Lin, Hong
(2018)
Reverse genetic approaches to explore new genes for rice pollen development.
PhD thesis, University of Nottingham.
Abstract
In plants, normal anther and pollen development involves many important biological events and complex molecular regulatory coordination. Understanding the gene regulatory relationships during male reproductive development is essential for fundamental biology and crop breeding.
In this work, a rice gene co-expression network was developed for anther development (RiceAntherNet) that allows prediction of gene regulatory relationships during pollen development. RiceAntherNet was generated from 57 microarrays derived from rice anther tissue across all developmental stages. The microarray datasets from 9 rice male sterile mutants, including msp1-4, ostdl1a, gamyb-2, tip2, udt1-1, tdr, eat1-1, ptc1 and mads3-4 mutants were used to explore and test the anther development network. Among the changed genes, three clades showing differential expression patterns were further constructed to identify genes associated with pollen formation during anther development. Consistent with the functional predictions, in Clade 1, 7 reported genes (OsABCG15, OsLAP5, OsLAP6, DPW, CYP703A3, OsNP1 and OsCP1) were confirmed to participate in pollen wall formation in rice. Furthermore, Clade 1 contained 12 genes with predicted orthologs in Arabidopsis that have been reported as key genes during pollen development in Arabidopsis and may play similar roles in rice. A website is also provided to present plots of the expression profiles for gene characterisation. This will assist in determining the key relationships between genes involved in pollen development, thus enabling characterization of critical genes associated with these regulatory networks. 9 candidate genes predicted by RiceAntherNet were chose for further study.
Mutants of these genes were obtained by CRISPR-Cas9 editing system. Phenotyping of these mutants indicate their function in rice pollen wall formation, which provides additional evidence that RiceAntherNet is a valuable tool for identifying novel genes during anther and pollen development in plants. The reliability of RiceAntherNet offers the possibility of using reverse genetics approaches for novel gene characterization.
TIP2 was shown to be involved in cell differentiation at early stages of pollen development and previous studies suggested the presence of a TIP2-mediated transcription cascade that regulates tapetum development in rice. In this study, downstream genes of TIP2 were predicted by reverse genetics approaches including microarray data analysis, gene expression patterns and gene promoter analysis. This work not only helps to explore the function of TIP2 and its regulatory network, but also offers the possibility of using reverse genetic approaches for novel gene characterization.
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