【作者】 王永勤；

【导师】 曹家树；

【作者基本信息】 浙江大学 ， 蔬菜学， 2003， 博士

【摘要】 白菜（Brassica campestris L. ssp. chinensis Makino）属十字花科芸薹属芸薹种白菜亚种，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种。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，雄性不育的机理及其应用研究一直深受人们的重视。雄性不育产生的机理十分复杂，涉及到小孢子发育的一系列结构基因和调节基因。要彻底搞清雄性不育产生的机制，必须搞清楚小孢子发育过程中的相关基因。从一对等位基因控制的雄性不育两用系入手，是解决这一问题的最佳途径。本研究以‘矮脚黄’白菜（B. campestris L. ssp. chinensis Makino var. communis Tsen et Lee cv. Aijiaohuang）隐性核雄性不育两用系（A/B line）为材料，从形态、生理生化、分子水平上，对可育株系和不育株系的时空表达特征进行研究，并分离与小孢子发育相关的基因全长，取得如下结果： （1）对‘矮脚黄’白菜核不育两用系中不育株系和可育株系田间生长状况、生理生化指标进行了比较研究。结果表明，不育株花药廋小，没有花粉或花粉畸形；莲座期不育株系和可育株系植株整齐一致，开花期不育株系株高显著高于可育株系，其主要原因可能如我们发现的，不育株花蕾抗氰呼吸强度低于可育株，与消耗能量少有关；可育株系与不育株系叶绿素含量、光合作用差异不显著；不育株系花蕾的IAA缺乏，而ZT盈余；不育株系的中花蕾和大花蕾过氧化物同工酶带强于可育株系，但小花蕾和不同时期的叶片未见差异；不同时期可育株系和不育株系叶片和大花蕾之间的细胞色素氧化酶同工酶未见差异，而可育株系中的小花蕾和中花蕾的两条特异的带强于不育株系；可育株系的中花蕾可溶性蛋白电泳表现出两条特异带，而不育株系的中花蕾只有一条特异带。 （2）以花柄为外植体，对白菜核不育两用系再生体系进行了研究，表明花柄外植体表面消毒需要75％酒精处理30s和0.1％升汞处理9min。不同浓度配比的6-BA和NAA对外植体芽的再生和再生曲生根有着不同的影响，不育株的最佳激素组合为2mg／LBA+0.2mg／L NAA，其诱导不定芽再生频率达75.0％；可育株的最佳激素组合为2mg／LBA+0.1mg／L NAA，其诱导再生频率达65.6％；生根培养基以1／2 MS培养基附加0.1mg／L NAA和7.5mg／LAgNO₃为最佳。 （3）对cDNA-AFLP技术体系进行了优化研究，表明cDNA-AFLP预扩增适宜退火温度为53℃，预扩增循环次数为20个循环，预扩增和选择性扩增PCR体系中Mg²⁺浓度均为2.3mM。对白菜核不育两用系进行cDNA-AFLP分析表明：可育株系和不育株系的花蕾之间表达存在差异，而莲座期叶片、开花期叶片和花茎之间基因表达无差异；两用系的叶片、花茎和花蕾三者之间的基因表达存在差异。对开花期的小蕾、中蕾和大蕾分析显示：22对引物中，3对引物在可育株中蕾和大蕾中有4条特异带，1对引物在可育株中蕾中有1条带表达丰度高于不育株中蕾。对其中表达丰度有差异的基因片段进行Northern验证，其结果与cDNA-AFLP结果一致。这说明cDNA-AFLP技术可以用于植物雄性不育基因表达差异的研究。 （4）利用A15T17引物对白菜核不育系两用系不育株系与可育株系表达差异进行cDNA-AFLP分 摘 要析，在可育株系中蕾和大蕾中发现一条特异表达条带（BCMFotl5T17）。Northern杂交证明该基因只在可育中蕾和大蕾中特异表达。利用 RACE技术得到该基因 cDNA全长，全长为 1683 hp；该基因全长 1983 hP，含有3个内含子，将该基因命名为 BCMF。经BLAST查询对比，该基因碱基序列与拟南芥中的一个未知功能基因有较高的相似性：与该基因有一定相似性的其它基因的功能全部未知。BCMPI的最大ORF从第76号碱基开始，第1419号碱基终止，1344个碱基编码447个氨基酸。 （5）用A18T16引物对白菜两用系不育株系与可育株系的表达差异进行CDNA－AFLP分析，发现可育株系的中蕾和大蕾中有两条特异表达的条带，分别命名为 BCMFA18T16．l和 BCMFA18T16．2。 利用RA＊E技术得到＊＊M卜A18＊16－ICDNA全长序列，其全长为1485 hp，将该基冈命名为BCMn。Northern杂交表明该基因只在可育中蕾和大蕾中特异表达。经BLAsT f询对比，其全序列与拟南芥多聚半乳糖醛酸酶M序列具有 83％（764ill52）的相似性JCMF最大Ony为 1263 hP，起始密码子ATG位于第67位，终止密码于为TAA，位于第1329位，其编码420个氨基酸。 利用RACE技术分离了BCMF－A18T16－2片段相关基因的全长，其长度为2077hp，将该基因命名为BCMH。RTPCR分析表明该基因只在可育中蕾和大蕾中特异表达。经BLAST查询对比，其全序列与大白菜的果胶甲酯酶 MA有 92％（1590／171）相似性。BCMF3最大 ORF为 1755 hP，起始密码于M位于第157位，终止密码子为TAA，位于第1911位，编码584个氨基酸。 （6）用A17T14引物对白菜核雄性不育两用系不育株系与可育株系的表达差异进行cDNA－AFLP分析，发现可育株系的中蕾和大蕾中有一条特异表达的条带（BCMPotl7T14）。RTPCR表明该基因只在可育更多还原

【Abstract】 Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino; syn. Brassica rapa L), originated from China, is one of the vegetable cultivars and possesses the largest cultivated area among all vegetable crops in China. Chinese cabbage-pak-choi has a long history of cultivation, and is affluent in resources of cultivars. Chinese cabbage-pak-choi is a typical allogamy plant and has redominant heterosis. Much attention has been_paid to research on the plant breeding and application of male sterility. Mechanism of male sterility is complex, which involved a set of structural and regulative genes in microspore development. The analysis of those genes associated with microspore development should contribute to elucidate the mechanism.We studied male sterility A/B lines of Chinese cabbage-pak-choi about their spatial and temporal expression characters at morphological, physiological and molecular levels. Differential bands from B line were isolated and four full-length cDNA sequences related to the development of microspore were cloned .The results were as follows:(1) Plants growth status, physiological and biochemical factors were compared between A line and B line of genie male sterile A/B line of Chinese cabbage-pak-choi. Results showed that anthers of sterile plants were small, and pollens were abnormal or even no pollen at all; during rosette stage the vegetative growth of the A line was consistent with the B line, but in the blooming stage, the growth_of the A line was remarkably higher than the B line. We deduced that the mainly possible cause of these results was that the floral buds of the male fertile plants need more energy to keep the higher level of cyanide-resistant respiration, because we found the cyanide-resistant respiration in the buds of the B line plants was significantly higher than the ones of the A line plants. There was no significant difference between the photosynthesis rates of leaves in different stages. IAA was absent but ZT was surplus in the buds of the sterile plants. The peroxidase isoenzyme bands of the middle-size and large alabstrums in the A line were more obvious than those_in the B line, but no apparent difference in little alabstrums and leaves. Two more obvious cytochrome oxidase isoenzyme bands existed in little and middle-size alabstrums in B line. However, there was no apparent different band of cytochrome oxidase isoenzyme of leaves and large alabstrums at different stages. Two specific bands in soluble proteins electrophoresis in the B line was found, but only one was found in the A line.(2) The study of plants regeneration from petiole explants of genie male sterile A/B line of Chinese cabbage-pak-choi was made. Results showed that petiole explants were surfacesterilized by rinsing in 75% ethanol for 30s followed by treated with 0.1%(w/v) aqueous mercuric chloride for 9 min was the best treatment; the concentration of 6-BA, NAA, and their ratio affected the inducing rate of adventitious shoot and root; the most suitable hormone combination to sterile explant was 2 mg/L BA and 0.2mg/L NAA, and inducing rate of adventitious shoot reached 75.0%; however, to the fertile explants, the combination was 2 mg/L BA, 0.1 mg/L NAA, whose adventitious shoot regeneration rate was 65.6%; the best root inducing medium comprised 0.1 mg/L NAA and 7.5 mg/L AgNC^.(3) Improving cDNA-AFLP technique and pjeamplified PCR conditions were_as follows: including 20 cycles and 2.3 mmol/L MgCl2, annealing at 53"C. Mg2+ concentration of selective amplification was also 2.3 mmol/L. cDNA-AFLP analysis revealed differential expression was showed in alabstrums and among leaves, floral stalk and alabstrums; however.no difference in rosette leaves, floral leaves and floral stalk leaves was found. Using three of the twenty-two pairs of primers, four differential bands were amplified in middle-size alabstrums and large alabstrums of the B line. With a pair of primers, expression abundance of a differential band in middle-size alabstrums of fertile line was更多还原