【作者】 张艳；

【导师】 王小佳； 李成琼；

【作者基本信息】 西南大学 ， 园艺学， 2010， 博士

【摘要】 结球甘蓝(Brassica oleracea L. var. capitata L.)杂交优势明显,利用细胞质雄性不育系对甘蓝等叶类蔬菜进行育种具有独特的优势。但甘蓝中尚未发现天然细胞质雄性不育系,生产上多通过转育外源不育细胞质获得甘蓝细胞质雄性不育系,研究甘蓝细胞质雄性不育性对核背景的响应特性,探寻不同细胞质不育类型其雄性不育性对核背景的响应特性,对丰富和完善细胞质雄性不育理论和确定选育方向,减少转育工作量有重要意义。本文以回交转育获得的多组同质异核细胞质雄性不育系为材料,首先利用各类细胞质雄性不育特异的基因鉴定引进不育材料的类型,然后观察各类细胞质雄性不育花器官形态对核背景的响应,而后再对其中三份材料进行多代连续回交转育,获得三组稳定的同质异核雄性不育系。通过石碏切片观察研究了三种不育细胞质花粉败育特征对不同核背景的响应,并利用半定量RT-PCR研究了线粒体上相关基因和花形态建成相关基因在花蕾发育不同阶段的表达情况；同时利用限制性内切酶酶切图谱分析了三种不育细胞质线粒体基因组结构在不同核背景下的变异情况；最后利用聚丙稀酰胺凝胶电泳分析不同核背景ATP、POD同工酶,从同工酶角度研究不同核背景的雄性不育系代谢差异,及细胞质对核基因的反向调控。主要研究结果如下：(1)国内目前对甘蓝上应用细胞质雄性不育类型缺乏分类鉴定,对各种不育细胞质类型的应用情况尚不明确,不育细胞质类型较为单一。本研究对从国内外多家科研机构引进的15份细胞质雄性不育材料进行鉴定,仅发现1份属于Nap型细胞质不育系,其余14份材料均属于Ogu胞质不育类型。(2)Nap型细胞质不育材料和两份Ogu细胞质不育材料花器官形态具有较大差异。Nap型细胞质不育材料花瓣呈喇叭状,雄蕊短小退化,花药形态多样,蜜腺退化。第一组Ogu细胞质不育材料花瓣皱缩,雄蕊心皮化,蜜腺退化；第二组Ogu不育花瓣较正常,雄蕊部分退化。Nap型花器官形态对不同核背景的响应表现3种类型,第1类与原材料相近或呈负响应,第2类表现为正响应,第3类花器官不同部位响应不一,呈现正或负响应。Ogu型花器官形态对核背景的响应主要表现为不变化或较弱的正响应。同一自交系对不同来源的不育材料影响的稳定性,因自交系不同而异,对于自交系K1、N4,不同来源胞质材料对其响应趋于一致,对自交系37、F1、G7的响应,在不同来源胞质材料间存在差异。(3)对Nap型胞质和Ogu型细胞质不育材料花蕾细胞学形态的研究表明,花药以及其中花粉的发育情况受到核背景的影响。Nap型细胞质不育系在不同核背景下败育类型分为无花粉囊分化型和有花粉囊分化型,有花粉囊分化的类型中有部分材料在四分体时期花粉停止发育,还有个别不育材料中四分体可以分离出小孢子,属于花药不开裂导致的雄性不育。第一组Ogu型不育材料花药有心皮化趋势,但是不同核背景下败育特征仍有较大差异,花粉囊分化的情况在不同核背景下表现很不一致。并且,在该组材料普遍雄蕊心皮化的情况下,编号为95材料仍具有较为正常的花药发育。第二组Ogu型不育系败育时期比较稳定,都集中于四分体时期,而花粉囊分化和绒毡层的发育异常方面,在不同核背景下略有差异。(4)通过对线粒体雄性不育相关候选基因在花蕾发育不同阶段和不同转育核背景下表达的研究,发现Nap型细胞质雄性不育材料中orf222, cox1, atp6 3个基因的变化趋势基本一致。cox1, atp6以及不育特异基因orf222均受到核背景的影响,在小中大花蕾中表达的规律,三个基因在不同核背景中表现不同,分为前期表达量高后期降低和发育不同阶段表达一致两种类型。这与材料败育特征表现有相关性。orf222, cox1, atp6表达量前期高后期降低的不育材料表现为无花粉囊型败育；而三个基因在花蕾发育过程中表达量稳定的不育材料表现为有花粉囊分化的败育类型。atpA、atp9在不同大小花蕾中和不同核背景下表达量无显著差异,即不受核背景的影响。Ogu型细胞质雄性不育系相关不育基因的表达则与Nap存在差异,且两类Ogu不育之间也不一致。79#来源的不育胞质中orf138在不同核背景下表达存在一定的差异,cox1, atp6 atpA、atp9的表达则不受花蕾发育阶段和核背景的影响。对于99#来源的第二组Ogu胞质,在orf138、cox1, atp6 atpA, atp9 5个线粒体基因的表达无明显差异,其表达既不受核背景影响,也不随发育阶段改变而发生变化。(5)对与花形态建成相关的AP3、PI、AG基因表达分析,表明AP3、AG在Nap型和Ogu型细胞质不育材料中的表达均表现为不受核背景的影响,并且在花蕾发育的三个阶段表达量保持稳定。但是PI基因在同质异核的不育材料间其表达量表现出了显著变化,推测PI在控制雄性不育系花器官形态发育上有重要作用。(6)经XhoⅠ、PstⅠ、BamHⅠ、EcoRⅠ四种限制性内切酶对三组不育材料线粒体基因组进行酶切后发现,13份Nap型细胞质同质异核不育材料之间,在四种限制性内切酶酶切图谱中没有差异。但两类ogu型不育中均检测到线粒体结构的变异,第一组Ogu型细胞质线粒体在8个材料中发现有1份材料出现了酶切图谱的变化；第二组Ogu型细胞质线粒体中8个材料中发现了3份不育材料出现了相同的酶切图谱上的变化。线粒体的结构受核背景的影响,并且趋向于在相同的位点发生重排。(7)对同质异核的不育材料ATP和POD同工酶的分析表明,Nap型细胞质雄性不育材料中ATP同工酶相对保持系,在花蕾发育后期酶活性显著降低,部分材料还表现出谱带的缺失。两组Ogu型细胞质不育材料在花蕾发育不同阶段ATP酶活性基本一致。但在花蕾发育后期同样有部分核背景下表现出谱带的缺失。表明ATP酶在不育材料中的变异程度不同核背景间有一定差异。编号为93#和107#的材料分别属于两组Ogu细胞质雄性不育系材料,两者具有相同的保持系,但是在不同细胞质条件下两者的POD同工酶谱带存在差异,Rf=0.787的酶带存在于93#中,但在107#中则缺失。并发现79#不育系中存在的Rf=0.382的核质互作产生的POD谱带,表明细胞质对POD同工酶组成有调控作用。更多还原

【Abstract】 Cabbage (Brassica oleracea L. var. capitata L.) is an important leafy vegetable crop with widely growing area only second to Chinese cabbage in China. Cabbage is typical allogamy plants and has obvious heterosis. Utilization of male sterile lines is becoming the trends in cabbage heterosis breeding. But cytoplasmic male sterility has not yet been found in cabbages. In application, cabbage CMS lines are always obtained by transmitting foreign sterile cytoplasm to cabbage. Studying on the response of cabbage cytoplasmic male sterility to nucleus background is significant to improve the mechanism of cytoplasmic male sterile and make it more convenient to determined the direction of breeding as well as reduce the breeding workload.Plant materials used in the parent study involved many alloplasmic lines which developed by hybridization between introduced CMS lines and different inbred lines, and then successive backcross with those inbred lines as male parents. The CMS types of introduced CMS lines were identified by amplified special cytoplasmic male sterile associated gene, then morphology response of the flowers to nuclear background was observed, after that, anther abortion stages and character were studied by the means of paraffin section method to detect the cytomorphology response of anther to nuclear background in alloplasmic lines developed from CMS lines 78、79、99 backcross with their maintainers. The expression of mitochondrial genes and flower morphogenesis-related genes were analysis using semi-quantitative RT-PCR in different developmental stages of bud. Mitochondrial structure was analyzed by restriction enzyme in and between the three group of CMS lines which with same sources of cytoplasmic and different nuclear background. Isozymes of POD, ATPase, were also analyzed in those materials. The main results are as follows:(1) Cytoplasmic male sterility materials in Brassica oleracea were lake of classification and identification at present. The application of every type of cytoplasmic male sterility material was also not so clear. Fifteen cytoplasmic male sterile materials introduced from home and abroad in Brassica oleracea was identified in this paper. Ogu CMS take up the most part in the materials investigated. Fourteen materials of 15 belong to this class. Only No.78 belongs to Nap CMS. The usage of sterile cytoplasm on radish (Ogu CMS) was overwhelmed.(2) There was an obvious difference between the Nap-type cytoplasmic male sterile and two Ogu cytoplasmic male sterile on floral organs morphology. Nap-type cytoplasmic male sterile petal was horn, and with degraded short stamens, anthers shapes varied greatly, degradation nectary. The first group of Ogu CMS petals wrinkled, and with pistillody stamens, degraded nectarys; the second group of Ogu CMS, their petals were normal, stamens were partly degraded. The response of Nap CMS to different nuclear background showed three types. The first type was similar with original material or show negative response; The second type was positive response; The third type was complicated, varying with floret parts. The Ogu CMS materials either showed a weak positive response to nuclear background or keep unchanged comparing to original material. Morphology response of different source of Ogu CMS to the same nuclear background was varied with unclear background. The inbred-line K1, N4 have similar impact on different cytoplasmic male sterile, but 37, F1 and G7 have different effect on different sources of cytoplasmic male sterile in flower morphology.(3) Cytomorphological analysis indicated that the abortion stages and characteristics of Nap CMS and Ogu CMS were affected by nucleus background. Nap-type cytoplasmic male sterile lines with different nucleus background were divided into two type, some materials were no pollen sacs differentiated, others abortion occurred after pollen sacs differentiated. As far as the later with pollen sac, some materials inhibition of pollen development occurred at tetrad stage, but microspores could be released from tetrads in several other materials, abortion in those CMS lines caused by anther failed to cracking.In the first group of Ogu CMS materials, their anther show a trend of Pistillody, but nucleus background had a deep affection on the performance of abortion. Absolutely, differentiation of anther were affected by nucleus background. In spite of anthers were pistillody in almost all CMS materials in this group, material coded 95 still had relatively normal anther development. In the second group of Ogu CMS materials, their abortion stages were relatively steady in different nucleus background, which were concentrated in tetrad stage. The abnormity of tapetal and anther development show some difference between different nucleus backgrounds in this group.(4) Expression analyze to CMS-associated mitochondrial gene at different development stages reveal that, as far as Nap CMS materials, expression level of orf222, cox1, atp6 always had same trend during bud development in same Nap CMS lines. Expression of the three genes (orf222, cox1, atp6) also affected by nucleus background. The expression tendency of the three genes were varied with nucleus background, which could been classified in two types:expression of these three genes were reduce in middle and large bud compared to that in small bud or show no difference among three stages investigated. This reveals some relationship with corresponding abortion character. Nap-CMS materials which expressed orf222, coxl, atp6 in small bud mostly had no pollen sacs differentiated. Whereas in these CMS lines which had pollen sacs development, orf222, coxl and atp6 express in similar abundance. atpA, atp9 shown no significant difference among three development stages of all the Nap-CMS materials investigated, so the expression of these two genes was not regulated by nucleus background.Performance of these mitochondrial genes in Ogu-CMS materials was different with that in Nap-CMS materials, and they were not identical between two groups of Ogu-CMS lines. In the first grope of Ogu-CMS materials whose cytoplasm sourced from No.79, the expression of orf138 show a little difference in different development stages in part of materials in this group. Expression of coxl, atp6 atpA, atp9 was identical among different development stage and different Ogu-CMS lines in this group.In the second grope of Ogu-CMS materials whose cytoplasm sourced from No.99, there were no obvious differences in such five mitochondrial genes orf138, cox1, atp6 atpA and atp9. Difference was detected neither in three development stages nor in different CMS lines. Expressions of these five genes were hardly affected by the nucleus background.(5) Expression analysis of genes which related to the development of floral organ was carried out by semi-quantitative RT-PCR, the results show that expression of AP3, AG were identical among three development stage of all the CMS materials, whether in Nap-CMS lines or in Ogu-CMS lines. However, the expression of PI show a significant change among these materials with different nucleus background, it suggests that PI may has an important role causing diverse abnormality of floral organ in CMS lines.(6) Mitochondrial DNA was isolated from three groups of CMS materials, restriction enzymes Pstl, Xhol, BamHl and EcoRl were used to digest the mtDNA. It was found that Restriction patterns of Mitochondrial DNA were identical among 13 Nap-CMS lines, However rearrangements of mitochondrial DNA were observed in both two groups of Ogu-CMS lines. In 8 Ogu-CMS lines of first group, one of them was distinct from others in BamHl-digested restriction map. It suggests that structure of mitochondrial genome have changed in this material, which may be result from its nucleus background.In 8 Ogu-CMS lines of second group, we found three CMS lines whose restriction map diverge considerably from those of other five CMS lines. It suggests that the structure of mitochondria genome was affected by the nucleus background in this group. The result also indicate that rearrangement tend to occur in same set, although the impact come from different nucleus background.(7) Isoenzymes of ATPase and POD were analyzed in this paper. The result show that, comparing to male fertile line, activity of ATPase reduced markedly in large-bud of Nap-CMS lines. In some of Nap-CMS lines, several band even have not been detected in large-bud. There was on significant difference in activity of ATPase in Ogu-CMS lines, whether first group or second group, but number of bands were digressed in middle and large bud in some Ogu-CMS lines. The absence of bands was varied with nucleus background.No.93 and No.107 belong to two groups of Ogu-CMS material respectively, but they had same maintainer. There was difference between these two lines in POD isozyme bands. The band Rf=0.787 was found in No.93, but obscene in No.107. In CMS line No.79, the band Rf =0.382 was detected which result from interaction of cytoplasm and nuclear. The results show the influence of cytoplasmic on the composition of POD enzyme.更多还原