【作者】 朱彦涛；

【导师】 李殿荣； 郭蔼光；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 细胞质雄性不育(CMS)是油菜杂种优势利用的重要途径之一,Polima CMS和陕2A CMS是两个经典实用的甘蓝型油菜(Brassica napus L.)细胞质雄性不育系,这两个著名的不育系在中国乃至世界油菜杂种优势的研究和利用中发挥了重要作用。有关这两个不育系的研究,过去主要集中在栽培、遗传以及杂种优势的利用方面,近年来,主要集中在不育机理和分子生物学研究方面。有关Polima CMS和陕2A CMS的深入研究,对进一步探索油菜CMS的分子机理,更好地利用这两个不育系为我国油菜科研和生产服务有重要意义。本论文对Polima CMS和陕2A CMS的国内外研究状况进行了综述,介绍了近年来这两个不育系的最新研究进展,并从形态解剖学、生理生化以及分子生物学方面对这两个不育系进行了比较系统的研究,为进一步深入研究这两个不育系提供了基本资料和依据。所取得的主要结果如下(为了描述方便,文中Polima CMS和陕2A CMS分别简写为PolA和陕2A,其相应保持系分别简写为PolB和陕2B):1.分别对PolA系统(PolA、PolB)和陕2A系统(陕2A、陕2B)及其核质互换系、核代换系的形态学特征如植株的农艺性状和花器的形态解剖结构进行了比较研究。结果表明:陕2A系统与PolA系统的幼苗形态、花器结构和植株农艺性状明显不同,说明这两个不育系统具有明显不同的遗传背景。在核质互换和同核代换的条件下,这两个不育系与其同核异质材料之间的幼苗形态、花器结构和植株农艺性状分别无明显差异,而这两个不育系与其同质异核材料之间则明显不同,说明植株的表型性状主要是由其核基因组所控制的。2.以PolA及其保持系PolB、陕2A及其保持系陕2B四个材料初花期的叶片、叶柄以及花蕾组织为材料进行酯酶(EST)同工酶和过氧化物酶(POD)同工酶的聚丙烯酰胺凝胶电泳。电泳结果表明,不育系与其相应保持系的叶片、叶柄的EST同工酶谱均无明显差异,但其花蕾的EST同工酶谱有一定的差异;PolA与陕2A两个不育系对应器官的EST同工酶谱表现为明显差异。不育系与其相应保持系的叶片、叶柄的POD同工酶谱均无明显差异,但其花蕾的POD同工酶谱却有明显的差异;两个不育系对应器官的POD同工酶谱也明显不同。因此,这两个油菜CMS系统有着不同的遗传背景。3.以PolA及其保持系PolB、陕2A及其保持系陕2B四个材料的幼苗叶片提取叶绿体DNA(cpDNA),并对cpDNA几个特异基因片段进行了PCR扩增。结果表明,5对油菜cpDNA特异基因的引物分别在四个材料中扩增出相同的一条带,而且扩增产物的大小与预期目的片段的大小一致。进一步对其中与光合作用有关的RubisCO大亚基(rbcL)基因进行了分子克隆和测序,结果显示,这四个材料的rbcL基因序列完全相同。采用4种限制性内切酶BamHⅠ、EcoRⅠ、HindⅢ和PstⅠ分别对这四个材料的cpDNA进行酶切和电泳检测,酶切片断的多态性比较分析结果也显示,四个材料之间的带型分别一致,酶切结果无明显差异。本研究的结果也反映了cpDNA的保守性。4.分别以PolA及其保持系PolB、陕2A及其保持系陕2B四个材料幼苗的根和叶提取线粒体DNA(mtDNA),并对所提取的mtDNA进行检测。结果表明,在同等条件下幼根所提取的mtDNA含量为3250ng/gFW,约为幼叶所提取mtDNA含量的6.5倍,显著大于叶片所提取的mtDNA,而且所提取的mtDNA纯度较高。琼脂糖凝胶电泳检测结果显示,油菜幼根所提取的mtDNA较幼叶所提取的mtDNA有更为清晰的条带。油菜线粒体基因的PCR扩增结果表明,幼根所提mtDNA扩增条带的重复性和稳定性也优于幼叶所提mtDNA。因此认为,用油菜幼根提取mtDNA是一条较好的途径。5.分别以与油菜育性有关的5个mtDNA基因片段设计引物,对PolA系统和陕2A系统四个材料的mtDNA进行了PCR扩增。结果显示:有3个基因的引物在四个材料中分别扩增出相同的一条带,且扩增产物与预期目的片段的大小相一致,其中基于orf474 and orf159基因的引物还在四个材料中扩增出另一条相同的分子量稍小的条带;基于orf158基因的引物未能扩增出预期的目的片段;基于orf224基因的引物在PolA和陕2A两个不育系中扩增出相同的一条带,且扩增产物与预期目的片段的大小相一致,测序结果表明二者DNA序列也完全相同,均为675bp。另外,基于orf224基因的引物在PolB和陕2B两个保持系中扩增出相同的一条分子量较小的条带,经测序确认为与orf224不育基因有一定同源性的DNA片段,其大小为323bp。因此,推测保持系和不育系的多态性片段可能与CMS的育性有关。6.采用基于油菜品种Westar的mtDNA全基因组的连续25个DNA片段的特异性引物,分别对PolA系统和陕2A系统四个材料的mtDNA模板的相应DNA区段进行了分段PCR扩增和电泳检测。结果表明,这四个材料mtDNA的组成结构和排列顺序与已知油菜品种Westar的mtDNA基因组序列相似,其mtDNA全长约为200kb。其中有11对引物在四个材料之间分别扩增出相同的1条带,而且扩增产物与预期目的片段的大小相一致,说明这些区段可能比较保守,这些区段的大小约占mtDNA全长的44%;有2对引物在四个材料之间分别扩增出相同的1条带,但其扩增产物与预期目的片段的大小不一致;有1对引物在四个材料中没有得到扩增产物。由于以上这些引物在四个材料之间扩增产物的带型分别相同(或没有得到扩增产物),因此预测这四个材料之间的同源性至少为56%。然而,其余一些引物在四个材料之间的扩增产物比较复杂,这些引物的扩增区域约覆盖mtDNA全长的44%,属于mtDNA重组的易变区。其中有些引物在不育系与保持系之间扩增出了差异片段,有些引物在PolA和陕2A之间扩增出了差异片段。但是,这些差异片段的生物学功能如何?以及这些差异片段是否与不育有关?还有待于进一步的深入研究。7.本论文从分子生物学(如cpDNA和mtDNA特异基因的PCR扩增、测序比对以及cpDNA限制性酶切分析)、生理生化(如EST和POD同工酶电泳)以及形态解剖学(如植株农艺性状考种和花器形态结构观察)三个不同水平上对PolA和陕2A两个不育系进行了比较系统的研究。总体结果表明:PolA和陕2A两个不育系的核背景明显不同,mtDNA有一定的差异因而细胞质也有所不同,但PolA和陕2A的不育基因orf224序列则相同。那么,这两个不育系的不育机理是否完全相同?陕2A的mtDNA上是否还有其它不育基因的存在?等等,还有待于进一步的深入研究。更多还原

【Abstract】 CMS is one of the important approaches in heterosis utilization in rapeseed. Polima CMS and Shaan2A CMS are two of the classic and effective CMS lines in Brassica napus L, and these two famous CMS lines have played very important roles in study and utilization of rapeseed heterosis in China even in the world. In early years, study on the two CMS lines was mainly focus on the aspects of planting, genetics as well as utilization of heterosis. Whereas in recent years, study on them was mainly concentrated on the respects of sterility mechanism and molecular biology. The further study on Polima CMS and Shaan2A CMS will have an important significance to probe into molecular mechanism of CMS and use the two CMS lines to service for scientific research and production in rapeseed. This paper summarized the study states of Polima CMS and Shaan2A CMS at home and abroad, introduced the new research progress on the two CMS lines in rapeseed in recent years, systemically researched the two CMS lines on the aspects of morphology and anatomy, physiology and biochemistry, and molecular biology, and afforded the information and basis to further research the two CMS lines in rapeseed. The main results were as follow(for convenience to describe, Polima CMS and Shaan 2A CMS in paper were shortened as PolA and Shaan2A, and their maintainer lines were shortened correspondly as PolB and Shaan 2B):1. Studying on the morphology characteristics such as agronomic traits of plants and modality and structure of flowers in PolA system(PolA, PolB) and Shaan2A system (Shaan2A, Shaan2B), their nuclear-cytoplasm exchanging materials, and their nuclear substitution materials, respectively, the results showed that seedling modality, flower configuration and plant agronomic traits were obviously different between Shaan2A system and PolA system, indicating that the two CMS systems had significantly different genetic backgrounds. In conditions of nuclear-cytoplasm exchanging and nuclear substitution, the two CMS lines with the materials of their same nuclear but different cytoplasm had not significant difference in seedling modality, flower configuration and plant agronomic traits, however the two CMS lines with the materials of their same cytoplasm but different nuclear had obvious difference, indicating that nuclear genome played a decisive role in plant’s surface traits.2. The polyacrylamide vertical board gel electrophoresis of Esterase(EST) and Peroxidase(POD) isozymograms were carried through the leaf blade, petiole as well as flower bud organization of 4 materials including PolA and its maintainer line PolB, Shaan2A and its maintainer line Shaan2B in the early flowering season in Brassica napus L. The results showed that between the two CMS lines and their corresponding maintainer lines, EST isozymograms of leaves and petioles had not distinct difference, respectively, but EST isozymograms of flower buds had some. And there were obvious distinctions between EST isozymograms of the two CMS lines, PolA and Shaan2A. The results of POD isozymograms in leaves and petioles,respectively, were not obvious in diversity between the two CMS lines and their corresponding maintainer lines, but the results of POD isozymograms in flower buds had clear difference, and there were obvious distinctions between POD isozymograms of the two CMS lines. Therefore, the two CMS lines had different genetic background.3. Chloroplast DNA(cpDNA) was extracted by seedling leaves from four materials including PolA and its maintainer line PolB, Shaan2A and its maintainer line Shaan2B, and several special gene fragments of cpDNA were amplified by PCR. The result was that the same one target band was found in horizontal submarine agarose gels when 5 pairs of the special gene primers of cpDNA were used respectively to the 4 materials in rapeseed, and amplified products were consistent with expected target fragments. Then cloned and sequenced the RubisCO large subunit(rbcL) gene which was related to photosynthesis. The result showed that the rbcL genes in the four materials were absolutely same. Restriction enzyme digestion and gel electrophoresis of cpDNAs were also performed respectively to the 4 materials using an excess of 4 kinds of enzymes including BamHⅠ, EcoRⅠ, HindⅢand PstⅠ. The results of restriction enzyme analysis indicated that the bands among 4 materials were same and the enzyme digestion products had no difference, respectively. The above results also reflected that cpDNA sequence was high conservative.4. Mitochondrial DNA(mtDNA) was extracted by tender root and leaf tissues from seedlings of four materials including PolA and its maintainer line PolB, Shaan2A and its maintainer line Shaan2B, and the extracted mtDNA was examined. The results showed that the mean content of mtDNA extracted from tender root tissue was about 3250ng/gFW and about 6.5 times of leaf tissue, significantly higher than that from leaf tissue in the same conditions, and mtDNA extracted from tender root had a higher purity. Results of the agrose gel electrophoresis also showed that mtDNA extracted from tender root had clearer bands than that from leaf. Moreover, the PCR amplifying results of mitochondrial genes showed that the repetition and stability of bands amplified from mtDNA extracted by tender root were much better than that by leaf. So tender root tissue is an ideal material for extracting mtDNA in rapeseed.5. Five sterility-related gene fragments of mtDNA in rapeseed were amplified by PCR to four materials of PolA system and Shaan2A system. The results showed that the same target bands were found by horizontal submarine agarose gels when 3 pairs of the special gene primers of mtDNA were used respectively to the 4 materials, and amplified products were consistent with the expected target fragments. Thereinto, another smaller band was also gotten with primer designed by orf474 and orf159 among 4 materials. The target bands were failed to be found out with primer designed by orf158. One same band was gotten with primer designed by orf224 bewteen PolA and Shaan2A, and amplified products were consistent with expected target fragments. The sequence-mensurating results showed that the sequences of PolA and Shaan2A were also the same at 675bp. In addition, a smaller band was also amplified with primer designed by orf224 bewteen PolB and Shaan2B, which was proved to be DNA fragments consistent with orf224 with 323bp by sequence-mensurating. So guessed that the fragments were relative to the fertility of CMS lines and their maintainer lines.6. With 25 pairs of special primers designed by continuous DNA fragments of the total genome of mtDNA from variety Westar in rapeseed, PCR amplification and Agarose gel electrophoresis were carried on the corresponding DNA section of mtDNA moulds of four materials of PolA system and Shaan2A system. The results showed that total lengths of mtDNAs of four materials were respectively about 200kb, whose framework and arrangement were similar with the known variety Westar. Thereinto, one same expected band was amplified respectively by 11 pairs of specific primers in 4 materials explained that the sections, which covered 44 percent of total genome of mtDNA, might be conservative sequences. Otherwise, one same band was amplified respectively by two pairs of primers in 4 materials, which were different from expected ones. Another pair of primers did not get the amplified products in 4 materials. The above primers respectively got the same band types(or did not get the amplified products) in PCR amplification in 4 materials, therefore forcasted that the homology of the 4 materials were at least 56 percent. However, the other primers had a complicated amplification result in 4 materials explained that these amplification regions, which covered about 44 percent of total genome of mtDNA, might be the variable regions of mtDNA recombination. Some of the primers amplified the different DNA fragments bewteen the CMS lines and their maintainer lines, and some of the primers amplified the different fragments bewteen PolA and Shan2A. Whereas, whether these different fragments have biological function and whether they have any relation with CMS need further study. 7. This paper systemically studied PolA and Shaan2A on three different levels: molecular biology(such as PCR amplification of special gene fragments of cpDNA and mtDNA, sequence-measuring, restriction enzyme analysis of cpDNA), physiology and biochemistry(gel electrophoresis of EST and POD isozymograms), and morphology and anatomy(investigation of plant agronomic trait and observation of flower structure). The total results confirmed that the nuclear genomes of the two CMS lines(PolA and Shaan2A) were obviously different, and their mtDNAs had certain differences thus their cytoplasms also had certain differences, but sequences of the sterility gene(orf224) from PolA and Shaan2A were same. Well then, whether or not were the sterility mechanisms of the two CMS lines completely same? whether or not were there other sterility-related genes on mtDNA of Shaan2A? and so on, these still need further research.更多还原