Phenotypic Observation And Gene Function Analysis On Double Mutant Osmads6-5/Osjag In Rice

More than half the world’s population depends on rice for survival. The development of floral organs plays a big role in determining the yield and quality of rice. Therefore, to find out the molecular genetic mechanisms of reproductive development is essentially important. Our laboratory has obtained two null-allele mutants Osmads6-5and Osjag from rice breeding materials. Loss of OsMADS6function causes lodicules, stamens and carpel completely transformed into lemma/palea-like organs. Whereas, loss of OsJAG function makes all floral organs abnormal, especially causing all of the stamens transformed into pistil-like organs. In order to better understand the mechanism of these two genes and their relationship, we constructed the Osmads6-5/Osjag double mutant and analyzed its morphology and the expression of the related genes. The results are as follows:1. Each whorl of floral meristem and the number of floral organs in spikelet of Osmads6-5/Osjag stay constant. However, the development of all four whorls of floral organs changed evidently. There exists partial similarities between the development of floral organs in Osmads6-5/Osjag and the single mutant of Osmads6-5and Osjag. The gull of Osmads6-5/Osjag is similar to the one in Osjag, The lemma and pelea of first whorl became narrower, harden and looked like crescent. The lodicule appeared like the one in Osmads6-5. The lodicule of second whorl transformed homologically into lemma/palea-like organs. The development process of the third whorl of stamen and the fourth whorl of pistil primordia are similar to the process of homological transformation from stamens and pistils to lemma/palea-like organs in Osmads6-5. Six stamen primordia and one pistil primordium developed into seven spikelet-like organs with multiple whorls of small gulls. We estimated the development process might be:Firstly, Loss of OsJAG function causes six stamen primordia in the third whorl transformedpistil-like. Subsequently, because of loss of OsMADS6and OsJAG function, these stamens and pistils in the fourth whorl developed into seven spikelet-like organs with multiple whorls of small gulls, which show a "flower in flower" phenotype.2. Fluorescence quantitative PCR results showed that:(1) Compared to the wild-type, in the early developing stage of inflorescence primordia in the double mutant Osmads6-5/Osjag, only class API OsMADS15gene expression level increased significantly, class B OsMADS4/16, class C gene OsMADS3/58and class E OsMADS7/8decreased largely. The expression level of other genes remains basically unchangd. This result is similar to the mutant Osmads6-5and indicates that OsMADS6shows a longer action time for regulating the development of floral organs than OsJAG (2) At the stage of floret primordia differentiation in spikelet, except that the expression of OsMADS15/1/19and OsJAG was up-regulated significantly and the expression of OsMADS14/43/58was down-regulated in the mutant Osmads6-5, the expression of OsMADS16/7/8in the mutant Osmads6-5and Osmads6-5/Osjag all descended greatly, the expression of DL all increased significantly.(3) The expression of gene OsMADS6in the mutant Osjag was up-regulated significantly, whereas the expression of gene OsJAG in the mutant Osmads6-5was down-regulated obviously. Furthermore, we use in situ hybridization analysis to identify the result of fluorescence quantitative PCR, which tests the expression of specific gene in the development of floral organs in Osmads6-5/Osjag. This result explains that, the expression of OsMADS3/58/16/7/8in spikelet of Osmads6-5/Osjag all decreased significantly compared to the wild-type, which was in agreement with the result of fluorescence quantitative PCR.Above all, as two important regulatory genes for the development of floral organs, OsMADS6and OsJAG respectively or jointly control the development process of all four whorls of floral organs in order. OsJAG plays a big role in the development of lemma/palea and stamen, whereas OsMADS6has an important function in the differentiation, development and floral meristem of lodicule and pistil.