| At present, rice, corn and rape have been achieved many more hybrids for planting large area relying on their male sterility line, and it brings very remarkable economic efficiency in practice. Now, a new chemical hybridizing agent named SQ-1 which can induce male sterile in any wheat at certain concentration developed by our research group in utilization of wheat heterosis. Although It helps us to cross wheat parents at random and getting a series of new varieties of hybrid wheat named “Xiza series”, for example “Xiza No. 1, Xiza No. 5, Xiza No. 9”, the pattern of physiological male sterility induced by SQ-1 remains unclear. To elucidate the mechanism of pollen abortion in PHYMS, we will discuss the relation of pollen abortion and the accumulation of energy material, the changes of pollen structure in substance metabolism perspective. Subsequently, the expression of small RNA in anther was identified by using high-throughput sequencing, and analysis the difference conservative mi RNA and its target gene function between the sterile and fertile material. Finally, the gene sequence which was related to physiological male sterile was obtained using of electronic cloning technology, and we made a preliminary study on the gene. The main results and conclusions were as follows:1. The physiological male sterile of 1376 are induced by a certain concentration SQ-1 at Feekes 8.5 with the male sterility rate up to more than 99%, but the plant phenotypic and active of pistils are not unaffected by SQ-1; Meanwhile,the irregular mitosis was found in microspores of the 1376-PHYMS, indicating that they had undergone abnormal mitosis,having only one or two nuclei in binucleate stage. Even a few microspores can develop to trinucleate stage that they were dot not like the normal karyotype.2. Out of structure of the pollen grains at trinucleate stage and distribution of nutrient substance(starch, protein and lipid) in anther and pollen grains from tetrad to trinucleate stage were detected by SEM and light microscopy respectively, these result indicating: Compared with CK-1376,the microspores of the PHYMS-1376 line were irregularly shaped and shrunken, and accumulated no sporopollenin precursors and lipids and proteins on their exine surfaces. The tapetum of the PHYMS-1376 line was degraded at the tetrad stage, indicating that this process occurred earlier than in the fertile plants. Further, metabolic substances(lipid particles, proteins, insoluble polysaccharides, and acidic polyanions) were abnormally distributed and accumulated in the PHYMS-1376 line, leading to an insufficient supply of nutrients for pollen development, and which resulted in reducing pollen volume. In addition, PHYMS-1376 line showed abnormal division of nuclei in the pollen and had considerably thinner intine, which might have led to the pollen abortion. Most importantly, this study revealed a new way by which pollen take up ubisch bodies or orbicules via intine endocytosis through the germinal aperture.3. Four small RNA libraries were constructed from CK-1376 and PHYMS-1376. We obtained clean reads 11351207, 11446429, 11671120, 11407681 and unique reads 3984769, 3812079, 4132493, 4792376 in four small RNA libraries, respectively. We identified 102 conserved mi RNA belonging to 78 mi RNA families. In addition,the difference conserved mi RNA of CK-1376 and PHYMS-1376 at the same stage were detected, mi R9774, mi R397, mi R159, rai R9652, mi R396, mi R9664, mi R9655, mi R9661, mi R9663, mi R160, mi R9658, mi R9774 and mi R9677 et al. many of which take part in development of pollen structure and metabolic substances process. Meanwhile,compared with targets of the difference conserved mi RNA of CK-1376 and PHYMS-1376 at the early uninucleate stage and binucleate stage, and then annotated and analysed the function of targets of the difference conserved mi RNA by GO and KEGG. Then, 4 and 94 targets of conserved mi RNA were annotated from at the early uninucleate stage and binucleate stage. The 50% target genes were took part in metabolic pathways at the early uninucleate stage. And, the ratio of plant-pathogen interaction and metabolic pathways at the binucleate stage were 37.23%和 17.02% respectively.4. We obtained the full sequence of F8-1 from wheat by homology cloning. The open reading frame sequence of the F8-1 gene was encoding 165 amino acid residues with a predicted molecular weight of 18.2759 k Da and an isoelectric point of 4.86. This protein was identified as a Secale cereale × Triticum durum F8-4 homolog with 100% identity. The amino acids represent a conserved domain in pollen Ole e I that contains the consensus sequence Q-G-R-V-Y-C-D-T-C-R. F8-1 protein was only expressed in anther during the binucleate and trinucleate stages. The expression of F8-1 gene of physiological male sterility 1376(PHYMS-1376) was lower than that of 1376 at both the binucleate and trinucleate stages. Subcellular localization showed that the F8-1 protein was expressed at the nucleus. In addition, down-regulation of the F8-1 gene resulted in an increase in pollen abortion. Therefore, F8-1 is inseparably linked with physiological male sterility. |
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