Functional analysis on sex-chromosome genes in haploid organism

在地茅（Marchantia polymorpha L.）的Y染色体上鉴定出64个基因，其中14个仅在雄性基因组中检测到。这14个基因在生殖器官中表达，而在营养菌体中不表达，表明它们参与了男性生殖功能。这个项目的目的就是为了证明这个假设。除1个基因外，其余基因均采用RACE法测定cDNA序列，并通过比较cDNA序列与基因组序列预测其启动子区域。为了便于转基因分析，我们开发了一种农杆菌介导的多形芽孢杆菌快速转化系统，利用孢子发育的未成熟菌体（Ishizaki等人，提交）。首先选取拟南芥UFO相关F-box蛋白编码基因M350E4.4作为模型案例。为了研究其表达模式，我们将一个携带M350E4.4预测启动子区域和β -葡萄糖醛酸酶基因（GUS）的构建体引入到未成熟的菌体中。在转基因菌体中未检测到GUS活性，这与我们之前的RT-PCR结果一致。3135054.4在性器官中的表达正在研究中。M350E4.4通过CaMV35S启动子异位表达、表达dsRNA沉默、过表达F-box结构域、过表达不含F-box结构域的编码序列等四种构建体来检测其功能。迄今为止所检测的转基因植物的菌体在形态上与野生型难以区分。目前正在研究转基因植物性器官的形态。

On the Y chromosome of the liverwort, Marchantia polymorpha L., 64 genes are identified, 14 of which are detected only in the male genome. These 14 genes are expressed in reproductive organs but not in vegetative thalli, suggesting their participation in male reproductive functions. The aim of this project is to prove this hypothesis.For all but one of the 14 genes, cDNA sequences were determined by RACE, and their promoter regions were predicted by comparing the cDNA sequences and genomic sequences. To facilitate transgenic analyses, we have developed a rapid Agrobacterium-mediated transformation system for M. polymorpha using immature thalli developed from spores (Ishizaki, et. al., submitted). First, the M350E4.4 gene, which encodes F-box protein related to Arabidopsis UFO, was selected as a model case. To examine its expression pattern, a construct which carries the predicted promoter region of M350E4.4 and the β -glucuronidase gene (GUS) was introduced to immature thalli. No GUS activity was detected in transgenic thalli, which is consistent with our previous result of RT-PCR. Expression of 3135054.4 in sexual organ is under investigation. Four types of constructs were tested to examine the function of M350E4.4: (1) ectopic expression by CaMV35S promoter, (2) silencing by expressing dsRNA, (3) overexpression of F-box domain, and (4) overexpression of the coding sequence without F-box domain. Thalli of transgenic plants examined thus far were morphologically indistinguishable from wild-type. Morphology of sexual organ of transgenic plants are being investigated.