采用cDNA-AFLP技术研究了甘蓝型油菜NCa不育系、保持系和可育F1花药败育前期的基因差异表达谱。用224对引物对不育系、保持系及育性恢复F1等3个材料的cDNA进行AFLP分析。回收、克隆可能与花药育性有关的差异条带，筛除假阳性，获得41条阳性差异条带（TDFs）；经测序、同源比对、功能预测，归并为35个非重复的TDFs。大多数TDFs在甘蓝型油菜中属首次报道。将已知功能的28个TDFs按其推测功能分为2大类10小类，82%的TDFs与细胞形态建成和降解、蛋白质合成、加工和转运、能量代谢、信号传导有关；基本涵盖花药发育相关的关键代谢过程。对其中7个TDFs基因进行了RT-PCR分析。结果表明，果胶甲酯酶基因和氨基酸通透酶基因都在不育系花蕾中大量表达，果胶甲酯酶基因在不育系叶片、保持系的花蕾、叶片中都没有检测到；而氨基酸通透酶基因有微量表达。驱动蛋白和乙醛脱氢酶主要在保持系花蕾中大量表达，而在不育系花蕾、叶片以及保持系叶片中都只有微量表达。玉米黄素环氧化酶（ZEP）和单脱氧抗坏血酸还原酶则在保持系花蕾、叶片和不育系叶片中大量表达，而在不育系花蕾中微量表达。花粉油体蛋白在不育系和保持系的花蕾中都有表达，不过保持系花蕾中的表达量约2倍于不育系花蕾；但是在两者的叶片组织中都没有检测到。本试验获得的差异表达基因对于了解油菜花药育性形成的调控机制提供了重要的分子信息。
Previous studies showed that NCa CMS was a novel CMS and would be of great importance for rapeseed hybrid breeding and production. In this article, differentially expressed genes during pollen abortion among NCa cms, maintainer line and fertile F1 were analyzed by means of cDNA-AFLP. 224 pair of AFLP primers were used to analyze the cDNA of the above 3 materials. The transcript-derived fragments (TDFs) were cloned, and then 41 TDFs were sequenced after identified as positive clones. Most of the TDFs were cloned firstly in Brassica napus. Gene function analysis revealed that 82% of TDFs were related with metabolism processes of (1) cell formation and degradation, (2) protein synthesis, processing and transporting, (3) energy metabolism and (4) signal transferring, involving all the key metabolism processes during pollen development. The differential expression of seven key genes among these TDFs in young leaves and floral buds of NCa cms and maintainer line were further analyzed by RT-PCR. The pectin esterase gene and amino acid permease gene abundantly expressed in floral buds of NCa cms while pectin esterase gene showed no transcript either in young leaves of NCa cms or in the two tissues of maintainer line, and amino acid permease gene slightly expressed in the corresponding tissues. However, kinesin gene and aldehyde dehydrogenase (ALDH) gene expressed abundantly in floral buds of maintainer line but just slightly detected in young leaves of maintainer line and both tissues of NCa cms. The transcriptions of ZEP gene and mono-dehydroascorbate reductase were reverse to that of amino acid permease gene. The transcription of pollen-special oleosin-like protein gene was detected almost twice in abundance in floral buds of maintainer line than in those of NCa cms, whereas not detected in young leaves of the two materials. As for the gene transcription in the floral buds, the differentially expressed genes cloned from the NCa cms were detected abundantly in its floral buds but almost no transcription in those of maintainer line, and vice versa, which demonstrated that the authenticity and efficiency of cDNA–AFLP applied to reveal and clone differentially expressed genes. Further studies were underway in order to investigate the relationship between these genes with NCa pollen abortion.
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