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Functional analysis on sex-chromosome genes in haploid organism

单倍体生物性染色体基因的功能分析

基本信息

批准号：
18510169
负责人：
YAMATO Katsuyuki
金额：
$ 2.64万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18510169/
关键词：
sex chromosomes haploid reproduction evolution bryophytes

项目摘要

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.

在地钱的Y染色体上，共鉴定出64个基因，其中14个仅在男性基因组中检测到。这14个基因在生殖器官中表达，但在营养体中不表达，表明它们参与雄性生殖功能。为了验证这一假设，本研究利用RACE技术测定了14个基因中除1个基因外的所有基因的cDNA序列，并通过比较cDNA序列和基因组序列预测了它们的启动子区域。为了便于转基因分析，我们建立了农杆菌介导的M. polymorpha使用从孢子发育的未成熟菌体（Ishizaki，et.例如，提交）。首先，选择编码与拟南芥UFO相关的F-box蛋白的M350E4.4基因作为模式病例。为了检测其表达模式，将携带M350E4.4的预测启动子区域和β -葡萄糖醛酸酶基因（GUS）的构建体导入未成熟的菌体。在转基因植株中未检测到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调查中。对四种类型的构建体进行了测试以检测M350E4.4的功能：（1）通过CaMV 35 S启动子的异位表达，（2）通过表达dsRNA的沉默，（3）F-box结构域的过表达，以及（4）不含F-box结构域的编码序列的过表达。到目前为止检查的转基因植物的菌体在形态上与野生型没有区别。转基因植物的性器官形态学研究正在进行中。