Abstract
BACKGROUND: As one of the main crops in the world, sterility of rice (Oryza sativa L.) significantly affects the production and leads to yield decrease. Our previous research showed that OsPUB73, which encodes U-box domain-containing protein 73, may be associated with male sterility. However, little information is available on this gene that is required for anther development. In the present study, we knocked out OsPUB73 by using the CRISPR/Cas9 system and studied the cytological and transcriptome of the gene-defect associated with pollen development and sterility in the rice variety (Taichung 65). RESULTS: The sequence analysis indicated that OsPUB73 was comprised of 3 exons and 2 introns, of which CDS encoded 586 amino acids including a U-box domain. The expression pattern of OsPUB73 showed that it was highly expressed in the anther during meiosis stage. The ospub73 displayed low pollen fertility (19.45%), which was significantly lower than wild type (WT) (85.37%). Cytological observation showed tapetum vacuolated at the meiosis stage and pollen exine was abnormal at the bi-cellular pollen stage of ospub73. RNA-seq analysis detected 2240 down and 571 up-regulated genes in anther of ospub73 compared with WT during meiosis stage. Among of 2240 down-regulated genes, seven known genes were associated with tapetal cell death or pollen exine development, including CYP703A3 (Cytochrome P450 Hydroxylase703A3), CYP704B2 (Cytochrome P450 Hydroxylase704B2), DPW (Defective Pollen Wall), PTC1 (Persistant Tapetal Cell1), UDT1 (Undeveloped Tapetum1), OsAP37 (Aspartic protease37) and OsABCG15 (ATP binding cassette G15), which were validated by quantitative real-time polymerase chain reaction (qRT-PCR). These results suggested OsPUB73 may play an important role in tapetal or pollen exine development and resulted in pollen partial sterility. CONCLUSION: Our results revealed that OsPUB73 plays an important role in rice male reproductive development, which provides valuable information about the molecular mechanisms of the U-box in rice male reproductive development.