【作者】 许忠民；

【导师】 巩振辉；

【作者基本信息】 西北农林科技大学 ， 蔬菜学， 2011， 博士

【摘要】 甘蓝(Brassica oleracea var. capitata L.)是世界普遍栽培的一种重要的叶类蔬菜,其为异花授粉作物,具有明显的杂种优势。利用雄性不育系生产杂交种已经成为甘蓝育种发展的一种趋势,而细胞质雄性不育系(Cytoplasmic male sterility,CMS)是甘蓝杂种优势育种的一种理想途径,具有重要价值,并己杂种优势中得到利用。我们发现一种新型甘蓝细胞质雄性不育系P451,通过多代回交转育,育成了不育性稳定,蜜腺正常,低温叶片无黄化和配合力高的甘蓝细胞质雄性不育系CMS451,并在生产上应用。为了明确CMS451不育机理,本研究对甘蓝CMS451从植物学、细胞学、生理生化和分子生物学等方面进行研究,试图探索甘蓝CMS451不育机理,为进一步更好地利用这一优良不育源奠定基础。主要研究结果如下：1.不育系与保持系形态比较研究表明,甘蓝CMS451不育系雄蕊花丝较短,花药较白、不能正常开裂,不能散出花粉,蜜腺正常。不育系花蕾发育较保持系慢,当花蕾长为6.5mm时花药逐渐变黄,饱满程度不如保持系。2.利用甘蓝不育系CMS451及其保持系分别作为母本和同一父本杂交,结果表明,二者作为母本的F1代植物学性状表现基本相似,没有显著性差异,但不育系F1单球重较高,中心柱较短,品质更好,说明不育系CMS451配合力强,优于保持系。通过利用甘蓝不育系CMS451大田制种表明,不育系蜜腺发达,能正常吸引蜜蜂,结实正常,分枝数和株高均优于保持系,产量较高,杂交种纯度达100%,制种成本较低。因此,甘蓝细胞质雄性不育系CMS451具有良好的育种和应用前景。3.小孢子细胞学观察表明CMS451不育系的花药败育时期为单核小孢子时期,此时绒毡层细胞与中层细胞逐渐分离,细胞液泡化并伸长膨大,营养物质消失,挤压小孢子,使小孢子只能挤成一团。随后,绒毡层细胞完全与中层细胞分离,并继续伸长膨大,接着绒毡层细胞逐渐降解,直至完全消失。随着绒毡层细胞降解,小孢子始终粘连在一起,也逐渐降解,形成一团染色很深的物质。到花药开裂期时,药室不能开裂释放花粉粒,小孢子彻底解体,其残余物留在药室形成黑色的条带。4.利用酶联免疫检测技术,研究花蕾不同发育时期内源激素含量的动态变化及激素之间的平衡关系。结果表明,不育系CMS451花蕾GA3和ZR含量均低于保持系,变化趋势与保持系基本相同；不育系IAA含量均高于保持系,变化趋势不育系为先降后升,保持系是持续下降；ABA含量在小孢子母细胞期不育系高于保持系,其它时期低于保持系。表明,在甘蓝花蕾发育早期不育系花蕾中IAA和ABA过度积累,ZR和GA3的亏缺可能是细胞质雄性不育发生的原因,而在花蕾发育后期则是不育产生的结果。5.游离氨基酸分析结果表明,不育系CMS451叶片和小花蕾中各种游离氨基酸含量与保持系略有不同,但总量相当,差异不明显；但大花蕾中游离氨基酸总量不育系显著高于保持系。甘氨酸、缬氨酸、异亮氨酸、亮氨酸和苯丙氨酸在甘蓝叶片、小花蕾和大花蕾中的游离氨基酸含量不育系均高于保持系,不育系均低于保持系的游离氨基酸为脯氨酸。据推测,不育系营养器官和生殖器官中甘氨酸、缬氨酸、异亮氨酸、亮氨酸和苯丙氨酸过度积累和脯氨酸匮乏,可能是导致甘蓝花药败育的重要原因。6.通过对甘蓝细胞质雄性不育系和保持系不同发育时期花蕾的活性氧指标及其主要抗氧化酶活性研究,结果表明,甘蓝花蕾不同发育时期,不育系花蕾中O2产生速率、H202和MDA含量均高于保持系花蕾,不育系花蕾中POD、SOD和CAT等酶活性均高于保持系。据此推测,不育系产生的活性氧过多,刺激植物产生大量的抗氧化酶进行清除,在花粉粒成熟期,不育系花蕾中SOD和CAT等酶活性降低,但H202和MDA却持续升高,不能完全清除达到平衡状态,导致膜中的蛋白质聚合和交联,以及类脂的变化,最终会造成生物膜损害和代谢失调,导致雄性不育。7.通过PCR扩增技术,在甘蓝CMS451不育系mtDNA上扩增出大小为594bp一条特异片段,该序列与Ogura胞质不育系特异片段Z12626序列的一段完全一致,Z12626包含有orfl38和orfl58两个开放阅读框。CMS451特异片段上游序列(1-228)与orfl38下游序列(189-417)完全一致；下游序列与orfl58上游序列完全一致,说明甘蓝不育系CMS451mtDNA包含有orfl38和orfl58两个特异片段,与OguCMS一致,因此甘蓝不育系CMS451来源于为OguCMS,并说明OguCMS不育相关基因具有保守性。更多还原

【Abstract】 Cabbage is a leafy vegetables widely cultivated all over the world, which is a cross-pollinated crops with significant hybrid heterosis. Using male sterile line obtain hybrid cultivar is a tendency of cabbage breeding. Cytoplasmic male sterility (CMS) is an ideal method for heterosis breeding, and it has an important and practical value on cabbage breeding. CMS451 cultivated by backcrossing cabbage cytoplasmic male sterile P451.CMS451 is fully express infertility, normal nectaries physiological function, no yellow leaves at low temperature and high combinationability. In order to ascertain sterile mechanism of CMS451, we have studied it from botany, cytology, physiology and biochemistry and molecular biology. It laid the foundations for furthermore utilize the beneficial of this excellent sterility source. The main results of the research are presented as follows.1. Morphological comparison between CMS451 and its maintainer showed that CMS451 has shorter filament and whiter anther. It could not crack and spill out pollen normally, but its nectary is normal. The bud of CMS451 develops slower than its maintainer. The anther became yellow gradually when the bud length grew to 6.5 mm. The plumpness of CMS451 was lower than its maintainer.2. CMS451 and its maintainer as female parent were crossed with same male parent, the results shows that F1 generation characters of botany be similar and there was no significant difference, it has the shorter head central axis, the more heavier head and the better quality. The combining ability of CMS451 was higher than its maintainer. The nectary of CMS451 was much more active, it has fertility and attract insects normally. The sent number and stub higher than maintainer, the yield is high and the purity of hybrid was 100%, so the cost of breeding was low. We believe that CMS451 has great application and development prospects in Cabbage breeding.3. Microspore cytological observation showed that pollen abortion of CMS451 happened at uninucleate stage. The tapetal cells and middle layer cells divided gradually, the cells were vacuolization and elongation, microspore squeezed together and nutritious substance was lost. Subsequently, the tapetal cells separate from the middle layer cells completely, and then the tapetal cells to disappear in the end. Microspore stick always together with the degradation of tapetal cells, formed into deep staining material. The mature pollen can not release from the dehisced anther lead to microspore gradual disintegrated, the remains of something that form a black band.4. Enzyme-linked immunosorbent assay (ELISA) method was employed to study the changes of endogenous hormones level and the balance between hormones in different developmental stages of the bud. The results showed that the content of GA3 and ZR in buds of male sterile lines CMS451 were lower than its maintainer line, they had same changing trends in male sterile line and its maintainer. IAA content of CMS451 was higher than its maintainer, trends of IAA had a change of descend firstly then ascend while it was continuous decrease in maintainer. The content of ABA in CMS451 was higher than its maintainer at microspore mother cell stage, and the other stage just the reverse. This shows that the reason of cytoplasmic male sterility was an excessive accumulation of IAA and ABA in buds of Cabbage male sterile lines and the deficiency of ZR and GA3 in the early developmental stage.5. Analysis of the free amino acid showed that the level of free amino acid in CMS451 leaves and small buds is slightly different from its maintainer, but the difference is not obvious in total amino acid level. In CMS451 large buds, the level of free amino acid was significantly higher than its maintainer. In the leaves, small buds and the large buds, the male sterile line is higher than maintainer for the content of glycine, valine, isoleucine, leucine and phenylalanine, just the opposite for proline. The reason of male sterility supposedly was an excessive accumulation of glycine, valine, isoleucine, leucine, phenylalanine and the lack of proline in the organs of reproduction and vegetation account for male sterility in Cabbage.6. The reactive oxygen and major antioxidant enzyme activity was analysed in Cabbage male sterile line and its maintainer, results shows that rate of (?) production, the content of H2O2 and enzymes activity of MDA, POD,SOD and CAT in male sterile line buds were higher than its maintainer during different developmental stages. It was presumed that the excess generation of active oxygen in male sterile line lead to the anti-oxidant enzymes level increased to remove them. In the buds of male sterile line, SOD and CAT activity decreased at stage of mature pollen grain, while H2O2 and MDA activity increases continuously, oxidant and anti-oxidant are not to maintain in a state of equilibrium so that proteins cross-linking in the plasma lemma, this state will eventually result in metabolic disorder and damage of biomembrane lead to infertility of male.7. PCR results demonstrated that the Cabbage CMS451 mtDNA amplified the 594bp specific band. The DNA sequence of CMS451 was coincident to specific band Z12626 of Ogura CMS, Z12626 contains two open reading frame orf138 and orf158.The sequence (1-228) of CMS451 and the sequence (189-417) of orfl38 were completely the same sequences, downstream sequence of CMS451 and upstream of orfl58 were completely the same sequences. These results indicated that the CMS451 mtDNA contain orf138 and orf158 in accordance with OguCMS, the origin of CMS451 is OguCMS; in addition, genes related to cytoplasmic male sterility are highly conserved.更多还原