山葡萄雄株性别CKX基因家族分析与VaCKX的性别转换功能研究

In order to reveal the molecular mechanism of the sex reversal of Vitis. amurensis Rupr (The sex of the male Vitis.amurensis flower can be converted to a hermaphrodite form after treatment with cytokinin.) at the transcriptional level, RNA-Seq technique was used to collect the transcriptome data of V. amurensis male flower buds after treatment with cytokinin. Differentially expressed genes (DEGs) encoded cytokinin oxidase/dehydrogenase (CKX) were found in this database and all up-regulated in the hermaphrodite flower buds. We hypothesize CKX family members may play an important role during the sex of V. amurensis male flower buds converted to a hermaphrodite form. In this study, the CKX family genes related with sex conversion were analyses by using the methods of gene cloning, bioinformatic analysis, subcellular localization, expression patterns of specific temporal and spatial, gene expression and regulation and function of transgenic plants. One or two CKX genes closely related with sex conversion were chosen for functional analysis by transforming into Arabidopsis. And the expressional level and their response to exogenous hormones in different flower organs were studied. These data may provide additional germplasm information for evaluation of VaCKX gene in the male V. amurensis, and laid the foundation for further mechanistic studies on V. amurensis male convert female. Moreover, the identification of VaCKX functional will be useful for generating superior varieties of V.amurensis.

在前期构建的山葡萄雄株花序性别转换关键期的转录组测序（RNA-Seq）数据库中，发现编码细胞分裂素氧化酶/脱氢酶（CKX）的6个差异表达基因（DEGs）在细胞分裂素CPPU处理后的雄株花序（已转变成两性花）中全部上调表达。根据测序结果我们初步分析了山葡萄雄株性别CKX基因家族与影响性别转换的关键基因。本项目以此为切入点，通过基因克隆、生物信息学分析、亚细胞定位、时空表达特异性分析、基因表达调控和转基因植株功能研究，对山葡萄雄株性别转换过程中CKX基因家族进行分析，从中选择1-2个与性别转换密切相关的关键基因（VaCKX）进行功能研究。揭示山葡萄VaCKX基因在雄株花序性别转换过程中的功能，为今后利用山葡萄雄株性别转化为雌花，获得可结实的山葡萄新种质提供基因依据。

外源细胞分裂素（CPPU）可以诱导山葡萄雄株发生性别转换，并使其具有开花结实的能力。本研究为探明细胞分裂素在性别转换中的作用，以RNA-Seq测序分析获得编码细胞分裂素氧化酶（CKX）的差异表达基因为基础，采用基因克隆和RACE技术，获得3个细胞分裂素氧化酶基因cDNA全长序列，命名为 VaCKX3、VaCKX5和VaCKX7。序列分析结果表明3个基因编码的氨基酸均具有 CKX 家族典型的 FAD（黄素腺嘌呤二核苷酸）结合域和 CK（细胞分裂素）结合域。亚细胞定位发现VaCKX3定位在细胞膜、细胞质上，VaCKX7定位在细胞核、细胞质和细胞膜上。组织特异性表达结果显示：VaCKX5的表达量在雄株花蕾分化早期最高，开花前降到最低，而雌株和两性株中表达量在开花前均较高；VaCKX基因在山葡萄根、茎、叶、花（雌蕊和雄蕊）和果实中均有表达，在雄蕊中VaCKX5的表达量显著高于其他组织器官，主要在雄蕊发育早期起重要作用。在雌蕊中VaCKX7的表达量显著高于其他组织器官，可能在性反转过程中雌配子的发育中起重要调控作用；外源细胞分裂素促进CKX基因的表达，表达量随激素浓度的增加先升高后降低。转入VaCKX3和VaCKX7的拟南芥阳性植株地上部均出现生长矮小、开花稍有延迟的性状，可能是内源激素IP和IPA含量显著下降所致。本研究通过生物信息学、不同时空表达模式以及转基因的分析，初步验证了VaCKX基因的功能，揭示了VaCKX的表达特性及其在山葡萄雄株性别转换中的作用，为深入研究CKX与葡萄的生殖生长发育提供了重要的启示性线索。