Functional analysis on sex-chromosome genes in haploid organism

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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.