【作者】 刘菁菁；

【导师】 尹佟明；

【作者基本信息】 南京林业大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 虽然性别决定和分化研究在动物中尤其是高等脊椎动物中已经相当明确，但是在植物中并没有确定。相反，植物由全能的分生组织细胞分化形成复杂的生殖器官——花，其变化超过植物体其他任何组织，正因为这种变化，使生物学家从很久以前就对植物的性别分化多样性展开研究。然而，在众多有性繁殖的植物物种中，大多数植物是雌雄混合，即植物个体是雌雄单花同株或雌雄同花。雌雄异株植物，即有单独的雌株和雄株，其拥有与动物相似的性别分化，但在被子植物物种中仅有4％。植物性别表型的决定因素是多种多样的，从玉米和黄瓜的激素调节代谢到地钱和麦瓶草的性染色体决定方式到凤尾蕨的费洛蒙串扰决定方式。根据推测性染色体演化可分为四个阶段：第1步：常染色体上出现一个单一的性别决定位点，没有基因重组抑制。第2步：常染色体演化成同态的性染色体X和Y，Y染色体上出现非重组区域。然后，非重组区域的扩大，X和Y染色体开始形态差异，但是仍然保持常染色质。最后，多态性染色体逐渐形成异色质区域，就像有两个不同的Y染色体的酸模，这意味着植物中也有类似哺乳动物SRY基因的演化过程一般发生突变和缺失。其演化进程也许会最终导致Y染色体退化。在哺乳动物的性别决定进化过程的步骤可以在植物中得到很好的诠释。然而黄瓜大多是雌雄同株的，但也可以是雌雄异株或雌雄同花，具有多种花性别类型。木瓜有三种性别类型，雌性，雄性和雌雄同花。而其他两种模型虽是雌雄异株的草本植物，但其性别由形态不同的性染色体来决定。杨树和柳树同属杨柳科，杨树是木本植物基因组研究的模式物种，其全基因组已完成测序。杨柳科植物比较基因组研究发现，杨属植物和柳属植物的基因组有相同的起源，且具有高度的同线性顺序。杨柳科植物是雌雄异株植物，由于其良好的基因组学研究基础，所以杨柳科植物是研究植物性别分化机制的理想遗传学材料。研究显示杨柳科植物的第19号染色体上存在性别决定位点，但并没有出现形态上有差异的性染色体，正处于性染色体形成的早期阶段。杨柳科的性状特征，填补了性别决定方式演化过程的空缺。杨树一般开花需要4-6年的时间，而原产我国的簸箕柳（Salix suchowensis）是当年开花的小灌木，因此簸箕柳更适合用于植物性别分化机制研究。在本文中，我们以簸箕柳为研究材料开展转录组基因表达差异比较分析，旨在发展控制柳树性别分化的候选基因，为阐明其性别决定机制提供信息基础。实验过程中还使用分子标记对簸箕柳家系进行了鉴定。我们筛选出条带清晰，多态性强的19对SSR引物，24条ISSR引物，14条RAPD引物。实验结果证明我们采摘的实验材料其亲缘关系很相近，并且大多数都是扦插材料，并非实生苗木。分别以簸箕柳雌、雄花序为材料，利用454焦磷酸测序得到了120多万条高质量的鸟枪序列（shotgun reads），共计467.96Mb,平均长度389bp，其中571,945条来自雄株，629,683条来自雌珠。这些序列拼装得到29,048个基因片段（contig）和132,709个低表达的单一序列（singleton）,共计161,757个可能的基因（unigene），测序深度为8.0257。根据毛果杨注释的基因序列，有21365条contig和39482条singleton和毛果杨基因序列高度同源。GO分类分析发现共有16308个基因至少有1个GO分类号。KEGG分析发现有6820个基因涉及291条生化途径。根据unigene定位到杨树基因组预测基因上的结果，我们将unigene融合成29,981条真基因（truegene）。我们对reads进行读数计算来查找在不同性别中的表达程度有差异的基因。找到的差异表达基因数为806个，都是只在单一性别（雌株或者雄株）中有过量表达的基因。根据染色体定位结果，有33条差异表达基因被定位到了第十九号染色体上，其中17条基因在雌性中高表达，16条基因在雄性中高表达。从两个不同性别的柳树花序中得到大量的EST序列，通过生物信息分析确定这两个不同性别类型花序中的差异基因和表达差异基因。这些EST序列为进一步了解植物性别决定过程的分子机制提供了非常有价值的信息，是丰富的资源，为将来的功能基因组学分析、分子标记发展和育种工作的开展奠定了基础。更多还原

【Abstract】 Although in animals, especially in higher vertebrates, the genetic mechanism of sexdetermination has been relatively clear, but the genetic mechanism of sex determination in theplant is not entirely clear. Flowers are the reproductive organs of angiosperms, which morevaried than any other group of organisms, because of this variation, interest in plant sexualdiversity has a long and venerable history in biology. However in many sexually reproducingplant species, the majority of plants are cosexuals. The dioecious system, with separate male andfemale individuals, is of course the rule in animals, but is found in only4%of angiospermspecies. The determinants of sexual phenotype in plants are diverse. The sex determinationevolution progress which has done in mammals is something can be tracing in plants. GenusPopulus and genus Salix are members of the Salicaceae. Populus is considered as a model genusfor genetic and genomic studies in forest trees with available resource of the genome sequence ofPopulus trichocarpa. According to the studies, high degree of synteny was found between willowand poplars. Studies about populous show there is a sex determinate location on the19thchromosome, while there is no such sex chromosome diverges in morphology. Shrub kind willowjust needs2years to finish the vegetative growth and enter the reproductive stage, which is muchfaster than the genus populous which needs several years to flower. All the characteristic ofSalicaceae shows a great model for the missing pieces of sex determinate evolution.We used molecular markers to identify the relationship between willows. We developed19pairs of SSR primers,24ISSR primers,14RAPD primers at last. Results showed that theexperimental materials we picked from Xinyi closely related, and most of them were cuttingmaterial.Totally,1,201,931high quality reads were obtained, with an average length of389bp and atotal length of467.96Mb. The ESTs were assembled into29,048contigs, and132,709singletons.These unigenes were further functionally annotated by comparing their sequences to differentproteins and functional domain databases and assigned with Gene Ontology (GO) terms. Abiochemical pathway database containing291predicted pathways was also created based on theannotations of the unigenes. Digital expression analysis identified806differentially expressedgenes between the male and female flower buds. And33of them located on the incipient sexchromosome of Salicaceae, among which,12genes might involve in plant sex determinationempirically. These genes were worthy of special notification in future studies.In this study, a large number of EST sequences were generated from the flower buds of amale and a female shrub willow. We also reported the differentially expressed genes between thetwo sex-type flowers. This work provides valuable information and sequence resources for uncovering the sex determining genes and for future functional genomics analysis of Salicaceaespp.更多还原