Study On The Molecular Mechanism And Utilization Of Recessive Genic Male Sterile Line S45AB In Brassica Napus L.

Rapeseed is one of the major oil crops in China and the utilization of heterosis is considered to be the most efficient way to increase yield, to improve resistance and tolerance. Male sterility is the most efficient way to use heterosis. In China, the recessive genic male sterile system (RGMS) has become one of the most important approaches for heterosis utilization for its characteristics of complete and stable sterility, and rich resources restorer and cytoplasm, however, the study about the mechanism of the RGMS line is far falling behind. S45AB, a RGMS two-type line in Brassica napus, was derived from male sterile mutant of canola variety Oro, genetic analysis indicated that two duplicate recessive genes named BnMs1 and BnMs2 controlled the male sterility (Pan et al.1988). However, the production of F1 hybrid seed and propagation of mother line require the removal of 50% of the fertile plants from the female line, which limit its wide application. As above, the objectives of this research are:(1) Combining cytological analysis with the molecular biology approach, we will elucidate the mechanism for the RGMS line S45A. (2) Furthermore, we use the transgenic method to estabilish the maintainer line of S45A for a primary study. The main results are as follows:1. The study of the molecular mechanism of the RGMS line S45A in Brasscia napus L.1.1 Scanning Electron Microscopy was used to investigate exine formation and structure in the wild type and the mutant. The results showed the pollen and exine pattern formation in the mutant were significant aberration and collapsed. Moreover, in contrast to the reticulate exine pattern in S45B pollen grains, the aborted pollen grains exhibited a smooth pattern in the mutant. This indicated that the sporopollenin deposition and exine patterning were defective in the S45A mutant. We also noted the cell length and width in the mutant anther epidermis were reduced compared with that in the wild type. Instead of the normal outermost linear-shaped surface of the wild-type anther, undeveloped cuticle/epicuticular structures were observed in the mutant anthers.1.2 Transmission Electron Microscopy was used to gain higher-resolution images of developing anthers in the mutant and compare to corresponding images from the wild type. At the meiosis stage, there were no distinct differences between the wild type and the S45A mutant anthers. The abnormal development of pollen exine formation was first observed at the tetrads stage. The probacular did not form or formed probacular did not expand in the mutant anther. Later, after the microspores released from tetrads, free microspores of the mutant contained thinner walls that were devoid of the pronounced exine wall seen in the wild type. Microspores development were stopped at the uninucleate stage and was followed by vacuolization and breakdown and the development of tapetum was also abnormal in the mutant. The lipid accumulation pattern was changed in the mutant tapetum. Furthermore, the tapetal cells of the mutant was evidently vacuolated and breakdown. These analyses showed that the defect in the S45A pollen development is primarily due to the lack of sporopollenin deposition, and exine formation soon after the post-meiosis stage of the microspores and severe defects in tapetal cell development are also observed. In the mutant, the thickness of the outer epidermis cell wall and cuticle was reduced greatly compared with those in the wild type which indicated that the abnormal synthesis/supply of lipophilic molecules for anther epidermal cuticle formation occured in the mutant anther.1.3 TUNEL assay showed the abnormal tapetal cells that failed to degenerate in the S45A mutant, suggesting that the failure of the tapetal cell degradation in the mutant is due to a defect in the PCD.1.4 Previously, Dr Yi has defined the BnMsl gene to the region of 21.2kb which contain four candidate genes, two of them could be related to pollen development. Complementation test indicated that the P450 gene, BnCYP704B1, can rescue the male sterility and was as the retorer gene for the S45A mutant.1.5 The expression analysis including RT-PCR, mRNA in situ hybridization showed that BnCYP704B1 was strongly, specifically, and transiently expressed in the tapetal cell layer of developing anthers. At the early meiosis stage, clear but relatively weak expression was detected. At the tetrad stage and the microspore stage, the BnCYP704B1gene was expressed in the tapetum at its maximal level. After the microspores released from the tetrads, the hybridization to tapetal cells weakened and disappeared during pollen maturation. Promoter-GUS assay indicated that the BnCYP704B1 gene was a tapetum expressed gene whose products were required for microspore development.1.6 We measured the compositions of the chloroform-extractable cuticular wax and the aliphatic cutin monomers in the anthers of both the wild type and S45A mutant.The results showed that the total wax amount per weight of the anther in the S45A mutant was very close to that of the wild type, however, the cutin amount was reduced significantly.1.7 We searched public databases using BLAST with the BnCYP704B1 sequence as a query and two genes, CYP704B1 of Arabidopsis and rice CYP704B2, showed high similarity with the BnCYP704B1. Biochemical analysis indicated that CYP704B1 and CYP704B2 catalyzedω-hydroxylation of mid-long chain fatty acids that may serve as building blocks in sporopollenin. Thereafter, we infer that BnCYP704B1 may encode a hydroxylase which involves in sporopollenin synthesis.1.8 Comparative sequence analysis showed that the first mutation site of the two mutations was particularly in the S45A mutant and it caused the protein to lose its function and finally male sterility in the S45A.1.9 The subcellular localization analysis indicated that the BnCYP704B1 is an endoplasmic reticulum localized protein and may function in the hydroxylation of fatty acids.1.10 We examined fatty acid composition in the flower buds of the wild type and the S45A mutant.The results showed that the total fatty acids reduced in the S45A mutant and the mutant only possessed 50% of the total fatty acids of the wild type plants. Taken together, we can infer the molecular mechanism of the two genes controled RGMS line. The BnCYP704B1 gene is not only involved in anther cutin and pollen exine formation, but also influenced the tapetum development. The loss of BnCYP704B1 function caused abnormal lipid metabolism and defects in the PCD pattern in the tapetum of the S45A mutant. Both development of the pollen and anther cuticle were seriously influenced, resulting in complete male sterility and abnormal anther development.2. The primary study on establishing the maintainers for the RGMS line S45A using the BnCYP704B1gene.2.1 Two vectors were constructed to estabish the maintainer line:vector one used a chemical inducible promoter to induce the expression of the BAX gene at the seedling stage; vector two used the same promoter to replace the local promoter of the retorer gene BnCYP704B1.2.2 Both vectors above were used to transform the RGMS line S45A, for vector one, 15 plants were obtained. Of these plants,2 events were identified the hopefully phenotype by germination on the paper and soil analysis and both envents could be used for further analysis. No any positive plant was gained for the vector two.