| Radish (Raphanus sativus L.) is a very important, worldwide cultivated vegetable and horticultural crop. Since Ogura CMS (cytoplasmic male sterility) line was first reported in radish in 1968, a lot of works have been carried out on searching for new CMS lines and studing their pollen abortion mechanism. On the one side, the cross breeding can improve the production and the ability of anti-disease or other resistances by using CMS lines; The sterile mutants also can be introduced into the brassica to develop new CMS lines. On the other side, CMS lines are excellent materials to study the cytoplasmic inheritance, the interaction of the nuclear and the cytoplasm, and the pollen development. So, it is a hot point of the currant molecular and genetic engineer research.Up to date, the studies of development and the structural alternations of the male gamete and tapetum of the CMS line is an important basis of classification of the CMS line and analyzing the pollen abortion mechanism. In order to study the molecular mechanism of pollen abortion of the CMS line, the cDNA-AFLP technology was been used to explore the differences of gene expression between the fertile and the sterile anther development.In this research, the materials, including the fertile 805B, DH303B, and the sterile 805A, DH303A, were used to study the structural changes of the tapetum and the pollen during the anther development at the cellular or subcellular level. Moreover, the transportation or metabolism of the polysaccharide in the 805A CMS line was detected. Initial research was made to explore the structural changes of tapetum and the pollen between the fertile DH303B and the sterile DH303A anther development at the cellular level.We also explored the differently expressed genes between the fertile 805B and the sterile 805A during the anther development. We cloned some differently expressed genes, and obtained genes ontology annotations for the Arabidopsis genome from the Arabidopsis Information Resource and NCBI database. The main results were as follows:During the pollen development of the sterile 805A, based on the initiated time of the pollen abortion, the degeneration of the anther locules could be divided into two types:the type 1, only one locule, in which the pollen abortion started at the PMC stage, and completely degenerated at the meiosis stage. In the type 2, including the other three locules, the abnormality of the tapetum appeared at the PMC (pollen mother cell stage), and the tapetum entirely disorganized until the late uni-nucleate stage or the early bi-cellular stage. Contrary to the tapetum, the pollen abortion started at the late tetrad stage and completely disappeared at bi-cellular stage, and resulted in the four empty locules of the anther without dehiscence.In the type 1 locule, the characteristics of the structural change during the pollen abortion were as follows:at the early PMC stage, the cytoplasm contraction and the plasmolysis appeared in the pollen mother cell, and a few large vacuoles appeared in the tapetal cells. At the late PMC stage or before the meiosis, the inclusion of the PMC or the microspore was almost disappeared, while the inner structure of the tapetal cells completely degenerated; until the early uni-nucleate stage, all the tapetum disorganized with a mass of microspores materials gathered in the locule centre; Whereafter, the microspores entirely degenerated with the empty locule left. In the type 2 locules, the characteristics of the structural change in the pollen abortion was as follows:at the PMC stage, the microspore developed normally, but few large vacuoles appeared in the tapetal cells; at the tetrad and uni-nucleate stage, more large vacuoles also appeared in the tapetal cells and microspore while a part of microspores lost their inclusion completely, meanwhile, hypertrophy of the tapetum, which might be caused by the vacuolation inside, became vary clearly; at the late uni-nucleate and the early bi-cellular stage, the tapetum degenerated completely, and inclusion of the microspore disappeared entirely; finally, all these changes led to the pollen abortion. The complex behavior of the CMS 805A pollen abortion implied the complexity of its male sterile mechanism.The characteristics of the metabolism or transport of the polysaccharide during the sterile 805A anther development were as follow:at the PMC stage, much more polysaccharide was deposited in the anther wall and parenchyma surrounding the vascular bundles; at the uni-nucleate stage, these polysaccharide granules dissolved gradually in the fertile anther; however, these polysaccharide granules still existed at the same time; in the fertile anther, the granules completely disappeared at the late uni-nucleate stage, while many of those existed in the sterile anther, and the color of the inner layer of the locules wall was darkly compared to the fertile anther. The results suggested that the deficiency of the polysaccharide metabolism and transport might be an important factor inducing pollen abortion.In the DH303A anther, all the microspores gathered at the center of the locules, while the cytoplasm was concentrated, and the plasmolysis appeared followed in many microspores At the tetrad stage. At the meantime, the tapetum was uniform, and the normal structure of tapetal cells could be detected. At the mid-microspore stage, the cytoplasm of some microspores degenerated completely. At the same time, the cytoplasm of the tapetal cells was dense, and the structure of them had no difference with the former stage. Otherwise, more and more microspores lost their cytoplasm, and they were gathered in the center of the locules by the serious hypertrophy and vacuolation of tapetum. Soon after, the tapetum degenerated with the abortion of microspores. Finally, the inner structure of the four locules of the anther was all disappeared with without dehiscence. The result suggested that the pollen abortion was not induced by the degeneration of the tapetum, but the inner factor of themselves.We have the mixed cDNA, taken from the three development stages of the fertile and the sterile anther, as the template to selective PCR amplificated with 128 pairs E+2/M+3 combinations. We have detected 2556 TDFs, and discovered the obvious difference of the gene expression among three anther developmental stages of the fertile and the sterile. The results suggested that the abnormality of genes expression during the anther development of the fertile and the sterile appeared at the early stage of the sterile anther development, and implied the sterile mutant or mutants disturbed the regulation of gene expression of the early anther development.The differently expressed genes could be classified into eleven differential expression types. These patterns might reflect the abnormal gene expression of the sterile anther development, and indicted that the disordered expression of many important genes induced the disturbance of related physiological process, furthermore lead to abnormal anther development and pollen abortion of the sterile.Based on homology based candidate gene method, we have got the conserved sequence of 216 TDFs. Analyzed by using the biosoftware,41 TDF was no similar sequence or unknown sequence, present 18.9% of all the TDFs; 35 TDFs was related to transcriptional gene, present 16.2% of all the TDFs; 23 TDFs was unknown protein, about 10.6%; 22 TDFs encoded genes related to the signal transduction, about 10.2%; 15 TDFs, about 6.9%, was related to the genes of DNA synthesis, cell growth and division; 15 TDFs was related to cell normal metabolism, about 6.9%; 13 TDFs, about 6%, was related to cell biosynthesis, and some other TDFs encoded functional genes. These results reflected that the development and differentiation of the sterile anther abortion might be inteferenced with the abnormity of the transcription and signal transduction, and partly explaned the characteristics of the sterile anther degeneration.We also discussed some genes differentially expressed during the anther development, and the role of these genes in the sterile anther degeneration.
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