| Rapeseed is one of the most important resourse of edible oil, vegetable protein andfeeding protein, and also can be used in morden industry. With the improvement ofpeople's life, we need to improve the yield and quality of rapeseed. So, utilization ofbeterosis to breed the high yield and high quality rapeseed hybrids has become the mainaim for rapeseed breeding. Dominant genie male sterility (DGMS) is an importantapproach to utilize the heterosis of Brassica napus, and the remarkable advantage ofwhich lies in its stable and complete sterility. Furthermore by using the homozygoustwo-type line, temporarily maintainer line and restorer line, the "three-line" system hasbeen established, which could produce 100% sterile population and not need to remove50% fertile plants during F1 seed production. However, there are still some difficulties inapplying the DGMS for hybrid seeds breeding, such as developing new resources ofhomozygous two-type lines and restorer lines, and we still know little about itsmechanism of fertility control. So for better utilization of the DGMS, it is necessary to dosome further investigation of it.In this paper, the homozygous DGMS two-type line, Rs1046AB, was used as material,and the differentially expressed genes between the fertile plants and sterile plants wasisolated by some mRNA differential display technology (DDRT-PCR, cDNA-AFLP, SSHand cDNA microarray). Here are the main results and conclutions of the research:1. DDRT-PCR, three anchored primers and 26 random primers, totally 78 primercombinations were used to screen the differentially expressed genes between fertileplants and sterile plants of Rs1046AB, and finally 26 differentially expressed bandswere isolated (20 bands were up-regulated in fertile plants, 6 bands were up-regulatedin sterile plants). Subsequently, the result of Northern blotting demonstrated that allthese differentially expressed genes were negative.2. cDNA-AFLP, totally 256 primer combinations were used, and the result showed thatevery primer combination could produce about 20 bands. And only 23 (8.98%)polymorphic primer combinations were isolated, which could produce 32differentially expressed bands. Sequence analysis was performed by Blast, after theredundant sequences were removed; only 28 differentially expressed genes wereidentified. 11 genes could find homologous sequences in GenBank (NCBI); the other17 genes without any homology (or function unknown). Bioinformatic analysisdemonstrated that these unigenes were participated in 8 biological processes, including metabolism, transcription, cell cycle, protein fate, cellularcommunication/signal transduction mechanism and flower development etc., theseprocesses were all related to male gametogenesis. So for those no homologie genes,they maybe novel genes involved in male gametogenesis. Subsequentle, Northernblot further confirm the credibility of the result of cDNA-AFLP.3. Two subtracted libraries were constructed by SSH. In the forward-subtracted library,tester cDNA was from fertile plants while in the reverse-subtracted library was fromsterile ones. Every library contained 3072 clones, after amplification and purification,more than 2000 single-band clones were got with an average size of 500bp.4. Besides the above purified clones, the external control (a PCR-amplified fragmentfrom theλ, control template DNA fragment), internal controls (housekeeping genesactin,β-tubulin and BNACBP) and negative controls (TFR and pUC19) were all usedto constreted cDNA microarray in Takara (Dalian). The purified cDNA inserts wereprinted in duplicate while the control cDNAs were spotted 48 times randomly indifferent locations on each slide. Finally 1369 differentially expressed genes wereisolated in the forward-subtracted library (ratio≥2), while in the reverse-subtractedlibrary; there were only 12 differentially expressed genes (ratio≥2). After removingredundant sequences by dot blotting, about 400 sequences were sequenced, and theyrepresented 216 unigenes (212 were up-regulated in fertile buds, and the other 4 wereup-regulated in sterile ones). Of the 212 up-regulated genes in forward-subtractedlibrary, 178 homologous sequences could be divided into 17 groups excluding thosethat encode the unclassified proteins; and the other 34 genes had no homology in thedatabases at the National Center for Biotechnical Information (NCBI). Genesinvolved in metabolism; cell rescue, defense and virulence; biogenesis of cellularcomponents and protein fate were the four largest groups, possessed 11.11%, 7.69%,6.55% and 5.70% respectively. Subsequently 9 clones were selected for Northernblotting; eight results of RNA blotting were consistent with the expression dataobtained by cDNA mieroarray analysis, demonstrating the credibility of microarraydata. But as to the four genes in reverse-subtracted library, only one result waspositive.5. In sterile plants of Rs1046AB, the cytological data demonstrated that at the earlystage of meiosis all cells contained condensed nuclei, suggesting that they weredegenerating. Although 31% cells could escapep from the state of nucleicondensation at about tetrad stage according to the normal PMC, the meiosis process stopped again at pachytene stage according to the chromosome state. With pachyteneor zygotene like chromosome separated randomly, nucleoplasmic and micronucleicould be found in some cells. Even when the "microspore's analogue" appeared, thenuclei condensation and escaped cells still could be found in the anthers. Accordingto the result of cDNA microarray, it could be presumed that the possible reason forthis phenomenon was: DNA mistakes activated some checkpoints during meiosis, butin sterile plants, DNA repair system could not work properly, so chromosomesbecame unstable. The result of RT-PCR of 4 ESTs, isolated from cDNA microarray,related to DNA repair demonstrated that 3 of 4 ESTs had different expression patternbetween two materials, 2 ESTs was not expressed in sterile plants, and the other onereduced a lot, moreover all these three could only be detected in flower buds.6. In this study, KOBAS was used to identify the most statistically significantlyenriched pathways of the genes from the forward-subtracted library with the defaultgolden criteria. Of 212 sequences, 41 (19.34%) were assigned to 47 KO terms.According to the p-values, three pathways (nitrogen metabolism, nitrobenzenedegradation, and starch/sucrose metabolism) may play important function in theprocess of male, especially the pathways of nitrogen metabolism and nitrobenzenedegradation (False Discovery Rate (FDR)＜0.05).7. 15 ESTs were selected to investigate their temporal and spatial expression pattern.Temporal expression pattern was confirmed by Northern blotting. It could be foundthat 9 ESTs were only expressed in fertile plants, and the other 6 ESTs could bedetected in both materials, but their expression patterns were quite different. Afterquantification by Quantity One?, it could be found that in fertile plants most ESTswere expressed in flower buds 1-3mm in length, and in sterile plants some geneswere totally suppressed, some genes were expressed in a delayed pattern. It wasmeant that in the sterile plants, not only the expression level, but also the expressiontime of some genes were affected. The investigation of spatial expression pattern wasperformed by RT-PCR, the results demonstrated that 6 ESTs were specificlyexpressed in flower buds; 6 ESTs were expressed with preference, among them, 5ESTs were preferentially expressed in flower buds; and the other 3 ESTs could bedetected with the same expression level in all organs.
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