| The study of male sterility has important role on protecting genetic diversity and enhancing adaptability of species. It is also widely used to improve hybrid seed production and study the interactions between nucleus gene and cytoplasm gene. Anther is an important organ that influences the development of pollens. The study on the structure and development of male sterile anther is helpful to explore the reason of pollen abortion and the mechanism of male sterility in plants.In this study, a recessive mutant was obtained from an indica rice cultivar Zhongxian 3037 by 60Co～γ irradiation, and was named mil3. Compared to the wild-type plant, the remarkable characteristic of the mil3 mutant was sterility. Except for this mutational phenotype, no obvious morphological aberrations were detected on the vegetative growth and development period. A closer examination showed that the mil3 plants were devoid of pollen grains, while the female gamete fertility was almost normal. These results indicated that the mutation in MIL is responsible for the male sterility in rice. Then, we cloned the MIL3 gene and studied the reasons for the sterile phenotype of mil3 mutant. The main results are as follows:1. Map-based cloning was performed to isolate the MIL3 gene. A F2 population was derived from crossing between the homozygous male-sterile mil3 mutants and a japonica variety, Balilla.150 sterile plants were selected from this F2 population and used for genetic mapping. The MIL3 gene was primarily mapped between the sequence-tagged site (STS) markers S2 and S3.300 sterile plants of F3 and new 9 adjacent STS markers were selected for further mapping, and the candidate gene region was located to a narrow region about 40 kb. In this region, there are nine putative genes. Sequencing analysis revealed that eight genes have the identical sequences in wild type and mil3, except for the candidate gene. There was a single nucleotide insertion at position 496 of the ORF(Open reading frame)of this gene, as well as one nucleotide substitutions at position 496 and 499, respectively, resulting in the translation frame shift after that. The candidate gene is annotated as an oxidoreductase gene.2. We obtained the full-length sequence of MIL3 by 3’RACE-PCR and 5’RACE-PCR. Then we constructed complementary vector pC3-HB, containing the upstream of initiation codon ATG, the downstream of the termination codon and the ORF of the gene, and negative control vector pC3-HBC. Then we transformed pC3-HB and pC3-HBC into the callus of heterozygous plants. Genotypic identification and phenotypic analysis confirmed that MILS is the target gene.3. The abnormal anther development of mil3 mutant resulted in the degradation of the pollen:Meiosis progression was normal in the pollen mother cells (PMCs) of the mil3 mutant. Fresh anther observation indicated that, along with the anther development, microspores increased in size in both the wild type and mil3. However, microspores of the mutant had been wrinkled after tetrad formation and the following stages. The semi-thin transverse sections of anthers revealed that, after microspores releasing, the tapetum in mil3 did not degraded, which was different from that observed in wild type. During the corresponding stages, there was remaining abnormal tapetal cells with expansion irregular morphology in mil3. Finally, the mutant microspores underwent degradation with no pollen production.4. The effect of MILS deficiency on the expression of other genes:The expression pattern of MIL3 detected by qPCR showed that the highest expression level is in the late development stage of the anther. The qPCR analysis for the genes related to tapetum formation and development revealed MIL3 acts downstream of MSP1, UDT and TDR and upstream of DTC1 and OsCP1. The expression level of CYP704B2 related to sporopollenin formation have no obvious change. Taken together, these results indicate that MILS plays an important role in process of rice tapetal development pathway. |
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