【作者】 周锦；

【导师】 刘胜毅；

【作者基本信息】 中南民族大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 油菜是我国重要的油料作物,在我国的油料生产和食用油供给中占有极其重要的地位。油菜也是杂种优势利用普遍的一种作物,其雄性不育的分子机理及其应用基础的研究深受人们重视。本文基于甘蓝型油菜双显性雄性核不育近等基因系材料的芯片表达谱分析结果,分别选择了与糖代谢途径相关的两个基因GAPC(glyceraldehyde-3-phosphate dehydrogenase, cytosolic)和AT2G36580 ;与脂肪酸代谢途径相关的两个基因LACS9(long-chain acyl-CoA synthetase 9)和ACAT2(acetoacetyl-COA thiolase 2)以及花药特异表达基因PABP(polyadenylate-binding protein)和LTP12(lipid transfer protein 12),采用荧光定量PCR方法对其在根、茎、叶、花、角果、雌蕊、雄蕊中的表达模式进行分析,进一步阐明了甘蓝型油菜雄性核不育的分子机理。克隆了甘蓝型油菜花药特异基因Bn_PABP,通过相关生物信息学软件对PABP家族序列进行了分析,对基因结构和功能进行了预测和探讨,以期帮助深入理解甘蓝型油菜雄性核不育的分子机理。研究获得的主要结果陈述如下:1.糖代谢途径相关基因GAPC和AT2G36580在甘蓝型油菜各个组织中均有表达,花蕾发育过程中它们均在可育株四分体至单核期(败育中期)表达量最高,分别是可育株减数分裂期(败育前期)的5.06倍和1.69倍,而在不育株花蕾发育前期和中期被抑制;雌雄蕊中的表达分析表明它们均在不育株雄蕊中被抑制,而在可育株雄蕊中表达量高,分别是不育株雌蕊(对照)的9.11倍和4.47倍。2.脂肪酸代谢途径相关基因LACS9和ACAT2在角果中高量表达,花蕾发育过程中它们在不育株花蕾发育前期和中期被抑制,在可育株花蕾发育中期表达量高,分别是可育株花蕾发育前期的3.28倍和1.39倍;雌雄蕊中的表达分析表明它们均在不育株雄蕊中被抑制,而在可育株雄蕊中表达量高,分别是不育株雌蕊的2.03倍和3.43倍。3.花药特异表达基因PABP和LTP12在花中特异高量表达,在不育株花蕾发育时期被抑制,在可育株花蕾发育前期和中期表达,后期略有差异。雌雄蕊中的表达分析表明它们在在不育株雄蕊中基本不表达,而在可育株雄蕊中特异表达,分别是可育株雌蕊的6.78倍和9.45倍。4.上述基因表达模式的分析表明这六个基因的表达模式具有一定的共性:在可育株花蕾发育过程中,它们在前期和中期表达量上升,后期表达量下降,而在不育株花蕾发育前期和中期它们都被显著抑制。雌雄蕊中的表达分析表明,它们都在可育株雄蕊中高量表达,而在不育株雄蕊中表达量低。这些结果表明,这六个基因尽管属于不同的类别,但它们都同雄性核不育表型密切相关,值得进一步对其功能进行研究。5.通过生物信息学及分子生物学手段对甘蓝型油菜PABP基因进行了分析。首先,克隆获得了全长DNA序列。翻译后发现其包含651个氨基酸,分子量大小约71598.4,等电点约为8.38。通过与拟南芥PABP家族基因比对作出了进化树,甘蓝型油菜Bn_PABP与拟南芥PABP家族中的PABP5最为相似,氨基酸同源性达到了76%。通过生物学软件Clone Manager5对Bn_PABP同拟南芥PABP5进行了基因结构分析,发现它们具有类似的结构,即都具有PABP家族的特征保守区域和4个RNA结合区域,三维结构模拟显示二者功能位点结构高度相似。更多还原

【Abstract】 China is a major country for plant oil consuming and importing. Rape is the main oil-bearing crop considering its growing areas and developing speed. In addition, Rape heterosis is becoming wide accept way to research the molecular mechanism of genic male sterility. In this study, based on the results of gene expression in Brassica napus double-dominat cytoplasm male sterile line by microarrays, we select six genes, GAPC and AT2G36580 are related to glucose metabolism, LACS9 and ACAT2 are involved in fatty acid metabolism, PABP and LTP12 are anther specific genes. Our purpose is to clarify the molecular mechanism of genic male sterility of Brassica napus. So we decide to explore all the chosen genes’expression model in roots, stems, leaves, flowers, pods, pistils and stamens by realtime PCR. Bn_PABP was cloned and sequenced, then gene structure and function were analysed by bioinformatics software. In all, we expect to understand the molecular mechanism of Brassica napus genic male sterility. The results as follows:1. In glucose metabolism, GAPC and AT2G36580 were expressed in all organizations. in fertile buds They exhibit the highest expression level at the stage of tetrad to uninucleate, which are 5.06 and 1.69 times compared to that at the meiosis stage. Furthermore, GAPC and AT2G36580 in fertile line stamen had a higher expression levels Nearly 9.11 and 4.47 fold than in sterile line pistils.2. In fatty acid metabolism, LACS9 and ACAT2 were highly expressed in pods. They were strong expressed in fertile buds, uninucleate stage, which are 3.28 times and 1.39 times respectively higher than that at the meiosis stage. However, they were significantly lower expressed in sterile buds. Further analysis of LACS9 and ACAT2 in stamens and pistils showed that expression level in fertile line stamens were 2.03 and 3.43 fold higher than that in sterile line pistils.3. PABP and LTP12 are anther specific genes. That is to say, both of them were highest expressed in flowers especially at the stage of tetrad to uninucleate in fertile buds But they were obviously lower expressed in sterile buds. Analysis of PABP and LTP12 in stamens and pistils showed that expression levels in fertile line stamens were 6.78 and 9.45 fold higher compared to that in pistils of sterile line.4. Analysis of gene expression profile indicated that all six gene expression patterns have some identities, such as the expression level of fertile buds raised up from the stage of tetrad to uninucleate and declined at the trinucleate stage, while they were significantly lower expressed in sterile buds from tetrad to uninucleate stage. Further analysis of these genes in stamens and pistils suggest that all of them were highly expressed in fertile line stamens and less expressed in sterile line stamens. All the results verify that these genes are related to genic male sterility , although they belong to different types of metabolic pathway. It would be worthwhile to study more detail to identify their functions.5. Bn_PABP gene was analysed by bioinformation and molecular methods, total length of DNA sequences has been cloned and translated. It encods 651 amino acids, which molecular weight was 71598.4, and isoelectric point was 8.38. Bn_PABP has a high similarity with PABP5 in PABP family of Arabidopsis thaliana because Brassica Napus and Arabidopsis are phylogentically very close related. The homology of protein was 76%. Comparing these genes structures by Clone Manager 5, we found they had similar structure which had four RNA recognize motifs and a PABP superfamily motif. Simulation of three-dimension showed that they were highly similitude in function locations.更多还原