| The genic male sterile (GMS) system has been widely applied for heterosis utilization in Brassica napus because of its stable and complete male sterility, rich sources of cytoplasm and wide spread of restorer lines. In this study, S45AB, a near isogenic line, which has been sub-matted for 25 generations and the fertility was controlled by BnMsl, was used as material. Semi-thin anther sections were used to compare the anther and pollen development of S45AB. Expression profile analyses between S45A and S45B buds were carried out by SSH libraries and microarray. According to the bioinformatic analysis of differentially expressed genes, predicted metabolism pathways were deduced for the male sterility. Main results are as follows:Until meiosis of the pollen mother cell, no differences were observed between the male sterile S45A and fertile S45B anther tissues. The tapetum cell elongated rapidly and was arranged tightly in tetrad stage, cross section of the uninucleate microspore was an approximate quadrangle in the S45B line but almost circular in the S45A line due to the complete lack of exine. After release of microspore from the tetrad, compared to the normal developmental microspore in the S45B line, the development of the microspore was blocked at the uninucleate stage and was followed by vacuolization, and then the microspores resulted in breakdown. Meanwhile, the tapetum cell deteriorated with evident vacuolization and soon collapsed in the S45A line. It is clear that the abnormal tapetal development cause the male sterility.Forward and reverse subtracted cDNA libraries were constructed by Suppression subtractive hybridization (SSH). Both cDNA libraries were rich in upregulated genes of S45B and S45A respectively. Both libraries contained 1536 clones, respectively. The positive clones were obtained by reverse northern blot and were sequenced, the sequences analysis showed that the clones represented 71 unigene, and four of them, Atlg30330, At1g71170, At5g10400 and At5g52160 were expressed specifically or abundantly in the buds.The microarray analysis showed that 69 genes were upregulated and 46 genes were downregulated in the S45B line compared with those in S45A. Functional categories of all upregulated genes from subtracted libraries and microarray in the S45B line were carried out using MIPS (http://mips.gsf.de/proj/funcatDB/search_main_frame.html), these genes were divided into 18 categories, mainly involved in protein fate (28.60%), protein binding (12.20%), cellular transport (36.80%), interaction with the environment (11.40%), subcellular localization (13.10%) and unknown functional gene (17.20%), only a few genes (1.63%) involved in metabolism regulation.Real-time PCR was used to validate and further investigate the expression differences of 25 upregulated genes in S45B line and 5 upregulated genes in the S45A line. Based on the expression pattern differences, the genes,23 upregulated in the S45B, were divided into three clusters. The bioinformatic analysis indicated that 4 genes of cluster A, At3g06860, At3g51590, At4g00040, and At5g48880, were involved in the lipid/fatty acids metabolism. It is deduced that the disorder of the lipid/fatty acids metabolism blocked the biosynthesis of sporepollenine, which caused the microspore abortion.According to the expression pattern revealed by Real-time PCR based on the genes from male sterility mutants including A6, CALS5, DEX1, MYB103, MS1, MS2 and NEF1 and the differences of phenotypes among S45A and other Arabidopsis male sterility mutants, a model was proposed to show the possible role of BnMsl in the regulation networks of anther development. |
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