【作者】 崔辉梅；

【导师】 曹家树；

【作者基本信息】 浙江大学 ， 蔬菜学， 2004， 博士

【摘要】 白菜（Brassica campestris L. ssp. chinensis Makino; syn. B. rapa L）属十字花科芸薹属芸薹种栽培亚种，原产我国，栽培历史悠久，品种资源十分丰富，是中国蔬菜作物中栽培面积最大的一类。白菜在我国农业生产和人民生活中占有非常重要的地位。白菜与芜菁（B.campestris L. ssp. rapifera （Matzg.）Sinsk）均为典型的异花授粉作物，具有明显的杂种优势。因此，雄性不育系的获得及其应用研究一直深受人们的重视。本研究通过甘蓝型油菜Ogura胞质雄性不育（cytoplasmic male sterility，CMS）系与‘矮脚黄991-6’、‘新选992-24’白菜自交系和‘耐病98-1’芜菁自交系的种间杂交和连续回交，获得了稳定的白菜、芜菁Ogura胞质雄性不育系及其相应的保持系。在获得不育系的过程中，对不同回交世代材料进行形态学、细胞学、生理生化学、分子生物学研究，取得如下结果： （1）对不同回交世代和相应回交亲本（保持系）的田间生长状况进行了比较研究。结果表明，3份材料的F₁植株在形态、育性方面整齐一致，完全不育，形态偏向于甘蓝型油菜，生长势强。每种材料的BC₁在植物学表型上主要有两种类型，一种生长势偏向甘蓝型油菜，生长势强；一种生长势偏向回交亲本，生长势弱。两种类巧不育度和不育率均达到100％。在‘矮脚黄991-6’、‘新选992-24’的BC₂中就有群体较整齐一致，偏向轮回亲本的株系，而‘耐病98-1’的BC₂群体还不整齐。到BC₃时3份材料群体已基本趋向轮回亲本，群体整齐一致，并从中选育出叶色正常不黄化、蜜腺正常、不育性稳定、经济性状良好且各具特点的白菜Ogura CMS系‘OguXX00-4-7A’、‘OguAJH00-14-17A’和芜菁Ogura CMS系‘OguNB 00-1-1-1A’，及其相应的保持系‘OguXX 00-4-7B’、‘OguAJH00-14-17B’和‘Ogu NB 00-1-1-1B’。利用获得的两个白菜OguraCMS系分别与‘早皇白012-08’、‘矮抗青011-16’、‘黄芽菜025-44’、‘J7809-011-15’四个白菜自交系杂交配制F₁，两个白菜（Ogura CMS系与3个白菜自交系获得的F₁表现出较强的杂种优势，初步证明所获得的两个不育系‘OguXX 00-4-7 A’和‘OguAJH 00-14-17A’可用于将来的生产制种。 （2）在Ogura雄性不育系转育过程中，对3份材料的各回交世代的花粉母细胞减数分裂行为进行了观察分析。结果表明，F₁花粉母细胞减数分裂染色体构型为10Ⅱ+9Ⅰ。BC₁生长势强的群体中‘新选992-24’和‘耐病98-1’染色体构型多为10Ⅱ+9Ⅰ，‘矮脚黄991-6’的染色体构型是10Ⅱ和10Ⅱ+7Ⅰ占优势。BC₁生长势弱的群体中‘新选992-24’和‘耐病98-1’染色体构型多为10Ⅱ+7Ⅰ，‘矮脚黄991-6’的染色体构型多为10Ⅱ+1Ⅰ。BC₂生长势强的群体中‘新选992-24’的染色体构型多为10Ⅱ+1Ⅰ，‘矮脚黄991-6’多为10Ⅱ。BC₂生长势弱的群体中‘新选992-24’ 摘 要和‘矮脚黄 991－6’多为 10II，‘耐病 98－l’多为 10if＋6I、10II＋SI。对 3种材料 BC3代根尖体细胞染色体数进行观察，结果显示在‘新选992－24’利‘矮脚黄991－6’的BC。群体中染色体数为 20的细胞已达到 100％，‘耐病 98－l’的 BC。中染色体数为 20的细胞达到 78．120。同时对BCI代不同染色体数在减数分裂1的分离规律进行了研究。形态学和细胞学研究表明，AACC染色体组物种与AA染色体组物种杂交，在较强的选择压下，经少数几代回交即可迅速获得后代性状相对稳定的不育系。 门）对获得的白菜、芜育Ogura CMS系及其相应的保持系的小抱子形成的细胞形态学研究表明，虽然这3个不育系核背景各异，但其花药败育的时期基本相同，均具有单核败育型的特征。小抱于到单核期后不再向前继续发育，而是迅速退化以至解体，开花前彻底败育。不育系的花药在减数分裂的早期绒毡层细胞中已出现液泡，有肥大趋势，而正常花药绒毡层的发育在减数分裂过程中达到高峰，四分体时出现退化迹象，开花前完全解体。不同的 Ogura CMS系虽然核背景不同，但花药败育的时期基本相同，说明 wura CMS胞质工作关系稳定，受核背景影响小。 （4）以 Ogura雄性不育系转育过程中，各材料的厂和 BC。与相应轮回亲本的＊＼花、花蕾为试材，进行了可溶性蛋白（SDS－PAGE）、过氧化物酶同工：酶（POD）、酯酶同工酶（EST）、a．淀粉酶（l－AMY）电泳分析。结果表明，FI和 BCZ与相应轮回亲本的同工酶、可溶性蛋白质存在谱带增减，酶活性强弱变化和 EST同工酶的器宫特异表达等差异，EST同工酶是h、BCZ和相应轮回亲本间差异最大的同工酶，a－AMY在两者之间没有明显的差异。 （5）利用 PCR技术，从甘蓝型油菜 Ogura雄性不育系及获得的不同回文世代的白菜、芜青Ogura材料基因组中，扩增了与Ogura胞质雄性不育相关的oyt38基因。通过T。连接酶将纯化的PCR产物与载体 pGEM－T连接转化至受体大肠杆菌 DHQ中，经 EcOR酶切分析，证明重组质粒中含有 Orf］38全基因，经核着酸序列分析，与报道的 Ogl38中的 A类型序列完全一致。 （6）利用 cDNA－AFLP技术对两种白菜 Ogura的不同回交世代 BCI、BC。与其亲本花蕾期更多还原

【Abstract】 Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino; syn. B. rapa L), originated from China, is one of the vegetable cultivars possesing the largest sowing area among all vegetable crops in China. There is a long history about the cultivation of Chinese cabbage-pak-choi, therefore its resources of cultivars is very affluent. Chinese cabbage-pak-choi and turnip (B. campestris L. ssp. rapifera (Matzg.) Sinsk ) are typical allogamy plants and have obvious heterosis. So far much attention has been paying to the research on breeding of the male steriles line and basis for application in Chinese cabbage pak-choi. In this research the Ogura CMS (cytoplasmic male sterility) lines of Chinese cabbage-pak-choi (B. campestris L. ssp. chinensis Makino var. communis Tsen et Lee) and turnip were obtained through interspecific hybridization between Ogura CMS line in B. napus and Chinese cabbage-pak-choi and turnip, and successive backcrosses with Chinese cabbage-pak-choi and turnip as male parents. In the course of acquiring cytoplasmic male sterility lines, Studies were performed on morphology, cytology, physiology, biochemical and molecular biology in various backcross generations. The main results are as follow:(1) Comparative analysis has been made among backcross generations and their maintainer lines on field growth condition. The results of plant morphology in field indicated that three F1 plants had the same morphological and sterile properties, which similar to B. napus with strong growth. There were mainly two morphologically type in BC1 plants. One type had strong growth similar to B. napus, the other type had weak growth similar to the parent of backcross. Male sterility occurred in all the plants of the BC, generations. Some lines in ’Xinxuan 992-24’ and ’Aijiaohuang 991-6" BC2 generation had the same growing characteristics as their recurrent parents, while the BC2 plants of ’Naibing 98-1’ were phenotypically intermediate or similar to the B. napus parent. In ’Naibing 98-1" BC3 generation plants had the same morphological character as their recurrent parents. The ’OguXX 00-4-7A’ , ’OguAiH 00-14-17A’ and ’Ogu NB 00-1-1-1 A’ CMS lines and ’OguXX 00-4-7B’ , ’OguAJH 00-14-17B’ and ’Ogu NB 00-1-1-lB’ maintenance lines were obtained. The CMS lines seedlings did not show chlorosis at low temperature. All the plants of the CMS lines had normal nectary and male gametes were highly sterile and female gametes were fertile. F1 obtained from ’OguXX 00-4-7A’ , ’OguAJH 00-14- 17A’ as female parents, and ’ZHB012-08’ , ’AKQ011-16’ , ’HYC025-44’ and ’ J7809-011-15’ as male parents respectively. The results indicated that three F1 have heterosis, andthe CMS ’OguXX00-4-7A’ and ’OguAJH 00-14-17A’ could be used to produce F1 hybrids.(2) The chromosome meiotic behavior of F1 hybrids and their backcross generations between B. napus L. CMS line and B. campestris L. have been analyzed. The chromosome configuration of F1 hybrid were 10II+9 I . In the plants with strong growth plant in BCi, the chromosome configurations of ’Xinxuan 992-24’ and ’Naibing 98-1’ were 10 II+9 I , and the chromosome configurations of ’Aijiaohuang 991-6’ were mostly 10II or 10II+7 I . In the plants with weak growth in BC1, the chromosome configuration of ’Xinxuan 992-24’ and ’Naibing 98-1’were 10 II +7 I , and the chromosome configurations of ’Aijiaohuang 991-6’ were mostly 10II+1 I . In the plants with strong growth plant in BC2, the chromosome configurations of’Xinxuan 992-24’ and ’Aijiaohuang 991-6’ were 10II+1 I and 10II, respectively, and the chromosome configurations of ’Aijiaohuang 991-6’ were mostly 10II. In the plants with weak growth in BC2, the chromosome configurations of ’Xinxuan 992-24’ and ’Aijiaohuang 991-6’ were 10II, and the chromosome configurations of Naibing98-1 were mostly 10II +6 I and 10lI+5.The chromosome number in BC3 generation of ’Aijiaohuang 991-6’ and ’Xinxuan 992-24’ were 20. The percentage of cell with 2n=20 was 78.12% in BC3 generation of ’Naibing 98-1’. These results sugges更多还原