹家树；

【作者基本信息】 浙江大学 ， 蔬菜学， 2007， 博士

【摘要】 利用植物雄性不育特性来选育雄性不育系是作物杂种优势利用中简化制种程序、降低制种成本的重要手段。十字花科作物是杂种优势利用最为普遍的一类作物，雄性不育系(简称不育系)是其生产一代杂种的理想系统。但植物雄性不育产生的机理极其复杂，至今尚未完全解开。花粉败育是植物雄性不育发生的表型体现，弄清花粉发育的全过程和分子机理是研究雄性不育的基础和关键所在。花粉发育涉及众多基因的表达调控，从转录组入手分析花粉发育突变体是获得花粉基因动态表达的途径之一，并可获得较大数目的花粉发育相关基因，有助于在整体水平上理解花粉发育的特征和花粉发育的分子调控机制。本文在获得共享同一保持系(核不育两用系的可育株系)的白菜(Brassica campestris L．ssp．chinensis Makino，syn．B．rapa ssp．chinensis)‘矮脚黄’核不育(‘Aijiaohuang’genic male sterility，ajhGMS)两用系‘Bcajh97-01A／B’、‘Polima’核质互作不育(polima genic-cytoplasmic male sterility，polG-CMS)系‘Bcpol97-05A’以及’Ogura’细胞质不育(Ogura cytoplasmic male sterility，oguCMS)系‘Bcogu97-06A’材料体系的基础上，采用扩增互补脱氧核糖核酸片断长度多态性(complementary deoxyribonucleic acid amplified fragment length polymorphism，cDNA-AFLP)及拟南芥基因芯片分析其花蕾转录组差异，对花粉基因表达谱进行系统分析，研究不同雄性不育遗传模式的基因突变导致下游系列基因表达变化的异同，为建立不同雄性不育遗传模式植物花粉基因表达谱的基本框架奠定基础，并鉴定出一批白菜花粉发育相关基因。进而采用反义RNA或RNA干涉(RNAinterference，RNAi)技术，对细胞壁合成与调控相关的两个多聚半乳糖醛酸酶(polygalacturonase，PG)基因BcMF2(Brassica campestris male fertility 2)和BcMF9(Brassica campestris male fertility 9)，以及一个未知功能的新基因BcMF10(Brassica campestris male fertility 10)进行功能验证，从形态学、分子生物学、细胞学等方面入手鉴别三个基因在白菜花粉发育过程中的作用机制。同时，从基因结构、表达特点、作用方式等方面，比较了白菜花粉表达的PG基因家族成员BcMF2、BcMF9和BcMF6(Brassica campestris male fertility 6)的异同。取得的主要结果如下：(1)形态和细胞学观察发现‘Bcajh97-01A’为雄性减数分裂的胞质分裂突变体mmc，对应基因MMC可能编码特异作用于雄性减数分裂的蛋白质。形态和细胞学观察发现，ajhGMS两用系不育株系‘Bcajh97-01A’与可育株系’Bcajh97-01B’的差异仅表现在花粉花药发育上，‘Bcajh97-01A’小孢子在减数分裂末期的胞质分裂中出现异常，不能形成胞间壁而导致四分体形成及后续的花粉发育各过程受阻，最终引起花粉败育。遗传学分析表明该不育性状由单基因位点控制，因此，‘Bcajh97-01A’实际上是一个受单基因位点控制的雄性减数分裂的胞质分裂突变体mmc(male meiotic cytokinesis)，其对应基因MMC可能编码一个特异与雄性减数分裂核分裂完成后的胞质分裂相关的蛋白质。(2)利用cDNA-AFLP技术结合拟南芥基因芯片分析白菜不同不育系之间及与其共同保持系之间的花蕾转录组差异，发现不同遗传模式不育系花蕾基因表达有相似之处，但不尽相同。利用cDNA-AFLP技术及拟南芥ATH1基因芯片，对白菜ojhGMS‘Bcajh97-01A’、polG-CMS‘Bcpol97-05A’和oguCMS‘Bcogu97-06A’，以及它们共同的保持系‘Bcajh97-01B’进行了花蕾转录组比较分析，发现不同的不育基因作用下均引起花蕾正常转录组的巨大变化，不育基因下游众多基因的表达受到抑制，也有相当一部分的基因被激活或表达上升，但作用的不育基因不同，引起的下游基因表达变化也不尽相同。与同一保持系‘Bcajh97-01B’相比，检测到在‘Bcajh97-01A’花蕾中上调表达基因93个、下调表达基因158个；在‘Bcpol97-05A’花蕾中上调表达基因174个、下调表达基因212个；在‘Bcogu97-06A’花蕾中上调表达基因196个、下调表达基因242个。其中。在三种不育系中共同下调表达的基因有37个，上调表达的24个；‘Bcajh97-01A’和‘Bcpol97-05A’共同下调和共同下调表达的基因各5个；‘Bcajh97-01A’和‘Bcogu97-06A’共同下调表达基因为70个，共同上调表达基因9个；‘Bcpol97-05A’和‘Bcogu97-06A’共同下调表达基因7O个，共同上调表达的基因45个。(3)通过差异表达基因功能分类，发现许多花粉花药发育相关基因功能未知，白菜不同不育系与保持系的花蕾转录组差异分析扩充了植物花粉表达或特异表达基因的数目。对在三种不育系与其共同保持系花蕾转录组中检测到的差异表达基因进行功能分类，发现约50％的基因功能未知。基于三种不育系和保持系花蕾的差别仅表现在花药发育和花粉形成上，在不育系和保持系花蕾中差异表达的基因最可能与花粉花药发育相关。我们发现的这些差异表达基因在一定程度上扩充了植物花粉花药表达和特异表达基因的数目。(4)基于基因功能分类对不同不育系与保持系花蕾基因表达差异进行的分析表明，不同遗传模式花粉花药转录组尽管具有相似的模块，但具有不同的组成特征。对差异表达基因进行功能分类发现，不育系与保持系差异表达的三组基因可分为大致相同的功能类别，但每一功能类别在每组差异基因中所占的比例不尽相同。在三种不育系花蕾中下调均较多的已知功能的基因与转运通道、蛋白质代谢、电子传递和能量途径、防卫和胁迫反应相关，上调均较多的基因与转运通道、转录调控和普通新陈代谢相关。除此之外，‘Bcajh97-01A’与‘Bcogu97-06A’在花粉花药基因表达上具有更大的共性，在两者花蕾中均下调表达的基因中，细胞壁合成与调控基因排在第一位，细胞骨架蛋白及信号转导相关基因也占了较大的比例，而转录相关基因的比例较小；两者下调表达的基因中，蛋白质代谢相关基因均比较多。‘Bcajh97-01A’与‘Bcogu97-06A’的花蕾转录组的组成特征与拟南芥花粉转录组特征较一致。相比之下，‘Bcpol97-05A’花蕾转录组的组成成分与其他两种不育系有较大的差别。突出表现在细胞壁合成与调控基因、细胞骨架蛋白基因以及信号转导相关基因在不育系花蕾中占较大的比例，而保持系与其相比，这三种类别基因的表达并不占优势，反而更少。由此可见，不同遗传模式花粉花药转录组尽管具有相似的模块，但组成特征各异。(5)时空表达模式分析发现三种不育系与其共同保持系花蕾差异表达的基因具不同的表达动态。对27条在三种不育系和保持系花蕾中差异表达的片段进行了时空表达模式分析，发现大多数差异片段所代表的基因表达在小孢子有丝分裂前后开始被检测到，在花粉成熟过程中继续积累，但在不同组织和发育阶段中的表达水平不尽相同。此外也有在花粉发育早期就检测到表达的基因。我们的研究结果证实了花粉花药发育中的基因表达调控是极其复杂的。在花粉花药发育的不同时期表达或不表达，以及表达水平有序地发生变化都是与花粉花药正常发育、实现其功能所需相对应的。我们的研究为明确白菜花粉花药发育不同时期的基因群作出了有益的探索。(6)表达和功能分析发现BcMF2可能是一个与花粉内壁发育相关的新PG基因。对本实验室前期从‘Bcajh97-01B’花蕾中分离得到的PG基因BcMF2进行时空表达模式分析，发现其转录本最早在四分体时期的花蕾中开始出现，随后在单核花粉粒时期的花蕾中表达量达到最高，之后，随着花粉发育的进行表达量逐渐降低直至花粉成熟，表明其属于花粉表达“早期”基因。利用反义RNA技术研究BcMF2的功能，发现BcMF2表达受抑制的转基因植株花粉体外正常萌发率大幅度降低。约80％的花粉管生长到一定程度后顶端膨大形成泡状结构，花粉管能穿过柱头组织，但在花柱道中的生长停止。进一步观察显示转基因植株花粉畸形，萌发沟的数目及分布不规则，萌发沟在形成过程中位置和数目紊乱，花粉粒内壁异常增厚。推测BcMF2表达受抑制影响了花粉内壁中果胶的动态代谢平衡而导致萌发沟及花粉粒内壁发育的异常。BcMF2与已知的在花粉发育早期起作用的PG基因没有序列及表达特点的相似性，因此它可能是一个新的在花粉发育早期表达的PG基因。(7)表达和功能分析发现分离获得的另一个白菜PG基因BcMF9可能参与花粉内壁和外壁的发育。分离获得在可育株‘Bcajh97-01B’花蕾中特异表达的差异片段BBS13／BPO023的全长序列BcMF9，发现该基因具有典型的PG基因结构特征。分子进化分析表明其属于花粉表达或特异表达的PG基因类群。时空表达模式分析显示BcMF9属于花粉表达“晚期”基因，因其表达最早出现在四分小孢子中，在单核小孢子中开始增强，然后一直持续较高水平的表达直到花粉成熟。BcMF9的转录本亦在绒毡层中出现，从四分体时期开始直到绒毡层降解退化，一直在绒毡层细胞中维持很强的表达信号。转反义BcMF9的植株花粉离体正常萌发率明显降低，约81％的花粉萌发时花粉管爆裂。花粉管能穿过柱头组织，但不久便停止生长。扫描电镜和透射电镜观察显示，BcMF9表达受抑制产生外壁网纹浅的畸形花粉，花粉粒萌发沟处内壁增厚异常，萌发沟的位置和数目紊乱，花粉发育晚期外壁的覆盖层和基粒棒部分脱落，含油层外溢，说明BcMF9可能参与花粉壁的发育。此外，发现转反义BcMF9植株花药绒毡层降解速度加快，绒毡层发育发生异常前的小孢子发育正常，而异常后的花粉外壁发育受影响。推测BcMF9表达受抑制可能破坏了绒毡层细胞的程序性死亡过程，启动了另一个死亡途径，加速了绒毡层的解体。绒毡层正常程序性细胞死亡(programmed cell death，PCD)的破坏，导致其向花粉外壁输送物质过程的改变，从而影响了花粉壁的正常发育。但由于BcMF9编码的蛋白在其N端有一段信号肽序列，而它的表达首先出现在四分体时期的绒毡层和小孢子中，且在绒毡层中的表达信号要比在小孢子中强。因此推测其功能发挥还有另一个可能：BcMF9在绒毡层表达，表达产物分泌到花粉囊腔，进而在花粉壁的发育中直接起作用。(8)比较分析发现PG基因家族成员BcMF2、BcMF6和BcMF9在花粉发育中可能扮演各自不同的角色。从基因序列特征、表达模式特点、基因进化关系及生物学功能等方面比较三个花粉发育相关的PG基因BcMF2、BcMF6和BcMF9的异同，发现尽管3个PG基因的表达所出现的发育阶段一致，但三者在各个发育阶段的表达量及表达变化的趋势各不相同。三者在十字花科物种中的进化特点也不完全一致。3个PG基因的表达受抑制引发了类似的结果，但并不尽相同，说明它们在花粉发育中所起的作用及作用模式可能各异。(9)分离获得在‘Bcajh97-01B’和‘Bcpol97-05A’花蕾中高水平表达的基因BcMF10，序列特征分析提示其可能与信号转导途径相关，RNAi研究发现BcMF10表达受抑制导致花粉萌发异常。分离获得在‘Bcajh97-01B’和‘Bcpol97-05A’花蕾中高水平表达，但在‘Bcajh97-01A’和‘Bcogu97-06A’花蕾中沉默的基因片段BBS31／BPO079的全长序列BcMF10。利用生物信息学分析发现预测的BcMF10氨基酸序列中具有可能与细胞增殖、分化和PCD相关的蛋白激酶C磷酸化位点，参与调控信号转导相关的蛋白激酶的酪氨酸磷酸化位点，及其它与细胞内信号转导、蛋白定位以及黏附等过程有关的位点。推测BcMF10编码的蛋白可能在花粉发育过程的信号转导中发挥重要作用。利用RT-PCR(reverse transcriptase PCR，反转录PCR)和原位杂交分析了BcMF10的时空表达特点，发现其转录本最早出现在‘Bcajh97-01B’花药发育早期的花粉母细胞和绒毡层细胞中，随后在减数分裂时期的小孢子和绒毡层中表达量进一步提高，之后随着发育的进行，表达量逐渐降低，但表达部位没有改变，在花粉成熟期消失，属于花粉表达“早期”基因。通过构建含BcA9绒毡层特异启动子的BcMF10 RNAi表达载体，利用转基因阻断内源BcMF10的表达，发现75％左右的转基因植株花粉不能正常萌发，约15.6％的花粉能长出花粉管，但花粉管长到一定长度时顶端爆裂，正常萌发率只有10.5％，说明BcMF10表达受抑制影响了花粉的正常萌发。但与通常发现的花粉管一伸出萌发孔即发生爆裂的现象不同，BcMF10表达受抑制植株花粉管能长到一定长度，然后将内容物从顶端喷出，花粉管生长停止，推测BcMF10也可能与花粉壁的发育相关。根据BcMF10预测蛋白质的结构特征及其在早期绒毡层细胞和小孢子中共表达的特点，可以认为其可能作用于细胞壁发育过程中绒毡层与花粉的信号传递过程。更多还原

【Abstract】 The application of male sterility becomes an important way in heterosis breeding to simplify the seed production procedures and reduce the production cost. The utilization of hererosis is prevalent in cruciferae crops breeding, and the male sterile line is a useful system for their hybrid seed production. However, the occurrence and the mechanism of plant male sterility still remain mysteries that have not been unveiled thoroughly. For male sterility mainly manifests in pollen abortion, to make clear of the whole process of pollen development and the mechanism underlined thus become the basis point for understanding of male sterility. Pollen development is regulated by a complex regulatory network that comprises a wide variety of genes. Transcriptional profiling on the mutant compared with its wild-type is a successful approach to investigate the dynamic gene expression during pollen development and to detect genes involved in this biological process, and ultimately to help to understand the characteristic of pollen development and the corresponding molecular mechanism. In this study, we established three different male sterile types, ’Aijiaohuang’ genic male sterility AB line (ajhGMS ’Bcajh97-01A/B’), ’Polima’ genic-cytoplasmic male sterility (polG-CMS ’Bcpol97-05A’) and ’Ogura’ cytoplasmic male sterility (oguCMS ’Bcogu97-06A’) in Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis. These male sterile lines share a common maintainer line. Then, we conducted profiling comparisons between flower buds of these male sterile lines and their maintainer line using complementary deoxyribonucleic acid amplified fragment length polymorphism (cDNA-AFLP) technology together with ATH1 genome array analysis. By analyzing the changes in expression pattern of genes acting downstream due to the gene mutation in different male sterile plants, plenty of candidate genes involved in pollen development were identified, and thus laid a foundation for establishing the profiles of gene expression in different types of male sterile lines. After that, three genes were selected for further characterization, including two putative polygalacturonase (PG) genes, BcMF2 {Brassica campestris male fertility 2) and BcMF9 {Brassica campestris male fertility 9) which may be related to cell wall biosynthesis and regulation, and one novel gene, BcMFW {Brassica campestris male fertility 10) with unknown function. Anti-sense RNA or RNA interference (RNAi) technique were used to investigate the function of the three genes, and morphologic, molecular biology as well as cytology analysis were carried through in functional assignment. Comparison of gene structure, expression pattern and biological function of three members, BcMF2, BcMF6 {Brassica campestris male fertility 6) and BcMF9 in PG gene family were also performed in this study. The results obtained are as follows. (1) Observation on the pollen development in male sterile line ’Bcajh97-01A’ suggests that it’s a mutant of male meiosis cytokinesis in B. campestris, and the corresponding gene, MMC may encode a protein acting specifically in male meiosis. When observed the morphology and cytology character of the ajhGMS ’Bcajh97-01A/B’ we found that the only detectable difference between the male sterile plants ’Bcajh97-01A’ and the fertile plants ’Bcajh97-01B’ was the formation of mature pollen grains. Just after telophase II, the cytokinesis of male meiosis is disturbed in ’Bcajh97-01A’ anther, and the formation of intersporal wall does not occur, which in turn results in the failure of mature pollen formation. Genetics analysis indicated that the male sterility was controlled by a single recessive allele. These results suggest that ’Bcajh97-01A’ plant is indeed a mutant of male meiosis cytokinesis, and the corresponding mutated gene MMC may be directly involved in male meiotic cytokinesis.(2) Investigations on the difference among the flower buds transcriptional profiles of male sterile lines compared with the common maintainer line both by cDNA-AFLP and ATH1 genome array analysis, show that gene expression in the flower buds of these various male sterile types are similar but not entirely identical. We conducted expression profiling comparisons between each male sterile line, ajhGMS ’Bcajh97-01A’.polG-CMS ’Bcpol97-05A’ and oguCMS ’Bcogu97-06A’ and the common maintainer line of B. campestris both by cDNA-AFLP analysis and hybridizing with the ATH1 genome array from Arabidopsis genome. The outcomes indicate that the mutation of the gene which controls the fertility of each plant have caused dramatic changes in expression pattern of genes acting downstream. Expression of numerous genes are blocked or weakened, otherwise that of some other genes are activated or enhanced. But gene expression changes in different male sterile line are not identical due to the different gene mutation. Compared to the maintainer line ’Bcajh97-01B’, 93 genes are up-regulated and 158 are down-regulated in ’Bacjh97-01A’, 174 genes are up-regulated and 212 are down-regulated in ’Bcpol97-05A’ 196 genes are up-regulated and 242 are down-regulated in ’Bcogu97-06A’, and thereinto, 37 genes down-regulated and 24 up-regulated simultaneously in three male sterile types are included. There are also many genes sharing common expression pattern between the male sterile lines, including 5 genes down-regulated and 24 up-regulated simultaneously in ’Bcajh97-01A’ and ’Bcpol97-05A’ 70 genes down-regulated and 9 up-regulated simultaneously in ’Bcajh97-01A’ and ’Bcogu97-06A’ 70 genes down-regulated and 45 up-regulated simultaneously in ’Bcpol97-05A’ and ’Bcogu97-06A’(3) Function classification of differentially expressed genes reveals that most of the genes involved in pollen and/ or anther development are function-unknown. Expression profiling comparisons of different male sterile lines and their maintainer line increase the number of pollen and/ or anther-expressed and/ or specific gene. We classified the differentially expressed gene detected in the flower buds transcriptome of the male sterile lines and their common maintainer line into several categories, based on the putative function as well as gene ontology annotations derived from homologies. About 50% of these genes have an unknown or hypothetical function. On account of the fact that each of these male sterile plant only differed from the fertile plants line by the failure of pollen formation and anther development, we conclude that the differentially-expressed genes in the flower buds between the male sterile lines and the maintainer line are most likely to act in pollen and/ or anther development. Those differentially-expressed genes with unknown of hypothetical function may represent new candidate proteins involved in pollen and/ or anther development. If this is true, the number of genes expressed and/ or specifically expressed in pollen and anther will be increased to some extent for the identification of these genes.(4) Based on the function classification, it is found that pollen and/ or anther transcriptome from different plants with unlike genetic pattern possesses similar ’module’ but unique composition. When classified the differentially-expressed genes into functional categories, we found that there were similar categories in three individual groups of differentially-expressed genes derived from the comparison between each male sterile line and its maintainer line. However, the proportions of the same category in three groups are not entirely identical. The major categories of genes down-regulated simultaneously in all the male sterile lines are involved in transporter and channel, protein metabolism, electron transport or energy pathways and defense mechanisms and stress response; up-regulated simultaneously in those are involved in transporter and channel, transcription and general metabolism. Further more, many similarities were found between the gene expression in pollen and/ or anther of ’Bcajh97-01A’ and ’Bcogu97-06A’ Genes related to cell wall biosynthesis and regulation is ranked in the first place of the down-regulated genes both in ’Bcajh97-01A’ and ’Bcogu97-06A’ and genes related to cytoskeleton and signal transduction also occupy a higher proportion. Otherwise, few genes involved in transcription have been detected in the down-regulated genes both in ’Bcajh97-01A’ and ’Bcogu97-06A’ In the up-regulated genes in both of these two transcriptome, those related to protein metabolism account for a higher proportion. The transcriptome composition features of flower buds in ’Bcajh97-01A’ as well as in ’Bcogu97-06A’ are more similar to that of Arabidopsis pollen. In contrast, there is lesser similarity between the transcriptome of flower buds of ’Bcpol97-05A’ and the other two. The most distinguished difference is the proportion of genes involved in cell wall biosynthesis and regulation, cytoskeleton and signal transduction. These categories occupy higher proportion in the male sterile pollen of ’Bcpol97-05A’ but a lower proportion in the fertile pollen of ’Bcpol97-05B’ Thus it can be seen that though there is similar module in the pollen and/ or anther transcriptome of the male sterile line with different genetic pattern, each of them possesses unique composition.(5) Spatial and temporal expression pattern analysis demonstrates that each gene related to pollen and/ or anther development has a distinct expression dynamic. We investigated the expression pattern of 27 fragments derived from the transcriptome comparison above. Although most of the transcripts were first detected around the time of microspore mitosis and accumulate continuously as the pollen matured, each transcript had a unique expression level at different developmental stages. The expressions of some other fragments were detectable at the earlier stage of pollen development. These results from the expression pattern analysis validate the complexity of gene expression and regulation during pollen and/ or anther development, and demonstrate that activation or silencing and orderly changes of expression level are consistent with the requirements for normal development of pollen and/ or anther and thus for achieving their function. This study also made a relevant attempt to assign genes to different developmental stages of pollen and anther in B. campestris.(6) Expression pattern and functional analysis of BcMF2 indicate that it may encode a novel PG involved in pollen intine deposition. Spatial and temporal expression pattern analysis was performed for BcMF2, a gene encoding PG isolated from flower buds of ’Bcajh97-01B’ previously. BcMF2 transcript was first detected in the flower buds at stage 3, which corresponded to the tetrad stage of microspore development, though it was comparatively weak. The signal then became stronger up to the flower buds at stage 4, which corresponded to the uninucleate stage, and then declined in the flower buds at stage 5 full of mature pollen grains. This expression pattern considered it an "early" gene in pollen. Functional analysis of BcMF2 by anti-sense technique revealed that it might be involved in pollen wall development. The germination of pollen from transgenic plants with anti-sense BcMF2 was affected. About 80% transgenic pollen formed a balloon-tipped pollen tube during in vitro germination. Though the pollen tube could penetrate through the stigma, it stopped growth in the style. Further investigation on the configuration and the development of transgenic pollen revealed that all the pollens were malformed, and the number of germination furrow as well as its distribution was anomalistic. The intine of transgenic pollen thickened unconventionally during pollen development. It’s a hypothesis that the silence of BcMF2 may result in the disturbance in pectin metabolism in the pollen wall and in turn leads to the aborted intine and germination furrow. For there is no similarity found in sequence and expression pattern between BcMF2 and the known PG gene acting at the earlier stage, BcMF2 is considered as a novel gene encoding PG whose transcript is accumulated at the earlier stage.(7) Another PG gene named BcMF9 in B. campestris was isolated and characterized as a gene playing roles both in intine and exine formation. BcMF9 was amplified from a fragment, BBS13/BPO023 detected in the flower buds of ’Bcajh97-01B’ Alignment against the published sequences revealed the presence of highly conserved domains and features among plant polygalacturonases in the deduced amino acid sequence of BcMF9, and molecular phylogenetic analysis put it into the cluster of pollen-expressed PG. BcMF9 was considered as a "late" gene in pollen, because its expression signal was first detectable in the microspore at the tetrad stage, and became stronger at the uninucleate stage and maintained till pollen matured. Its transcript was also detected in the tapetum cell from tetrad stage to the degeneration of tapetal cells. Functional analysis of BcMF9 by anti-sense technique revealed that it may play roles both in intine and exine formation. The in vitro germination percentage of transgenic pollen was dramatically reduced. About 81% pollen tubes burst during germination. The pollen tube could penetrate through the stigma, but it stopped growth in the style soon after that. Further investigation revealed that silence of BcMF9 led to the produce of malformed pollen with a flat meshwork on the face and anomalous germination furrows. The intine of transgenic pollen thickened unconventionally during the pollen development and at the late stage, the bacula and the tectum fell off from the exine, which subsequently resulted in the overflowing of tryphine. These results strongly indicate that BcMF9 may play an important role in pollen wall formation. Furthermore, we found that degeneration of tapetum was accelerated in the transgenic anther. Microspore development was normal before the abnormity in tepetum occurring, but soon after the abnormity occurring, it was defected in exine formation. We speculate that the silence of BcMF9 disturbes the normal process of programmed cell death (PCD) in the tapetal cells, and starts an alternative cell death process which accelerated the degeneration process. Break of the normal PCD in tapetum may alter the releasing of tapetal wall materials onto the pollen grains and thus affect the complete deposition of pollen wall. Alternatively, BcMF9 may act directly in the formation of pollen wall. It may be expressed in the tepetum and then is secreted into the locule and located on the microspore during the pollen development, for the putative signal peptide in BcMF9 protein and the unique expression pattern of BcMF9.(8) Comparison of BcMF2, BcMF6 and BcMF9, three members of the PG gene family reveals that they may play different role in pollen development. The gene structure, expression pattern, gene evolution and biological function of three PG genes, BcMF2, BcMF6 and BcMF9 were analyzed. Though it was found that these PG genes accumulated in the same developmental stages, the expression level in each stage and the dynamics of expression of them are different. The evolutions of them in family Cruciferae are not coincident, and the silences of them result in similar but not identical phenotype. These results illuminate that the function and the pattern of gene activity of BcMF2, BcMF6 and BcMF9 may be different.(9) A novel gene named BcMFlO predominantly expressed in flower buds of ’Bcajh97-01B’ and ? ’Bcpol97-05A’ but silenced in those of ’Bcajh97-01A’ and ’Bcogu97-06A’ was isolated and characterized. This gene was amplified from a fragment, BBS31I BPO079 detected in the comparison of flower buds transcriptome of male sterile lines and the maintainer line. Bioinformatical analysis of the sequence revealed several patterns which were involved in protein location, cell adhesion and signal transduction associated with cell proliferation, differentiation, and apoptosis. It is supposed that BcMF10 may represent a new candidate protein involved in signal transduction. RT-PCR and in situ hybridization was performed to investigate the expression pattern of BcMF10, and the result indicated it was an "early" gene in pollen. In ’Bcajh97-01B’ BcMF10 has its transcript began to be detectable in pollen mother cell and tapetum at the early developmental stage, and became strong during the meiosis. Then, the expression decreased gradually up to the mature pollen. However, the location of its transcript has not changed. Functional analysis by RNAi technique revealed that silence of BcMF10 blocked the normal germination of pollen. About 75% transgenic pollens could not germinate in vitro, and 15.6% grew with a burst tip. But the burst pollen tube could grow to some extent and then spray the content, which distinguished it from the other burst tube observed generally with an immediate burst soon after germination. We suppose that BcMF10 may encode a protein synthesized in the tapetum and secreted out of the cell which plays a role in the secretion of nutriments or cell wall materials for the pollen grains.更多还原