芦笋雌、雄花发育中基因差异表达分析及应用研究

The famous vegetable, Asparagus officinalis, which has high values of both nutrient and medicinal, was cultivated worldwide. Asparagus industry has been developed very quickly in China recently. With characteristics of being viable of supermale (YY) and existing occasional fertile andromonocious (XY) flowers, the dioeciously Asparagus (XX for male, XY for female) was regarded as important model plant for elucidating mechanism of sex determination and its sex chromosome evolution. Currently the researches on sex determination of A. officinalis were mainly focused on fine mapping of M locus on genome followed with gene(s) cloning, however, due to pericentromeric location in L5 chromosome and existing recombination suppression of M between X and Y chromosome, the previous conception of inherited like single gene in M locus became controversial. Therefore for understanding the mechanism of asparagus sex determination, research works of cloning gene(s) of sex determining based on mapping should be continued, however new strategies and methods should be developed to solve this problem as well. We select to cultivate the offspring, which were obtained from andromonocy selfing of asparagus Var. Guelph Millennium, in green house. Both male and female flowers, which were collected in different development stage were analyzed to find the differential expression of CDSs, microRNAs and proteins by using both RNA-Seq and proteomics methods. After their differential expression was further confirmed with q-RT-PCR, their temporal and spatial expression patterns were tested by RT-ISPCR detection, the candidates of sex determining genes were chosen for cloning and followed DNA sequencing analysis. With available sequences of candidate genes, the DNA polymorphism were detected in both male and female asparagus, and the specific molecular markers for male and/or female were selected for developing convenient protocols for quickly detecting sex ,especially supermale, in asparagus seedlings. The study will be benefit for elucidating plant sex evolution and breeding "all male" asparagus as well.

具重要营养与保健作用的名贵蔬菜芦笋，其产业在我国发展迅速。芦笋具两性分化、超雄存活、存在雄花两性花同株等特征使其为性别决定机制及其进化研究的珍贵材料。当前芦笋性别决定研究集中在控制雄性发育M位点的图位克隆上。M位点因重组抑制，连锁分析虽表现为单基因控制，但有争议，故阐明芦笋性别决定机制，除继续图位克隆外，新的方法将应用来解决这一难题。本研究选芦笋品种"格尔夫"两性株自交后代温室栽培，收集不同发育的雌、雄花为材料，通过RNA-seq和蛋白组学方法分析雌、雄花间差异表达CDSs，microRNA 和蛋白质；经q-RT-PCR验证差异表达；RT-ISPCR确定差异表达基因在发育花器官时、空表达等来获得性别决定候选基因；基于候选基因的序列，分析雌、雄株间DNA多态性，筛选雌、雄专一的分子标记，开发苗期性别，尤其是超雄的鉴定方法。本研究将为阐明芦笋性别决定机制，加快"全雄"芦笋育种进程提供指导。

雌雄异株的芦笋处于性染色体起源的早期阶段，为阐明其性别分化基因，本研究以超雄材料（DH0086）进行芦笋基因组的测序组装与注释，以格尔夫和Venlim品种两性株自交的不同性别株为材料，进行RNAseq分析RNA和microRNA差异;用iTRAQ进行蛋白组分析，结果表明93.7%的组装序列被放置在10个连锁群（对应10个染色体）。其中29.3%组装序列已被确定在染色体上方向和顺序， 而6.2%的基因组组装序列不能被分配到连锁群上。基于芦笋雌株的重测序与比对，位于连锁群1上3 Mb~4.5 Mb为雄专一Y区（MSY），MSY预测39基因，其中1-209，1-215，1-204和 1-207为优选性别决定基因。RNASseq分析显示雌、雄差异表达基因富集到转录因子、信号转导、配子发育，激素等.其中MYB 、MADS类转录因子， LRR蛋白，泛素修饰途径等参与性别分化；芦笋编码并表达111个microRNA，但未有MSY区编码或调控MSY区基因的microRNA。 蛋白组分析发现，JA合成相关的亚油酸13S单加氧酶，是唯一MSY区编码雌性偏向蛋白，其他雌性偏向蛋白则富集到分子伴娘，亮氨酸重复，指结构等蛋白和核酸与蛋白降解酶类等；雄性趋向基因富集到ACC氧化酶，ABA相关蛋白及ATP依赖蛋白裂解酶，DNA聚合酶及泛素修饰降解系统等。利用植物化学方法，从芦笋中分离鉴定37化合物 (4为新化合物)，涉及甾体、黄酮类、苯丙素、含硫等化合物。其中含量3-4mg/kg干重的Asparagin A，是甾体皂苷，具有较强抗肿瘤活性。结合基因组和转录组数据，成功筛选出鉴定性别相关的STS标记用于苗期性别（包括超雄）的筛选。研究获得的候选基因为芦笋性别分化的分子机制阐明提供基础，筛选的性别筛选STS标记，将缩短芦笋“全雄”育种中超雄株筛选进程，推动芦笋产业的发展。